HK1015275A - Connective tissue growth factor - Google Patents

Description

Connective tissue growth factor

The completion of the invention is supported by government with grant numbers: GM37223, funded by the national institutes of health. Accordingly, certain rights are also made to the government of this invention. Background

Technical Field

In a broad aspect, the invention relates to the field of growth factors, and in a minor aspect, the invention relates specifically to Connective Tissue Growth Factor (CTGF), polynucleotides encoding such factors, and methods of using CTGF.

RELATED ART

Growth factors are a class of secreted polypeptides that stimulate target cells to proliferate, differentiate, and form organisms in tissues undergoing growth and development. The action of growth factors relies on binding to specific receptors, thus initiating intracellular signaling processes. Some of the more extensively studied growth factors include Platelet Derived Growth Factor (PDGF), insulin-like growth factor (IGFI), transforming growth factor beta (TGF-beta), transforming growth factor alpha (TGF-alpha), Epidermal Growth Factor (EGF), and Fibroblast Growth Factor (FGF).

PDGF is a positively charged, thermostable protein found in the alpha-granules of circulating platelets and is known to be a mitogen and chemotactic agent for connective tissue cells such as fibroblasts and smooth muscle cells. Due to these activities of PDGF, this molecule is thought to be a major factor involved in the normal healing process of wounds and to play a role in the pathological process of certain diseases such as atherosclerosis and fibrotic diseases. PDGF is a dimeric molecule consisting of an a chain and a B chain. These two chains form either heterodimers or homodimers, and all combinations of dimers isolated to date are biologically active.

In investigating the effects of various growth factors on tissue regeneration and repair, PDGF-like proteins were discovered. These proteins have the same immunological and biological activities as PDGF and can be blocked by PDGF-specific antibodies. These new growth factors may play an important role in the normal development, growth and repair of human tissues. Therapeutic agents derived from these molecules may be used to accelerate normal or abnormal growth processes involving connective tissue in certain clinical situations such as wound healing. When these growth factors are involved in the pathological process of the disease, therapeutic measures against these proteins can be used to control or improve the uncontrolled tissue growth.

The formation of new and regenerated tissues requires the mutual regulation of a variety of genes that produce regulatory and structural molecules involved in the process of cell growth and tissue construction. Transforming growth factor beta (TGF-. beta.) appears to be a very important regulatory component in this process. TGF- β is released by platelets, macrophages and neutral cells, which appear in the early stages of the repair process. TGF-beta can be used as a growth stimulating factor of interstitial cells and can also be used as a growth inhibiting factor of endothelial cells and epithelial cells. The growth stimulatory effect of TGF- β appears to be mediated by an indirect mechanism, including autocrine growth factors such as PDGF BB or PDGF AA or Connective Tissue Growth Factor (CTGF).

The activity possessed by many members of the TGF- β superfamily suggests their potential application in the treatment of cell proliferative diseases such as cancer. In particular, TGF-. beta.has been shown to be a potent growth inhibitor of a variety of Cell types (Massague, Cell 49, Vol. 437, 1987), MIS has also been shown to inhibit the growth of human endometrial cancer in nude mice (Donahoe, et al, Ann. Surg., Vol. 194, p. 472, 1981), and inhibin. alpha.inhibits the development of ovarian and testicular tumors (Matzuk, et al, Nature, Vol. 360, p. 313, 1992).

Many members of the TGF- β family are also important mediators of tissue repair. TGF-. beta.has been shown to have a significant effect on collagen formation and is responsible for the pronounced angiogenic response in neonatal mice (Roberts, et al, Advance in the American national academy of sciences (Proc. Natl. Acad. Sci. USA 83, 4167, 1986.) osteoplastic proteins (BMPs) induce new bone growth and are effective in the treatment of bone fractures and other skeletal defects (Glowacki, et al, Lancet. 1, 959, 1981; Ferguson, et al, Studies in and related to clinical orthopedics (Clin. Orthoped. Relat. Res.), 227, 265, 1988; Johnson, et al, Studies in and related to clinical orthopedics (Clin. Orthoped. Relat. Res.), 230, 257, 1988).

The isolation of growth factors and their encoding genes is important for the development of diagnostic and therapeutic agents for various connective tissue related diseases, and the present invention provides such an invention.

Summary of The Invention

Various cell types produce and secrete PDGF and PDGF-related molecules. In an attempt to identify the type of PDGF dimer present in the growth medium of cultured endothelial cells, a novel growth factor was discovered. This previously unknown factor, designated Connective Tissue Growth Factor (CTGF), is immunologically and biologically related to PDFG, but is the product of another distinct gene.

In a first aspect of the invention, a connective tissue cell polypeptide growth factor is provided. The polypeptide is a cell mitogen and chemotactic agent.

In a second aspect, the invention provides a polynucleotide encoding connective tissue growth factor, characterized by encoding a protein that (1) exists as a unimer having a molecular weight of about 36-38kD, and (2) binds to a PDGF receptor.

In another aspect, the present invention provides a method for accelerating wound healing in a patient by applying to the wound an effective amount of a composition comprising CTGF.

In yet another aspect, the present invention provides a method for diagnosing a pathological condition in a patient suspected of having a pathology characterized by a cell proliferation disorder, the method comprising: (1) obtaining a sample suspected of containing CTGF from the patient, (2) determining the level of CTGF in the sample, and (3) comparing the level of CTGF in the sample to the level of CTGF in normal tissue.

The invention also provides a method of ameliorating a disease characterized by a cell proliferative disorder by treating the disease site with an effective amount of an agent that binds CTGF.

The present invention identifies a TGF- β reactive or regulatory element located in the 5' untranslated nucleotide (about-154 to-145) segment of the CTGF gene. Based on this element identification, the invention thus provides a method for identifying a composition that affects CTGF expression, the method comprising incubating a composition comprising the composition with a TGF- β regulatory element (T β RE) and adding TGF- β factors that modulate T β RE, and determining the effect of the composition on CTGF expression. Accordingly, the present invention provides a method for discovering a drug for treating fibrotic diseases.

Brief Description of Drawings

FIG. 1A shows the structural composition of the CTGF gene. Exons are indicated as boxes, where the filled-in regions of the gene correspond to open reading frames.

FIG. 1B shows a comparison of the nucleotide sequences between the CTGF promoter and the fisp-12 promoter, with the same nucleotides being marked with asterisks. TATA boxes and other consensus sequences are also indicated and shaded. The transcription start point is indicated at position + 1.

FIGS. 1 C.1-1 C.3 show the complete nucleotide sequence of the CTGF structural gene and the deduced amino acid sequence, as well as the 5 'and 3' untranslated sequences.

FIG. 2 shows Northern blot analysis. A illustrates that short-term application of TGF-. beta.causes delayed induction of CTGFmRNA. The confluent culture of human skin fibroblasts is incubated by DMEM-ITS culture solution which contains 5 mug/ml of insulin, 5 mug/ml of transferrin and 5ng/ml of selenium, and TGF-beta is added after 24 hours of culture. After 1 hour of treatment with 10ng/ml TGF-. beta.the cells were washed with PBS and incubated with DMEM-ITS for various times as indicated in the figure. B illustrates the effect of Cycloheximide (CHX) on CTGF mRNA induction. Lanes a and H are 4H and 24H untreated control cells, respectively. Lane B: 4 hours cycloheximide (10. mu.g/ml); lane C: 4 hours, reacting cycloheximide for 2 hours, and adding TGF-beta for 1 hour at the 1 st hour to react; lane E: RNA was prepared as in B, but 24 hours after cycloheximide addition; lane f: 24 h TGF-. beta.s (10. mu.g/ml); lane G: RNA was prepared as in D, but 24 hours after cycloheximide addition and 22 hours after TGF-. beta.removal. C illustrates the effect of protein synthesis inhibitors on CTGF mRNA induction. Cells were treated with puromycin or anisomycin for 4 hours. TGF-. beta.was added 1 hour after addition of the protein synthesis inhibitor, and total RNA was isolated after 3 hours of continued cell incubation. The transcription of CTGF was analyzed by Northern blotting as described in the examples.

FIG. 3A shows deletion analysis of CTGF promoter-luciferase constructs. Known consensus sequences are indicated in the figure. NIH/3T3 fibroblasts were transfected with this construct, 10ng/ml of TGF-. beta.was added to activate the cells, and cell extracts were prepared 24 hours later. The relative degree of induction was expressed as a fold higher than in control cells without induction and normalized with β -galactosidase activity obtained from co-transfection of control plasmids and CTGF constructs. These studies were repeated 6 times with similar results. The data presented in the figure are the mean of two transfections of the indicated construct from a single experiment.

FIG. 3B shows the response of an enhancer-free SV40 promoter element-luciferase reporter construct containing the TGF- β region of the CTGF promoter to TGF- β. The CTGF promoter region shown was cloned upstream of the enhancer-free SV40 promoter in either forward or reverse orientation. Cells were tested for luciferase activity 24 hours after treatment with 10ng/ml TGF-. beta.s. These experiments were repeated 4 times with similar results. The data presented in the figure are the mean of two transfections of the indicated construct from a single experiment.

FIG. 4 shows the competitive gel mobility shift assay for studying TGF- β response elements in the CTGF promoter-205 to-109 regions. A nucleotide fragment consisting of the-205 to-109 region of the CTGF promoter is marked at the end³²P was used in a competitive gel mobility change assay, competing oligonucleotides are shown schematically. The bands of specific gel mobility changes are indicated by arrows. The sequence diagram used shows the sequence positions corresponding to NF-1-like and TIE-like elements. The numbered fragments in the diagram represent the number of lanes in the competitive gel mobility shift assay, with the specific nucleotide sequence indicated above the lanes (i.e., 3, -205/-150). The amount of non-labeled competitor used was more than 250 times the number of molecules of the labeled fragment. Only oligonucleotides containing the-169-150 region can act as specific competitors. Neither NF-1 nor TIE-like regions were competitive in this assay.

FIG. 5 shows the interference test of methylation of the CTGF promoter regions-205 to-109. FIG. 5A shows the sequence analysis of the regions-205 to-109. Shown are the sequences of-200 to-113. Lane G is the G sequence of the complete marker probe, lane S is the sequence of the altered band and lane F is the sequence of the unaltered free probe in the same sample. Only the region from-157 to-145 contains the unmethylated G residue.

FIG. 5B shows sequence analysis of the-159-142 region with a smaller promoter fragment (-169-193). Lanes are as in Panel A. The competing G residues in this sequence are indicated by arrows. Filled circles indicate G residues detected by complementary strand analysis (data not shown). Symbol^*And # is used to indicate the direction relative to the sequence in the a diagram.

FIG. 6 shows a titration experiment of oligonucleotide competitive gel mobility change in T β RE. By using³²P-terminal-labeled human CTGF promoter fragments (-205 to-109) were tested for overlapping and non-overlapping oligonucleotides in the region containing the CTGF promoter-159 to-143 by a competitive gel mobility shift assay. Complete competition is achieved when the concentration of the complete fragment (-159 to-143) is 10ng, and the highest affinity is shown. All of whichThe fragment only contains a part of the sequence and has low competitive power, wherein the competitive power is the lowest in a region from-150 to-134. Lanes 14 and 15 are NF-1 and TIE elements, respectively, which were not competitive even at concentrations greater than 5000 times the number of labeled probe molecules.

FIG. 7 shows that T β RE point mutations reduce the induction of CTGF promoter by TGF- β.

FIG. 8A shows the effect of herbimycin, phorbol ester, cAMP and cholera toxin on TGF- β induced CTGF expression, detected using luciferase assay.

FIG. 8B shows photomicrographs of NIH/3T3 cells untreated, TGF- β treated, cAMP (8 bromocAMP) treated, or cAMP and TGF- β co-treated.

FIG. 8C shows the results of anchorage-independent growth inhibition by 8 bromocAMP and cholera toxin, and the reversal of TGF- β -induced anchorage-independent growth by CTGF against cAMP or cholera toxin.

Detailed Description

The present invention discloses a novel protein growth factor, which is known as Connective Tissue Growth Factor (CTGF). This protein may play an important role in the normal development, growth and repair of human tissues. The discovery of CTGF proteins and the cloning of cdnas encoding such molecules are of great interest because no growth factors having mitogenic and chemotactic activity for connective tissue cells have been previously discovered. CTGF has biological activity similar to PDGF, but CTGF is a gene product unrelated to the a or B chain gene of PDGF. CTGF is produced by endothelial cells and fibroblasts, which are present at the wound site, and thus it is possible that CTGF functions as a growth factor in wound healing.

Pathologically, CTGF is associated with diseases with connective tissue cell overgrowth, such as cancer, tumor formation and growth, fibrotic diseases (e.g., pulmonary fibrosis, renal fibrosis, glaucoma), and atherosclerosis. CTGF polypeptides may be used as a therapeutic agent in cases where there is poor healing of skin wounds or where it is desirable to accelerate the normal healing process. Furthermore, antibodies directed against CTGF polypeptides or fragments are valuable as diagnostic tools that facilitate the detection of diseases in which CTGF is a pathological factor. In therapeutic aspects, the antibodies or antibody molecule fragments may also be used to neutralize the biological activity of CTGF in diseases in which CTGF induces tissue overgrowth.

The major biological activity of CTGF polypeptides is their mitogenicity, or ability to stimulate proliferation of target cells. The end result of this mitogen activity in vivo is the growth of the target tissue. CTGF also has chemotactic activity, which means that cells interact with specific molecules and as a result chemically induced motility of the cells is induced. Preferably, the CTGF of the invention has mitogenic and chemotactic activity towards connective tissue cells, however, other cell types may also be responsive to CTGF polypeptides.

The CTGF polypeptides of the present invention are characterized by being present in monomeric form and having a molecular weight of about 36-38 kD. CTGF is secreted by cells and is active when it interacts with receptors on a responding cell. CTGF is antigenically related to PDGF, although there is little homology between the peptide sequences of both. The anti-PDGF antibody has a high affinity for the PDGF heteromer and the non-reduced form of the CTGF molecule, whereas its affinity for the reduced form of the two is 10-fold lower, and this reduced peptide has no biological activity. This suggests that there are regions of identical tertiary structure between the PDGF heteromer and the CTGF molecule, forming common epitopes.

The term "substantially pure" as used herein means that CTGF is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. The substantially pure polypeptide exhibits a single main band on a non-reducing polyacrylamide gel. The purity of CTGF polypeptides can also be determined by amino-terminal amino acid sequence analysis. CTGF polypeptides include functional fragments of the polypeptide, provided that such fragments retain the mitogenic and chemotactic activity of CTGF. Smaller peptides comprising CTGF biological activity are also encompassed by the invention. Furthermore, the preparation of more efficient CTGF molecules is also encompassed by the present invention, for example by site directed mutagenesis of CTGF cDNA.

The present invention provides an isolated polynucleotide encoding a CTGF protein. The term "isolated" as used herein means that a polynucleotide is substantially free of other polynucleotides, proteins, lipids, carbohydrates or other materials with which it is naturally associated. These polynucleotides include DNA, cDNA, and RNA sequences that encode connective tissue growth factors. It is understood that all polynucleotides encoding intact CTGF or a portion of CTGF are also included herein, so long as they encode polypeptides having the mitogenic and chemotactic activities of CTGF. Such polynucleotides include naturally occurring, e.g., allelic variants, purposefully engineered, e.g., mutagenized, and artificially synthesized polynucleotides. For example, site-directed mutagenesis may be performed on CTGF polynucleotides. The polynucleotides of the present invention include those degenerate sequences as a result of the genetic code. There are only 20 natural amino acids, most of which are defined by more than one codon. Therefore, all degenerate nucleotide sequences are included in the present invention as long as the amino acid sequence of CTGF is not functionally changed.

The term "polynucleotide" also refers to DNA, cDNA and RNA encoding untranslated sequences that flank the gene encoding CTGF structure. For example, one polynucleotide of the invention comprises a 5 'regulatory nucleotide sequence and a 3' untranslated sequence linked to a CTGF structural gene. A polynucleotide of the present invention comprising both 5 'and 3' untranslated regions is shown in FIG. 1C. The 5' regulatory region containing the promoter is shown in FIG. 1B.

The CTGF cDNA sequence contains an open reading frame of 1047 nucleotides with a start site at position 130 and a TGA termination site at position 1177, encoding a 349 amino acid peptide. The CTGF cDNA has only 40% sequence homology with the PDGF a and B chain cdnas.

The invention provides CTGF promoter nucleotide which is positioned between-823 bit and +74 bit, and TGF-beta regulatory element (T beta RE) which is positioned between-162 bit and-128 bit in the CTGF promoter sequence. Methylation interference test and competitive gel migration test locate a unique 13-nucleotide sequence between-157 and-145, and the unique sequence is a novel TGF-beta cis-regulatory element.

The CTGF open reading frame encodes a polypeptide containing 39 cysteine residues, indicating that this is a protein with many intramolecular disulfide bonds. The amino terminus of the peptide contains a hydrophobic signal sequence, suggesting that it is a secreted protein and that the peptide has two N-linked glycosylation sites at asparagine residues 28 and 225 of the amino acid sequence. CTGF is a member of a family of proteins including serum-induced immediate early gene products such as Cyr61 (O' Brien, et al, molecular Cell biology (mol. Cell. Biol.), Vol.10: 3569, 1990) and Fisp12(Ryseck, et al, Cell Growth and Differentiation, Vol.2: 225, 1991)/BigM2(Brunner, et al, DNA and Cell biology (DNA and Cell Biol.), Vol.10: 293, 1991); a v-src induced peptide (CEF-10) (Simmons, et al, Advance in the national academy of sciences, USA (Proc. Natl.Acad.Sci.USA), Vol.86: page 1178, 1989), and a putative oncoprotein (nov) (Joliot, et al, molecular cell biology, Vol.Cell.biol., Vol.12: page 10, 1992). A twisted gastrulation gene (tsg) functions to control the induction of mesodermal components in Drosophila dorsal/ventral embryogenesis and is distantly related to CTGF (Mason, et al, Gene and development, Genesand Devel, 8: 1489, 1994). The total sequence homology between CTGF polypeptide and the polypeptide encoded by CEF-10mRNA transcripts is 45% (Simmons, et al, Proc. Natl. Acad. Sci. USA, Vol. 86: page 1178, 1989); homology of 52% is achieved when the putative alternative splice region is missing.

The DNA sequences of the invention can be obtained by several methods. For example, DNA may be isolated using hybridization methods well known in the art. These hybridization methods include, but are not limited to: 1) probes are hybridized to genomic or cDNA libraries to detect common nucleotide sequences, and 2) antibody screening of expression libraries to detect common structural features.

Any gene sequence can be isolated from any organism using a screening procedure that relies on nucleic acid hybridization, provided that there is an appropriate probe. For example, oligonucleotide probes identical to a portion of the sequence encoding the protein can be chemically synthesized. This requires that the amino acid sequence of a short oligopeptide be known. The DNA sequence encoding the protein can be deduced from the genetic code, but the degeneracy of the code must be taken into account. When the sequence is a degenerate sequence, mixed addition reactions may be carried out. This involves denaturing a heterogeneous mixture of double stranded DNA. For such screening, hybridization is preferably performed on single-stranded DNA or denatured double-stranded DNA. Hybridization is particularly useful for detecting cDNA clones prepared from sources having very low amounts of mRNA sequences which are related to the polypeptide of interest. In other words, with stringent hybridization conditions aimed at avoiding non-specific binding, it is possible, for example, to make a specific cDNA clone visible by autoradiography by hybridizing the target DNA to a single probe which is completely complementary in the mixture (Wallace, et al, nucleic acids Research, Vol.9: page 879, 1981).

CTGF peptides having at least one epitope can be screened indirectly from a cDNA expression library, such as λ gt11, using antibodies specific for CTGF or antibodies to PDGF that cross-react with CTGF. Such antibodies may be of polyclonal or monoclonal origin, and are used to detect expression products, thereby suggesting the presence of CTGF cDNA.

The DNA sequence encoding CTGF may be expressed in vitro by transferring the DNA into a suitable host cell. A "host cell" is a cell in which a vector can be amplified and DNA can be expressed in the vector. The term also includes any progeny of the host cell. Since mutations are likely to occur during replication, it is apparent that not all progeny cells are identical to the parent cell. However, when the term "host cell" is used, these progeny cells are still included.

The DNA sequence encoding CTGF may be expressed in vivo in prokaryotic or eukaryotic cells. Methods for expressing DNA sequences having eukaryotic coding sequences in prokaryotic cells are well known in the art. Hosts include microorganisms, yeast and mammalian organisms.

Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are well known in the art. Such vectors are used to accommodate the DNA sequences of the present invention. In general, the use of expression vectors containing promoter sequences that promote efficient transcription of inserted eukaryotic genetic sequences is relevant to the host. Expression vectors typically contain an origin of replication, a promoter and a terminator, as well as specific genes, which provide phenotypic selection of transformed cells.

In addition to expression vectors well known in the art, such as bacterial, yeast and mammalian expression systems, baculovirus vectors can be used. One advantage of expressing foreign genes in such invertebrate virus expression vectors is that they can express high levels of recombinant proteins that are antigenically and functionally similar to their corresponding native proteins. Baculovirus vectors and suitable insect host cells for use with the vectors will be well known to those skilled in the art.

The term "recombinant expression vector" refers to a plasmid, virus, or other vector into which the CTGF genetic sequence has been inserted or otherwise introduced, as is well known in the art. These expression vectors contain a promoter sequence which promotes efficient transcription of the inserted genetic sequence in the host. Typically, expression vectors contain an origin of replication, a promoter, and a specific gene for phenotypic selection of transformed cells. Vectors suitable for use in the present invention include, but are not limited to, T7-based expression vectors for expression in bacteria (Rosenberg et al, Gene 56, p. 125, 1987), pMSXND expression vectors for expression in mammalian cells (Lee and Nathans, J.Biol.Chem., 263, p. 3521, 1988), and baculovirus-derived vectors for expression in insect cells. In the vector, the DNA fragment is operably linked to regulatory elements, such as a promoter (e.g., T7, metallothionein I, or polyhedrin promoter).

The vector may comprise a phenotypic selectable marker to identify the host cell containing the expression vector. Typical markers for prokaryotic expression vectors include antibiotic resistance genes for ampicillin (. beta. -lactamase), tetracycline, and chloramphenicol (chloramphenicol acetyltransferase). Typical markers for mammalian expression vectors include the adenosine denitrification enzyme (ADA) gene, the aminoglycoside phosphotransferase (neo, G418) gene, the dihydrofolate reductase (DHFR) gene, the hygromycin-B-phosphotransferase (HPH) gene, the Thymidine Kinase (TK) gene, and the xanthine guanine phosphoribosyltransferase (XGPRT, gpt) gene.

Isolation and purification of the polypeptide expressed by the host cell of the present invention may be carried out by any conventional method, for example, preparative chromatographic separation and immunological separation methods such as those involving the use of monoclonal or polyclonal antibodies.

Transformation of a host cell with recombinant DNA may be carried out by conventional methods well known to those skilled in the art. When the host is a prokaryotic cell such as E.coli, competent cells having the ability to take up DNA can be prepared by: cells in log phase growth were harvested, after which they were CaCl using procedures well known in the art₂The method treats the cells. Or by other alternative methods, e.g. MgCl₂Or the RbCl method.

When the host used is a eukaryotic cell, various DNA transduction methods can be employed. These include precipitation with calcium phosphate, conventional mechanical methods such as microinjection, insertion of liposome-encapsulated plasmids or transfection of DNA with viral vectors. Eukaryotic cells may also be co-transformed, wherein one gene is a DNA sequence encoding a polypeptide of the present invention and a second foreign DNA molecule encodes a selectable phenotype such as a herpes simplex thymidine kinase gene. Another approach is to transiently infect or transform eukaryotic cells and express proteins with eukaryotic viral vectors such as simian virus 40(SV40) or bovine papilloma virus. (Eukaryotic Viral Vectors), Cold spring harbor Laboratory, eds. Gluzman, 1982). Examples of mammalian host cells include COS, BHK, 293, and CHO cells.

Eukaryotic host cells may also include yeast. For example, the DNA may be expressed in yeast by inserting the DNA into an appropriate expression vector and introducing the vector thus constructed into a yeast host cell. Various shuttle vectors have been reported for yeast expression of foreign genes (Heinemann, J. et al, Nature, Vol. 340: p. 205, 1989; Rose, M. et al, Gene (Gene), Vol. 60: p. 237, 1987).

The present invention provides antibodies specifically reactive with CTGF polypeptides or fragments thereof. Although this polypeptide is cross-reactive with antibodies directed against PDGF, not all CTGF antibodies are reactive with PDGF. The present invention provides not only monoclonal antibody preparations of a single specificity, but also antibodies consisting essentially of pooled monoclonal antibodies with different epitopic specificities. Methods for preparing monoclonal antibodies from antigens containing fragments of the protein are well known in the art (Kohler, et al, Nature, 256: 495, 1975; modern techniques in Molecular Biology, eds., Ausubel, et al, 1989). For example, a method for selecting a CTGF-specific monoclonal antibody can be performed by screening a hybridoma culture supernatant that reacts with CTGF but not PDGF.

The present invention provides not only monoclonal antibody preparations of a single specificity, but also antibodies consisting essentially of pooled monoclonal antibodies with different epitopic specificities. Methods for preparing monoclonal antibodies from antigens containing fragments of the protein are well known in the art (Kohler, et al, Nature, 256 Vol. 495, 1975; modern techniques in Molecular Biology, eds., Ausubel, et al, 1989).

The term "antibody" as used herein includes both intact molecules and fragments thereof, such as Fab, F (ab')₂And Fv fragments. These antibody fragments retain some ability to selectively bind to their antigen or receptor and are defined as follows:

(1) fab, the fragment contains a monovalent antigen binding fragment of an antibody molecule, and is prepared by hydrolyzing intact antibody with papain to obtain an intact light chain and a portion of a heavy chain;

(2) fab', which is a molecular fragment of an antibody obtained by treating an intact antibody with pepsin, followed by reduction, thus obtaining an intact light chain and a part of a heavy chain; two Fab' fragments are available per antibody molecule;

(3)(Fab′)₂the antibody fragment is obtained by treating the whole antibody with pepsin without subsequent reduction; f (ab')₂Is a dimer of two Fab' fragments linked together by two disulfide bonds;

(4) fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and

(5) single chain antibody ("SCA"), defined as a genetically engineered molecule containing a light chain variable region and a heavy chain variable region linked by an appropriate polypeptide linker into a single chain molecule that is genetically fused.

Methods for preparing such fragments are well known in the art. (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference).

As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants are usually composed of chemically active groups on the surface of a molecule such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.

The antibodies of the present invention that bind to CTGF polypeptides can be prepared by using the intact polypeptide or the small peptide fragment of interest as an antigen for immunization. The polypeptides or peptides used to immunize an animal may be obtained by translation of cDNA or by chemical synthesis, and these peptides may be coupled to a carrier protein if desired. Such commonly used carriers to which peptides are chemically coupled include Keyhole Limpet Hemocyanin (KLH), thyroglobulin, Bovine Serum Albumin (BSA), and tetanus toxoid. The conjugated peptide can be used to immunize an animal (e.g., a mouse, rat, or rabbit).

If necessary, the polyclonal or monoclonal antibody may be further purified, for example, by binding the polypeptide or peptide used for preparing the antibody to a substrate, binding the antibody to be purified to the polypeptide or peptide on the substrate, and then eluting. Those skilled in the art will appreciate the numerous techniques commonly employed in the art of immunology for the purification and/or concentration of polyclonal antibodies as well as monoclonal antibodies (see, e.g., Coligan, et al, element 9, Current Protocols in immunology, Wiley Interscience, 1994, incorporated herein by reference).

Monoclonal antibodies that mimic an epitope can also be prepared using anti-idiotypic techniques. For example, an anti-idiotype monoclonal antibody raised against a first monoclonal antibody has a binding domain in its hypervariable region which is the "image" of the epitope to which the first monoclonal antibody binds.

The present invention provides a method for accelerating wound healing in an individual, such as a human, by applying to the wound an effective amount of a composition comprising CTGF, preferably purified CTGF. PDGF and PDGF-related molecules such as CTGF are involved in the normal healing process of skin wounds. The CTGF polypeptides of the invention are valuable as a therapeutic agent in cases where the healing of skin wounds is poor or where it is desirable to accelerate the normal healing process, such as burns. An important advantage of using CTGF protein to accelerate wound healing is due to the high percentage of cysteine residues of the molecule. CTGF or functional fragments thereof are more stable and less susceptible to degradation by proteases than PDGF and other growth factors known to be involved in wound healing.

CTGF is produced by endothelial cells and fibroblasts, both of which are present at the skin wound site. Thus, agents that stimulate CTGF production may be added to the composition for accelerating wound healing. Preferably, in the present invention, the agent is transforming growth factor beta (TGF-. beta.), but it is possible that other members of the TGF-. beta.family may also help to accelerate wound healing by inducing CTGF. The compositions of the present invention assist in wound healing, in part, by promoting connective tissue growth. The composition is prepared by combining pharmaceutically acceptable carrier materials such as inert gels or liquids, purified CTGF and TGF-beta.

The term "cell proliferative disorder" refers to a pathological state characterized by a persistent proliferation of cells resulting in an excessive increase in a certain cell population within a tissue. These cell populations are not necessarily transformed, tumorigenic, or malignant cells, but may also include normal cells. For example, CTGF may play a pathological role by inducing proliferative lesions in the intima of the arterial wall, leading to atherosclerosis in animals. The CTGF inhibitors or antagonists of the present invention may be used to neutralize the activity of CTGF associated with atherosclerosis in vivo, thereby replacing attempts to reduce risk factors for the condition, such as lowering blood pressure or lowering elevated cholesterol levels in the patient. CTGF antagonists may also be used to treat other connective tissue overgrowth-related disorders, such as various fibrotic disorders, including scleroderma, arthritis, alcoholic cirrhosis, keloids, and hypertrophic scarring.

The present invention provides a method for detecting an increased level of CTGF, which method is useful for diagnosing the presence or absence of a pathological condition characterized by a cell proliferative disorder. For example, a sample suspected of containing CTGF is obtained from a patient, the level of CTGF is detected, and the level is compared to the level of CTGF in normal tissue. CTGF levels can be determined by immunoassay, e.g., with anti-CTGF antibodies. Other modifications of these methods known to those skilled in the art, such as Radioimmunoassay (RIA), ELISA and immunofluorescence, can also be used to detect CTGF levels in a sample. In addition, the nucleic acid probe can be used for detecting and quantifying CTGF mRNA, and can also achieve the purpose of reflecting the CTGF level.

The invention also discloses a method which comprisesAn effective amount of a CTGF-reactive agent treats the disease site for amelioration of a disease characterized by a cell proliferation disorder. The term "ameliorating" means that the deleterious effects of the disease-inducing response are reduced in the patient being treated. When the disorder is due to hyperproliferation of cells, antagonists of CTGF are effective in reducing the amount of growth factors that bind to CTGF-specific receptors on the cells. Such an antagonist can be an antibody specific for CTGF or a functional fragment of an antibody (e.g., Fab, F (ab')₂). Furthermore, a polynucleotide containing the T.beta.RE region in the promoter may also be used as a CTGF-responsive agent by acting as a competitor for TGF-beta. Such treatment requires contacting the disease site with the antagonist. When the cell proliferation disorder is due to a decrease in the number of cell proliferations, a CTGF-reactive agent that has a stimulatory effect may be used to contact the site of the disease. For example, TGF-. beta.is such a reactive agent. Other formulations will also be well known to those skilled in the art.

When the cell proliferation disorder is associated with expression of CTGF, a therapeutic approach that directly prevents translation of CTGF information into protein may be employed. For example, antisense nucleic acids or ribozymes can be used to bind to or cleave CTGF mRNA. Antisense RNA or DNA molecules specifically bind to the RNA information of the target gene, thereby blocking the expression of the protein product of the gene. The antisense nucleic acid binds to the messenger RNA to form a double-stranded molecule that cannot be translated by the cell. Antisense oligonucleotides about 15-25 nucleotides in length are preferred because they are easily synthesized and have the same inhibitory effect as antisense RNA molecules. In addition, chemically reactive groups such as iron-containing ethylenediaminetetraacetic acid (EDTA-Fe) can be attached to the antisense oligonucleotide, which can cause cleavage of the RNA at the hybridization site. Antisense methods for inhibiting the in vitro translation of genes are well known in the art for use in these and other applications (Marcus-Sakura, analytical biochemistry (anal., biochem., vol.) 172: p 289, 1988).

Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a particular mRNA molecule (Weintraub, American Scientific (1990), Vol.262: page 40). In a cell, antisense nucleic acids hybridize with the corresponding mRNA to form a double-stranded molecule. Since the cell is unable to translate the mRNA in its double strand form, the antisense nucleic acid blocks translation of the mRNA. Antisense oligomers of about 15 nucleotides in length are preferred because they are easy to synthesize and they are less likely to cause problems than larger molecules when entering CTGF-producing target cells. The use of antisense methods to inhibit gene translation in vitro is well known in the art (Marcus-Sakura, analytical biochemistry, anal. biochem., Vol.172: p.289, 1988).

The use of oligonucleotides to prevent transcription is called a triple strategy because the oligomers wrap around the double helical DNA, forming a triple helix. Thus, such tripartite compounds can be designed to identify unique sites on selected genes (Maher, et al, Antisense Res.and Dev., vol.3: 227, 1991; Helene, C., Anticancer Drug Design, vol.6: 569, 1991), e.g., the T.beta.RE region of the CTGF promoter.

Ribozymes are RNA molecules that have the ability to specifically cleave other single-stranded RNA in a manner similar to that of DNA restriction endonucleases. By modifying the nucleotide sequence encoding these RNAs, it is possible to genetically engineer molecules which recognize and cleave specific nucleotide sequences in RNA molecules (Cech, journal of the American medical Association (J.Amer.Med.Assn.), Vol.260: page 3030, 1988). Due to its sequence specificity, the main advantage of this method is that only mrnas containing a specific sequence are inactivated.

There are two basic types of ribozymes, the tetrahymena type (Hasselboff, Nature, 344 Vol.: page 585, 1988) and the "hammer" type. Tetrahymena-type ribozymes recognize sequences of 4 bases in length, while ` hammer-type ribozymes recognize base sequences of 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will only occur in the target mRNA species. As a result, hammerhead ribozymes are preferred over tetrahymena ribozymes for inactivating a particular mRNA species, and 18 base recognition sequences are preferred over shorter recognition sequences.

The identification of CTGF gene promoter elements, and in particular the TGF-beta response/regulatory element (T beta RE) (5'-GTGTCAAGGGGTC-3' (sequence # 8); nucleotides-157 to-145) is the basis for a screening method for identifying compounds or compositions that affect CTGF expression. Thus, in another embodiment, the invention provides a method of identifying a composition that affects CTGF expression, the method comprising incubating components comprising the composition with a TGF- β responsive element of a CTGF promoter, wherein the incubation is conducted under conditions that facilitate interaction of the components; and determining the effect of the composition on CTGF expression. The method further includes adding TGF-beta or a TGF-beta family member reactive with T beta RE to the reaction mixture. Thus, the method allows the identification of TGF-beta inhibitors or anti-fibrotic compounds. The promoter regions preferably used in the screening assays described herein include nucleotides-823 to +74, however, smaller regions, including TGF- β response elements, may also be used in the methods of the invention (e.g., -162 to-128 or-154 to-145).

The observed effect on CTGF expression may be inhibitory or stimulatory. For example, the enhancement or reduction of CTGF activity can be measured using CTGF biological assays, as described in the examples herein (e.g., example 1 and example 2). Alternatively, a polynucleotide encoding both CTGF regulatory (promoter) and structural regions may be inserted into the expression vector. The effect of the composition on CTGF transcription can be detected, for example, by Northern blot analysis. Adding radioactive compounds to the mixture, e.g.³²P-ATP, and the amount of radioactive incorporation of CTGF mRNA was then determined.

Another method for identifying compositions that affect CTGF expression is: a reporter gene is operably linked to the TGF- β responsive region of the CTGF promoter, the components comprising the composition to be assayed, the construct containing the reporter gene and the TGF- β are incubated together, and expression of the reporter gene is then assayed. These reporter genes are well known to those skilled in the art and include, but are not limited to, the luciferase gene, the chloramphenicol acetyl transferase gene (CAT test) or the beta-galactosidase gene.

An inducer of Tss RE may be added before or after the addition of the composition to be tested. Preferably, the inducer is added after the composition is added. Preferably, the inducer for this region in the CTGF promoter is TGF- β, however, other members of the TGF- β family are also possible for inducing this element. Other members or factors of this family are well known to those skilled in the art.

The method of the invention is preferably carried out in an indicator cell. An "indicator cell" is a cell in which CTGF activation or a reporter gene can be detected. Examples of mammalian host indicator cells include pre-B cell lines, 70Z/3, Jurkat T, COS, BHK, 293, CHO, HepG2, and HeLa cells. Other cell lines can be used as indicator cells, as long as the level of the reporter gene can be detected. These cells may be recombinantly modified to contain an expression vector encoding one or more additional copies of the T β RE binding region, preferably operably linked to a reporter gene. These cells may also be modified to express CTGF, as described above.

Reporter genes are phenotypically identifiable markers for detecting stimulation or inhibition of CTGF activation. Preferred markers for use in the present invention include the LUC gene, the expression of which is detectable by the luciferase assay. Typical markers for prokaryotic expression vectors include antibiotic resistance genes for ampicillin (. beta. -lactamase), tetracycline, and chloramphenicol (chloramphenicol acetyltransferase). Typical such markers for mammalian expression vectors include the Adenosine Deaminase (ADA) gene, the aminoglycoside phosphotransferase (neo, G418) gene, the dihydrofolate reductase (DHFR) gene, the hygromycin-B-phosphotransferase (HPH) gene, the Thymidine Kinase (TK) gene, the xanthine guanine phosphoribosyltransferase (XGPRT, gpt) gene, and the beta-galactosidase (beta-gal) gene, with mammalian expression vectors being preferred in the present invention.

In another embodiment, the invention provides a method for treating a patient having a cell proliferative disorder associated with expression of a CTGF gene, the method comprising administering to the patient having the disorder a therapeutically effective amount of an agent that modulates expression of the CTGF gene, thereby treating the disorder. The term "modulation" refers to the inhibition or suppression of the expression of CTGF when it is overexpressed, and the induction of its expression when it is underexpressed. The term "therapeutically effective" refers to an amount of a CTGF agent effective to alleviate the symptoms of a CTGF-associated cell proliferative disorder.

The agent that modulates CTGF gene expression may be, for example, a polynucleotide. The polynucleotide may be an antisense nucleotide, a triplex agent, or a ribozyme, as described above. For example, an antisense nucleotide can be directed to a structural gene region or promoter region of CTGF.

The formulation also includes a polynucleotide comprising a T β RE of the invention. Preferably, this region corresponds to nucleotides-162 to-128 of the CTGF modulating polypeptide shown in fig. 1B. More specifically, the T β RE region corresponds to about-154 to-145 in FIG. 1B. These polynucleotides serve as competitive inhibitors or pseudo-substrates for TGF-beta or other growth factors that bind to Tss RE to induce CTGF transcription.

The recombinant expression vector such as chimeric virus or colloidal disperse system can be used to achieve the purpose of carrying and delivering antisense nucleic acid, triple preparation, ribozyme, competitive inhibitor, etc. Various viral vectors that may be used as gene therapy as indicated in the present specification include adenovirus, herpes virus, vaccinia virus, or preferably RNA viruses such as retrovirus. Preferably, the retroviral vector is derived from a murine or avian retrovirus. Examples of retroviral vectors into which a single foreign gene can be inserted include, but are not limited to: moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV) and Rous Sarcoma Virus (RSV). Many other retroviral vectors can insert multiple genes. All these vectors can deliver or incorporate genes that serve as selectable markers, and the cells so transduced are identified and propagated. The vector thus constructed has targeting specificity by inserting the desired polynucleotide sequence into the viral vector together with another gene encoding a ligand for a receptor on a particular target cell. For example, a retroviral vector may be rendered target specific by the insertion of a polynucleotide encoding a sugar, glycolipid or protein. Preferred targeting is to target the retroviral vector with an antibody. Those skilled in the art will know or readily appreciate that if the assay is appropriate, specific polynucleotide sequences can be inserted into the retroviral genome to allow targeted specific delivery of retroviral vectors containing antisense polynucleotides.

Since recombinant retroviruses are defective viruses, they need help to produce infectious viral particles. This can be provided, for example, by the use of helper cell lines which contain plasmids encoding all of the retroviral structural genes under the control of regulatory sequences within the LTRs. These plasmids lose a nucleotide sequence that enables the packaging machinery to recognize the RNA transcript for encapsidation. Helper cell lines for which the packaging signal is absent include but are not limited to ψ 2, PA317 and PA 12. Since no genome is packaged, these cell lines produce only empty virions. If a retroviral vector is introduced into these cells in which the packaging signal is intact, but the structural gene is replaced by another gene of interest, the vector can be packaged to produce vector virions.

Alternatively, NIH3T3 or other tissue culture cells were directly transfected with plasmids encoding the retroviral structural genes gag, pol and env by conventional calcium phosphate transfection. These cells are then transfected with a vector plasmid containing the gene of interest. The resulting cells release the retroviral vector into the culture medium.

Another targeted delivery antisense polynucleotide system is a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, microcapsules, microparticles, beads and lipid-based systems including oil-water emulsions, micelles, mixed micelles and liposomes. The preferred colloidal system in the present invention is a liposome. Liposomes are artificial membrane carriers that are used as delivery vehicles in vitro and in vivo. Studies have shown that Large Unilamellar Vesicles (LUVs) of 0.2-4.0 μm in size can encapsulate a significant portion of an aqueous buffer containing macromolecules. RNA, DNA and whole virus particles can be encapsulated inside a liquid and transported to cells in a biologically active form (Fraley, et al, trends in Biochemical sciences, Sci., Vol.6: page 77, 1981). In addition to mammalian cells, liposomes have also been used to transport polynucleotides to plant, yeast and bacterial cells. In order for liposomes to be effective gene delivery vehicles, they should have the following characteristics: (1) the target gene is efficiently wrapped without reducing the biological activity of the gene; (2) preferentially and substantially binds to target cells as compared to non-target cells; (3) efficiently transferring the aqueous contents of the vehicle to the cytosol of the target cell; and (4) accurate and efficient expression of genetic information (Mannino, et al, Biotechnology, Vol.6: page 682, 1988).

The composition of liposomes is generally a combination of phospholipids, particularly high phase transition temperature phospholipids, and is generally combined with a steroid, particularly cholesterol. Other phospholipids or other lipids may also be employed. The physical properties of liposomes depend on pH, ionic strength and the presence or absence of divalent cations.

Examples of lipids used for preparing liposomes include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly preferred are diacylphosphatidylglycerols, wherein the lipid moiety contains 14 to 18 carbon atoms, particularly 16 to 18 carbon atoms, and is a saturated lipid. For example, phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.

Targeting of liposomes can be classified according to both anatomical and functional factors. Anatomical classification is based on the level of selectivity, e.g., organ-specific, cell-specific, and organelle-specific. Targeting can be classified as passive or active. Passive targeting exploits the natural distribution propensity of liposomes, namely, to reticuloendothelial system (RES) cells of the sinusoidal capillary organ. In contrast, active targeting involves modification of the liposomes, coupling them with a specific ligand such as a monoclonal antibody, sugar, glycolipid or protein, or by modifying the composition or size of the liposomes for the purpose of targeting organs and cell types, rather than naturally occurring in a localized location.

The surface of the targeted delivery system can be modified in a variety of ways. In the case of liposome targeted delivery systems, lipid groups may be incorporated into the lipid bilayer of the liposome in order to maintain a stable association of the targeting ligand with the liposome bilayer. A variety of linking groups may be used for binding between the lipid chain and the targeting ligand. In general, compounds that bind to the surface of targeted delivery systems are ligands and receptors that allow the targeted delivery system to find and "actively track" the cells of interest. The ligand may be any compound that binds to another compound, such as a receptor.

Agents useful for modulating CTGF gene expression in the methods of the invention include those that cause an increase in intracellular cyclic nucleotides. For example, agents such as cholera toxin or 8 bromo-cAMP are preferred for treating patients suffering from cell proliferative disorders associated with CTGF gene expression. Preferably, the cyclic nucleotide that is elevated after treatment with the method of the invention is cAMP or a cAMP analog that is either functionally or structurally similar, or both. Those skilled in the art will know other agents that induce intracellular cAMP or analogs thereof, and these agents are useful in the methods of the invention.

The therapeutic formulations used in the methods of the invention may be administered parenterally by injection or by a prolonged stepwise infusion method. The route of administration may be intravenous, intraperitoneal, intramuscular, subcutaneous, intracavity or transdermal.

Formulations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsifiers. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, ethanol/water solutions, emulsions or suspensions, including saline and buffers. Parenteral vehicles include sodium chloride solutions, ringer's dextrose solutions, dextrose and sodium chloride solutions, lactated ringer's intravenous vehicles including body and nutrient supplies, electrolyte supplies (such as those formulated with ringer's dextrose solution), and the like. Preservatives and other additives may also be added, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases, to name a few.

The present invention also includes a pharmaceutical composition comprising a therapeutically effective amount of CTGF formulated in a pharmaceutically acceptable carrier. Such carriers include those listed above in connection with parenteral administration.

The examples set forth herein (see example 10) demonstrate that TGF-. beta.is cell type specific (e.g., fibroblast) for the induction of CTGF. Thus, inclusion of T β RE in the CTGF promoter region facilitates expression of the structural gene specifically in connective tissue cells. It is envisioned that any gene product of interest can be produced specifically in connective tissue cells, provided that its gene is operably linked to T β RE and TGF- β is present. For example, there may be a need to operably link PDGF or another growth factor to a polynucleotide containing a Tss RE, thereby allowing the specific production of PDGF or another factor in connective tissue cells. Alternatively, in the case where the level of CTGF or other factors produced is too high, it may be desirable to introduce antisense nucleotides to CTGF, for example, under the control of T β RE, thereby reducing the amount of CTGF produced in the cell.

The following examples are intended to illustrate the invention without limiting it. While these embodiments may be typical of application examples, other methods known to those skilled in the art may be used instead.

Example 1

Identification and partial purification of mitogen cells from PDGF-immune-related HUVE cells

Human Umbilical Vein Endothelial (HUVE) cells were isolated from fresh Human umbilical cord by collagenase perfusion (Jaffe, et al, Human Pathol., 18: 234, 1987) and maintained in culture at 199 in 20% FCS, 0.68mM L-glutamine, 20. mu.g/ml gentamicin, 90. mu.g/ml porcine heparin (Sigma, St. Louis, Mo.) and 50. mu.g/ml endothelial cell growth supplement (Sigma). The cells used for collecting the culture fluid are third generation cells. Cells with non-overlapping pebble morphology and positive staining for factor-III associated antigens are endothelial cells. NRK cells were obtained from American type Culture, NIH/3T3 cells as donated by S.Aaroson (NCI, Bethesda, Md.), both cells maintained in DMEM with 10% FCS, 20. mu.g/ml gentamicin. Fetal bovine aortic smooth muscle cells were obtained from tissue explants as previously described (Grotendorst, et al, Advance in national academy of sciences, Proc. Natl.Acad.Sci.USA), volume 78: page 3669, 1981), cells maintained in DMEM containing 10% FCS, 20. mu.g/ml gentamicin, and used for experiments at the second or third generation. Growth factor and antibody

Homogeneous human PDGF was purified from platelets by the previously described method (Grotendorst, Cell, 36: page 279, 1984). Recombinant AA, BB and AB chain dimer PDGF molecules were obtained from Creative biomoles, (Hopkinton, MA). FGF was obtained from Sigma. Purified PDGF or synthetic peptides containing amino and carboxyl sequences of mature PDGF a and B chain molecules are used to make antibodies in goats. The immunized goat was injected multiple times intradermally with 20 μ g of purified PDGF or 50 μ g of synthetic peptide in Freund's complete adjuvant. Immune sera were collected seven days after the 4 th booster (in Freund's incomplete adjuvant) and subsequent boosts. Immunoblot analysis showed that the anti-PDGF antibody did not have any cross-reactivity with TGF-. beta.s, EGF or FGF. The anti-peptide antibody has sequence specificity, has no cross reaction with other synthetic peptide sequences, and has no cross reaction with recombinant PDGF peptide without the specific antigen sequence. The above results were confirmed by Western blot and dot blot analysis. Antibody affinity chromatography column

Goat anti-human PDGF IgG (150mg) was covalently linked to 25ml Affi-Gel 10 matrix (BioRad) according to the protocol, at a final concentration of 6mg IgG/ml Gel. The chromatographic column was incubated with 1 liter of HUVE cell culture supernatant at 4 ℃ for 18 hours under stirring, and the HUVE cell culture supernatant was the culture broth collected after 48 hours of intensive culture of the cells. Thereafter, the gel was perfused into a chromatography column (5X 1.5cm), the column was washed with 4 volumes of 0.1N acetic acid (pH adjusted to 7.5 with ammonium acetate), and then the antibody-bound PDGF immunoreactive protein was eluted with 1N acetic acid. The determination method of the elution peak component is a bioassay method and an immunoblotting method, and each component is collected intensively.

Initial studies of PDGF-related growth factors secreted by HUVE cells removed serum-containing growth media from confluent cultures of cells, and replaced it with serum-free media. Periodically, a portion of the culture was removed and the proteins were detected by immunoblotting using antibodies specific for human platelet PDGF. The antibody has no cross reaction with other known growth factors, and can detect less than 500 microgram of dimer PDGF or 10 nanograms of reduced monomer A or B chain peptide by immunoblotting. HUVE cells were grown to confluence in 6-well plates. Growth medium was removed and cells were washed with PBS, after which 1ml serum-free medium was added to each well. After the cells are subjected to intensive culture for 6-48 hours, culture solution is taken, dialyzed against 1N acetic acid, and then freeze-dried. The samples were then electrophoresed in 12% PAGE, electroblotted to nitrocellulose membrane, reacted with anti-human PDGF antibody and developed. 5 nanograms of purified platelet PDGF were also used as a reference.

The results indicate that the cells constitutively secrete several molecules immunologically similar to platelet PDGF but with higher relative molecular weights (36-39kD), whereas platelet PDGF or A-chain or B-chain homodimers are expected to have molecular weights of 30-32 kD. Chemotaxis and mitogen assays performed with this serum-free conditioned medium showed that the total biological activity after a 48 hour incubation period was equivalent to 15ng/ml platelet PDGF. Incubation of this medium with 30. mu.g/ml of anti-human PDGF IgG neutralises approximately 20-30% of mitogen activity and similar levels of chemotactic activity.

The presence of several PDGF immunoreactive molecules in HUVE culture broth was unexpected, particularly since these molecules had a higher molecular weight than expected from the production and secretion of dimer molecules of the A and B chains by endothelial cells (Collins, et al, Nature, 328: pp.621-624, 1987; Stiaras, et al, J.Cell. physiol., 132: pp.376-380, 1987). In order to obtain larger amounts of PDGF-like protein for further analysis, HUVE cells were only maintained in culture with 20% fetal bovine serum, since these cells began to die 24 hours after incubation in serum-free or low serum culture. The PDGF immunoreactive protein was partially purified from serum-containing media using antibody affinity chromatography columns prepared from anti-human PDGF IgG and Affi-Gel 10 matrix (BioRad). Mitogen assays were performed using NRK cells as target cells (PDGF BB 5ng/ml, PDGF AA 10 ng/ml). HUVE medium was 250 μ l HUVE cell serum-free conditioned medium (48 hours) that had been dialyzed against 1N acetic acid, lyophilized, and resuspended in DMEM prior to addition to the test wells. The affinity purification fraction was 5. mu.l/ml of a concentrated mixture of the major components obtained from an Affi-Gel 10 affinity column. anti-PDGF IgG or non-immune IgG (30. mu.g/ml) was added to the samples and incubated at 4 ℃ for 18 hours before mitogen assay determination. The mean of triplicates of samples was determined with a standard deviation of less than 5%. The test was repeated at least 3 times, and the results were similar.

When partially purified proteins were used for chemotactic and mitogen activity assays, pre-incubation with anti-human PDGF antibody protein neutralized all biological activities. This indicates that the only biologically active molecule present in the partially purified culture broth protein is the PDGF immune-related molecule.

Immunoblot detection of partially purified protein with the same anti-PDFG antibody indicated the presence of high molecular weight molecules observed in serum-free conditioned medium. The major species secreted migrated to 36kD in the polyacrylamide gel, which fraction contained at least 50% of the total immunoreactive protein purified from the conditioned medium. The remaining immunoreactive species, which are mostly constituted by immunoreactive proteins, migrate to 37kD and 39 kD. Similar results were found for 35S-cysteine labeled proteins affinity purified using anti-PDGF IgG immunoaffinity chromatography columns. Less than 15% of the total affinity purified protein co-migrates with purified platelet PDGF or recombinant PDGF isoforms.

Preincubation with purified PDGF and antibody (300ng PDGF/2. mu.g IgG) blocked the binding of the antibody to all molecules, indicating that these molecules share common antigenic determinants with dimeric platelet PDGF. Interestingly, when antibodies were blocked with recombinant AA, BB or AB dimers, all three dimeric forms inhibited antibody binding to HUVE secretory proteins to the same extent, indicating that the antibodies recognized a common epitope present on all three PDGF dimers and HUVE secretory molecules. To ensure that the antibody-binding molecules detected by Western blotting were not derived from fetal bovine serum or other additives in the culture broth, a new unused antibody affinity chromatography column was prepared and the culture broth, which had never been used for cell culture, was treated exactly as conditioned medium. The components obtained from the column were not detected by immunoblotting for PDGF immunoreactive molecules nor any biological activity. Monomeric a chain (17kD) and B chain (14kD) peptides were visible on immunoblots when platelet PDGF or recombinant dimer was reduced with 200mM Dithiothreitol (DTT). HUVE molecules were treated in 100mM DTT sample buffer, resulting in a slower migration rate of the primary immunoreactive peptide on polyacrylamide gels. The vast majority of immunoreactive molecules migrate to 38-39kD, with lighter bands seen at 25kD and 14 kD. The amount of reduced protein used in the running gel is at least 10 times higher than the amount of non-reduced protein in order to allow detection of the reduced molecule. This is consistent with the affinity of the antibody for PDGF a chain and B chain peptide haplotypes. These data indicate that the main species of PDGF-related proteins affinity purified from HUVE cell conditioned media are monomeric peptides that migrate on acrylamide gels with an apparent molecular weight of 36kD when non-reduced and 38kD when reduced.

Example 2

Biological assay

Chemotactic Activity was determined using a Boyden Chamber chemotaxis assay using NIH3T3 or bovine hostsArterial Smooth Muscle (BASM) cells, as described in the literature (Grotendorst, et al, Advance in the national academy of sciences of the United states (Proc. Natl. Acad. Sci. USA), volume 78: pages 3669-3672, 1981; Grotendorst, et al, Methods in enzymology, volume 147: pages 144-152, 1987). Mitogen assays were performed in 96-well plates using Normal Rat Kidney (NRK) fibroblasts or NIH3T3 cells as target cells. Cells were seeded in DMEM with 10% FCS; NRK cells were cultured to 10-14 days after confluence for the assay, while 3T3 cells were incubated in serum-free DMEM containing 0.2mg/ml BSA for 2 days prior to use to allow for resting phase. Dilutions of the sample protein and known standards were added to the wells and plates were incubated at 37 ℃ with 10% CO₂Incubation for 18 hours under 90% air conditions, followed by addition of 5. mu. Ci/ml final concentration³H-Thymidine, and further incubation for 2 hours. Removing the culture medium, washing the cells, and determining DNA synthesis by scintillation counting of the cells incorporated in trichloroacetic acid precipitate³H-thymidine. Gel electrophoresis and immunoblotting

Unless otherwise stated, electrophoresis was performed on SDS-containing 12% polyacrylamide gels (Laemmli, U.K., Nature, 227: pp.680-685, 1970). Immunoblotting was performed by electroblotting the proteins onto nitrocellulose membranes, and incubating the membranes in 50mM Tris-HCl, pH 7.4, 100mM NaCl (TBS) containing 5% defatted dry milk powder at 25 ℃ for 1 hour to block non-specific antibody binding. The blocking solution was removed, and antibody (15. mu.g/ml) diluted in TBS containing 0.5% skimmed dry milk powder and 1. mu.g/ml sodium azide was added and incubated overnight at 25 ℃. The membrane was washed 5 times with 0.5% milk in TBS for 10 min each, and then incubated with affinity purified rabbit anti-sheep IgG (KPL, Gaithersburg, Md.) coupled with alkaline phosphatase for 1 h at 25 ℃ and the enzyme-coupled rabbit anti-sheep IgG was diluted 1: 1000 in TBS with 0.5% milk. The filters were washed 5 times in TBS for 10 min each and blots were developed with alkaline phosphatase substrate solution (0.1M Tris-HCl, pH 9, 0.25mg/ml nitroblue tetrazolium, 0.5mg/ml 5 bromo-4-chloro-3-indolyl phosphate). The major chemotactic and mitogenic activities are those produced by the 36kD peptide rather than the PDGF peptide

To determine whether the chemotactic and mitogenic activities observed in partially purified culture fluid proteins were derived from molecules containing PDGF a and B chain peptides or from molecular products not containing these sequences, affinity purified culture fluid proteins and recombinant PDGF AA and BB homodimers and AB heterodimers were serially diluted and subjected to biological assays. Sufficient samples were prepared so that a portion of each diluted sample was used for mitogen and chemotaxis assays and immunoblotting. The mitogen activity of HUVE affinity purified factor was comparable to that induced by all three recombinant PDGF dimers. The chemotactic activity is comparable to that of the AB heterodimer, which produces a response lower than that of the BB homodimer and higher than that of the AA homodimer. When the biological activity in a sample is compared to an equivalent amount of an immunoblot of the same sample, neither A-chain nor B-chain molecules are detected in the sample tested. These data indicate that the biological activity present in the anti-PDGF affinity purification moiety cannot be explained by molecules containing the a or B chain and suggest that the major PDGF-immunoreactive protein species present in these samples (36kD peptide) are biologically active but do not contain the amino acid sequences seen at the amino and carboxy termini of the PDGF a or B chain peptide.

Example 3

Receptor competition assay

NIH3T3 cells were cultured in 24-well plates (Costar) in DMEM containing 10% fetal bovine serum, 10. mu.g/ml gentamicin, and used for experiments when the cells were grown to confluence. The growth medium was removed and the cells were washed 2 times with serum-free DMEM containing 0.2mg/ml BSA, the plates were added with serum-free DMEM containing 0.2mg/ml BSA and placed on ice for 30 minutes. Test samples and controls were formulated in 0.2mg/ml BSA serum free DMEM containing 5-10ng/ml HUVE affinity purified protein and one of the serially diluted recombinant PDGF isoforms (concentration range 300 ng/ml-16 ng/ml). Add 1ml of sample to the well of the 24 well plate and incubate for 2 hours on ice on a bench shaker. After incubation, cells were washed 3 times with PBS on ice for 10 minutes each. The proteins bound to the cell surface were eluted with 5. mu.l of 1N acetic acid for 10 minutes. The acetic acid eluted samples were lyophilized, resuspended in 5mM HCl, electrophoresed on 12% polyacrylamide gels, and then immunoblotted onto nitrocellulose membranes for detection with anti-PDGF antibodies.

To confirm that endothelial cell molecules bind to cell surface PDGF receptors, competitive receptor binding assays were performed. Because the immunoblotting result of affinity purification of HUVE cell secretory protein shows that several PDGF immunoreactive molecules exist, it can not be used¹²⁵I-labeled PDGF competition assay, since this assay does not indicate which molecules in the mixture compete for labeled PDGF binding to receptors on the target cells. Since the molecular weight difference between the PDGF allotype and the major PDGF immune-related proteins secreted by HUVE cells, competition for receptor binding can be demonstrated by immunoblotting. Direct binding of the anti-PDGF immunoreactive peptide to NIH3T3 cells was confirmed by incubation of the anti-PDGF affinity purified protein (10ng/ml) with 3T3 fibroblast monolayer culture cells at 4 ℃ for 2 hours. Bound peptides were dissociated by washing the cell layer with 1N acetic acid and quantified by immunoblot analysis using anti-PDGF IgG. This data shows that the 36kD immunoreactive peptide binds to the cell surface of NIH3T3 and that this binding can be competed by increasing concentrations of recombinant PDGF BB added to the binding medium. These data suggest that CTGF peptide binds to specific cell surface receptors of NIH3T3 cells and that this binding can be competed by PDGF BB. RNA isolation and Northern blotting

Total RNA was isolated from monolayer cultured cells. The lyophilized RNA was resuspended in gel loading buffer containing 50% formamide, heated at 95 ℃ for 2 minutes, then loaded (20. mu.g total RNA per lane) onto a 2.2M formaldehyde, 1% agarose gel and run at 50 volts. The integrity of the RNA was determined by visualization of the 18S and 28S rRNA bands by ethidium bromide staining. After electrophoresis, RNA was transferred to nitrocellulose membrane by blotting with 10 × SSC buffer overnight. The nitrocellulose membrane was air dried and baked in a vacuum oven at 80 ℃ for 2 hours. Adding 5X 10 at 46 deg.C⁵CPM/ml ³²P-labeled probe was hybridized overnight. For Northern blotting, the entire plasmid is usually labeled as a probe. The labeling method is to label the reagent by using random primers of Boehinger Mannheim companyAnd (5) a box. After hybridization, the membrane was washed with 2 XSCC, 0.1% SDS at room temperature for 2 times 15 minutes each, 0.1 XSSC, 0.1% SDS at room temperature for 1 time 15 minutes, and finally 0.1 XSSC, 0.1% SDS at 46 ℃ for 15 minutes. Blotted on a Kodak X-omat film at-70 ℃.

Example 4

Library screening, cloning and assay

Standard Molecular biology techniques (Sambrook, et al, Molecular Cloning a laboratory Manual, 2 nd edition, Cold Spring Harbor laboratory Press, Col. Spring Harbor, N.Y.) are used for subcloning and purification of various DNA clones. Clone DB60 was selected from a lambda gt11 HUVE cell cDNA library by induction of fusion proteins and anti-PDGF antibody screening. The picked plaques were screened again and positive clones were re-plated at low titer and isolated.

The EcoRI insert obtained from clone DB60 was cloned into the M13 phage vector and single stranded DNA was obtained for making a clone of the reverse orientation insert. Then using the Sequenase kit (U.S. biochemical) and³⁵S-dATP (duPont) these M13 clones were sequenced by the dideoxy method. Both strands of the cloned DNA were completely sequenced using primer extension and GTP and ITP chemistry. A portion of the sequencing reaction was run on a 6% acrylamide (16 hours) and 8% acrylamide (6 hours) gel, dried under vacuum and autoradiographed for at least 18 hours.

cDNA fragment from clone DB60 to³²P-CTP labeling was used for rescreening HUVE cell cDNA. lamda.gt 11 library. Several clones were picked, of which the largest 2100bp was designated DB60R32 and subcloned into Bluescript phagemid. PstI, KpnI and EcoRI/KpnI restriction fragments were also subcloned into Bluescript. The sequencing of these subclones was carried out by the double-stranded plasmid DNA sequencing technique using the Sequenase described above. The 1458bpEcoRI/KpnI clone containing the open reading frame was subcloned into M13mp18 and M13mp19 using the primersElongation of the material and GTP and ITP chemistry completely sequenced both strands of DNA using single-stranded DNA sequencing techniques. Clonal expression and sequencing of connective tissue growth factor cDNA

To further characterize these PDGF-related molecules, a sufficient amount of CTGF protein is required for amino acid sequencing. However, because of the low CTGF concentration in the conditioned medium of HUVE cell culture, and the high cost and time-consuming techniques used to harvest and culture these cells, it is impractical to purify homogeneous proteins and sequence amino acids therefrom. Thus, anti-PDGF antibodies were used to screen HUVE cell cDNA libraries constructed in expression vector λ gt 11. More than 500,000 recombinant clones were selected, several clones showing strong signals against the anti-PDGF antibody were obtained during the selection, purified and subcloned into the M13 phage vector, and partial sequence data was obtained by single-stranded DNA sequencing. Query of the GenBank DNA sequence database indicated that there were two fragments of the picked clones containing the PDGF B chain cDNA open reading frame sequence. One of these clones is similar to the 1.8kb insert isolated previously with the c-sis cDNA probe, Collins et al (Nature, Vol.316: pages 748-750, 1995). The third 500bp clone was completely sequenced and the query for all nucleotide and amino acid sequences in GenBank did not find a homologous sequence (CEF 10 sequence was not available at that time). This clone was designated DB 60. The affinity purified protein completely blocked the binding between the anti-PDGF antibody and the fusion protein produced by clone DB 60. Preparation from DB60³²P-labeled probe for Northern blot analysis of 20. mu.g total RNA isolated from HUVE cells. The blot results indicated that the probe hybridized to a 2.4kp mRNA of sufficient size to produce a 38kD molecular weight range of protein seen when immunoblotted against affinity purified protein. The DB60 clone was used to rescreen the HUVE cell cDNA λ gt11 library, and the largest clone isolated contained the 2100bp insert designated DB60R 32. The probe prepared with the cloned DB60R 322100 bpEcoRI insert was also able to hybridize with a single 2.4kb messenger RNA in a Northern blot of total RNA from HUVE cells.

Example 5

In vitro transcription and translation

In vitro transcription reactions were performed using the 2100kb cDNA clone DB60R32 in Bluescript KS vector. The plasmid was digested with XhoI, which cleaves the plasmid at the 3' end of the vector multiple cloning site cDNA insert as a single cleavage site. The T7 promoter site located 5' to the cDNA insert was used for transcription. In vitro transcription was performed using a kit (Stratagene) supplied with Bluescript vector.

The in vitro translation reaction is performed by using nuclease-treated rabbit reticulocyte lysate and³⁵s-cysteine, the latter being formulated in a cysteine-free amino acid mixture (Promega) for labelling peptides. The reaction was run with a final volume of 50. mu.l per tube, containing³⁵S-cysteine 1mCi/ml (1200Ci/mMole, DuPont) and serial dilutions of mRNA obtained from in vitro transcription reactions (concentration range 50-500 ng). The reaction was incubated at 30 ℃ for 60 minutes. The reaction solution was divided into small portions, one of which was subjected to reduced or non-reduced 12% polyacrylamide gel electrophoresis, dried and subjected to autoradiography.

Bacterial expression of immunoreactive CTGF peptide was achieved by subcloning clone DB60R32 into the EcoRI site of the pET5 expression vector (student, et al, Academic Press, Vol. N.Y. 185: pp. 60-89, 1990) in both sense and antisense orientation (determined by restriction enzyme digestion analysis). Coli HMS 174 cells were cultured in M9 medium to OD6000.7, IPTG was added to the medium to give a concentration of 0.4mM, and the culture was continued for 2 hours. Centrifuging to precipitate the cells, cracking the cells, and centrifuging to remove the inclusion bodies. Cell extracts were divided into aliquots, and portions were subjected to 12% polyacrylamide gel electrophoresis and immunoblotting with anti-PDGF antibodies. The clone DB60R32 inserted the expressed protein in the sense orientation to produce anti-PDGF reactive peptides in the 36-39kD MW range, while the antisense control produced no immunoreactive peptides. Recombinant peptides produced in E.coli systems completely blocked the reaction of anti-PDGF with CTGF peptide present in conditioned media. Expression of CTGF in toads

For expression in toad oocytes, mature female xenopus laevis was obtained from Nasco (Fort Atkinson, WI) and fed at room temperature. Anaesthetizing toad by low-temperature treatment, and cutting ovary tissue by operation. Ovarian tissue was minced and digested with 0.2% collagenase (Sigma type II) diluted with calcium-free OR-2(Wallace, et al, Experimental zoology (Exp. Zool.), vol.184, pp.321-334, 1973) for 2-3 hours. Then, intact stage VI oocytes (Dumont, J.Morphol., 136: pp.153-180, 1972) 1.3mm in diameter were carefully selected and microinjected.

Stage VI oocytes (5-10 at a time) were placed on a concave plexiglass platform and drained of excess OR-2 solution. A sample containing 10ng RNA of about 50nl was injected into the oocyte animal pole just above the equator of the oocyte using a Leitz system microinjector. After injection, the oocytes were returned to OR-2 buffer containing 0.1% BSA and incubated at 25 ℃ for 24 hours. Live oocytes were collected and extracted by homogenization in 100mm NaCl, 10mm Tris pH 7.5 (20. mu.l/oocyte) using a Dounce homogenizer over 10 strokes. Thereafter, the cell homogenate was mixed with an equal volume of freon to remove pigments and lipids, and centrifuged at 10,000rpm for 30 seconds to separate the liquid phases. The upper aqueous phase was assayed for chemotactic activity by NIH3T3 cells as described above.

DB60R32 clone in vitro transcription preparation of RNA 10ng to toad oocyte injection leads to its fibroblast chemotactic activity. Control injected cells did not produce this activity. These results indicate that the open reading frame encoded by the DB60R32 clone has fibroblast chemotactic activity like CTGF.

Example 6

CTGF sequence analysis

The clone DB60R 322100 bp insert was initially sequenced by subcloning the PstI and KpnI restriction fragments into Blusecript and using a double-stranded dideoxy method. The results suggest an open reading frame of 1047 base pairs and mapped the DB60 insert to a larger cDNA. The EcoRI/KpnI fragment containing the complete open reading frame was inserted into M13mp18 and M13mp19 and the double stranded DNA was sequenced by single strand dideoxy by primer extension with GTP and the GTP analogue ITP. The nucleotide sequence of the open reading frame cDNA encoded a 38,000MW protein, confirming cell-free translation results and consistent with the size of the immunopurified peptide. A new query of the GenBank database showed that this cDNA has 50% homology with the nucleotide sequence of CEF-10mRNA, a immediate early gene induced in v-src transformed chicken embryo fibroblasts (Simmons, et al, Advance in American national academy of sciences (Proc. Natl. Acad. Sci. USA), Vol.86: pp. 1178-1182, 1989). The cDNA for the translated human CTGF shares 45% overall homology with avian CEF-10 and 52% if the putative alternative splice region is deleted. This region is located between amino acid 171 (aspartic acid) and amino acid 199 (cysteine) in the CTGF sequence.

Example 7

Cell culture for CTGF promoter region analysis

Human skin fibroblasts were cultured from skin biopsy specimens. NIH/3T3 cells and Cos 7 cells were obtained from the American type culture Collection (ATCC, Rockville, Md.). All cells were incubated at 37 ℃ with 10% CO₂And 90% air in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Calf Serum (FCS). The human skin fibroblasts used were all cells before passage 6. Growth factor

TGF-. beta.1 is awarded by Richard Assoaian (U.S. of Miami). Recombinant PDGFBB was obtained from Chiron (Emeryville, Calif.). Purified murine EGF was purchased from Sigma (st. RNA isolation and Northern blotting

The acidic thiocyanogen guanidine-phenol-chloroform extraction method previously reported (Chomczynski et al, biochemistryAngle of gravy (Birchem), volume 162: pp 156-159, 1987) isolating total RNA from cultured cells. Total RNA was electrophoresed on a 1.5% agarose/formaldehyde gel and transferred to nitrocellulose membrane. A1.1 kb fragment representing the CTGF open reading frame was obtained by PCR as a CTGF probe using specific primers HO 15 '-CGGAATTCGCAGTGCCAACCATGACC-3' (sequence # 3) and HO 25 '-CCGAATTCTTAATGTCTCTCACTCTC-3' (sequence # 4). By 1X 10⁶cpm/ml with [ alpha-32P ]]dCTP-labeled these probes were hybridized, and the probe labeling method was performed using a random primer DNA labeling kit (Boehringer Mannheim Biochemicals, Indianapolis, Inc.). Autoradiography was performed at-70 ℃ for 6-72 hours using X-ray film and intensifying screen. Genomic clone isolation and sequence analysis

Genomic DNA was isolated from human skin fibroblasts as previously described (Sambrook, et al, Cold Spring Harbor Laboratory Press, Vol. 9: 14-19, 1989). The CTGF gene fragment was amplified by PCR using 4. mu.g of genomic DNA as a template, using primers HO2 and HO 35 '-CGGAATTCCTGGAAGACACGTTTGGC-3' (sequence # 5). The PCR product was digested with EcoRI and subcloned into M13. Using the Sequenase kit (^*Sequence analysis of U.S. biochemical cprp, Cleveland, Ohio) by the dideoxy chain termination method (Sanger, et al, proceedings of the national academy of sciences of the united states (proc.natl.acad.sci.usa) showed a 900bp fragment with a 387bp intron in the middle. Using a lambda FIX^TMHuman genome library in II vector (Stratagene, LaJolla, Calif.), we used³²P-labeled 900bp genomic DNA fragment as a probe was screened at approximately 1X 10⁶And 3 recombinant phages are separated, and the CTGF gene is contained in the clones of the phages. Luciferase reporter gene assay

The CTGF promoter fragment containing nucleotides from-832 to +74, which is one of the human genomic clones, was first cloned into the Sacl-Xhol cloning site of pGL2-Basic vector (Promaga). This construct (PO) was used as template for PCR and deletion mutants were constructed with the following specific primers: p1 includes nucleotides-638 to +74, P2-363 to +74, P3-276 to +74, and P4-128 to + 74. All deleted fragments were sequenced to ensure no mutationsThe variants are introduced into the promoter fragment. Lipofectin in 6-well plates^The reagent (GIBCO BRL) was transfected into NIH/3T3 cells for 6 hours. Each transfection contained 2. mu.g of pSV-. beta. -galactosidase vector (Promega). Cells after transfection in the ITS-containing cells^TM(collagen Biochemical Products) for 24 hours, followed by addition of growth factors for 4 hours or 24 hours. Luciferase activity was measured by single photon monitoring using the luciferase assay system (Promega) and scintillation counter (Beckman LS 6000 SC). To normalize differences in transfection efficiency, Galacto-Light was used^TM(TROPIX, Inc.) chemiluminescence assays were performed to determine beta-galactosidase activity. Preparation of nuclear extract

Nuclear extracts were prepared as described by Abmayr and Workman (Current protocols in Molecular Biology), Vol.2, pp.12.1.1-12.1.9, Ausubel et al, Greene Publ, and Wiley Interscience, NY, NY). Briefly, cells were treated with hypotonic buffer (10mM HEPES pH 7.9, 1.5mM MgCl)₂10mM KCl, 0.2mM PMSF, 0.5mM DTT), homogenized with a glass dounce homogenizer at 10 strokes, and centrifuged at 3300 Xg for 15 minutes to separate the nuclei. The nuclear proteins were extracted by suspending the nuclei in an equal volume of extraction buffer (20mM HEPES pH 7.9, 25% glycerol, 1.5mM MgCl)₂0.8MKCl, 0.2mM EDTA, 0.2mM PMSF and 0.5mM DTT). The extract was dialyzed against 20mM HEPES pH 7.9, 20% glycerol, 100mM KCl, 0.2mM EDTA, 0.2mM PMSF and 0.5mM DTT before use. Protein concentration was determined using BCA protein assay reagent (Pierce). Gel mobility Change test

Fragments of the CTGF promoter were prepared by PCR or restriction enzyme digestion of the promoter fragment. Annealing the complementary single-stranded oligonucleotide to obtain the double-stranded oligonucleotide. All oligonucleotides and fragments were checked by agarose gel or polyacrylamide gel electrophoresis. Radiolabeled CTGF promoter fragments were prepared by end-labeling using Klenow enzyme (Boehringer Manheim) and polynucleotide kinase (Boehringer Manheim). The labeled fragments were subjected to 2% agarose before being used in gel mobility Change assaysPurification was performed by gel or 20% polyacrylamide gel electrophoresis. The binding reaction mixture contained 1. mu.g of nuclear extract protein in 20. mu.l of a reaction system containing 10mM HEPES pH 7.9, 5mM Tris, 50mM KCl, 0.1mM EDTA, 1. mu.g of poly (deoxyinosine-deoxycytidine) ° poly (deoxyinosine-deoxycytidine) (pharmacia), 10% glycerol, 300. mu.g/ml BSA and 10,000cpm³²P-labeled DNA probe. Non-labeled competitor DNA was added prior to the addition of labeled probe and incubated at 4 ℃ for 2 hours. Then, the labeled probe was added to the reaction mixture and incubated at 4 ℃ for 1 hour. Electrophoresis was performed with 50mM Tris, 0.38M glycine and 2mM EDTA using a 5% polyacrylamide gel. Methylation interference test

End-labeled double-stranded oligonucleotide fragments were prepared as described for the gel mobility shift assay. The oligonucleotide was methylated by reaction with dimethyl sulfate (Fisher Scientific) for 5 minutes at room temperature. DNA-protein binding and gel mobility modification assays were performed as described above using large doses of labeled probe (100K cpm) and nucleoprotein (20. mu.g). DNA was purified from the altered and unaltered bands and cleaved with piperidine (Fisher Scientific) and samples were electrophoresed in polyacrylamide DNA sequencing gel. The mobility changes and the sequence of the unchanged fragments were compared to the sequence of the complete probe, which was sequenced in the same way.

Example 8

Delayed induction of CTGF mRNA by short-term effects of TGF-beta

Most immediate early genes induced by growth factors, such as c-fos and c-myc, are very transiently expressed even if the growth factors are present in the culture broth. In contrast, CTGF transcription remains high after 24 hours after activation of cells by TGF-. beta.s (Igarashi, et al, molecular biology and cytology, mol. biol. cell, Vol.4: pages 637-645, 1993). This example examines whether long-term increases in CTGF transcription depend on the continued presence of TGF- β.

Confluent human skin fibroblasts were cultured in serum-free DMEM (DMEM-ITS) supplemented with insulin, transferrin and selenium for 24 hours, followed by addition of TGF-. beta.s. After 1 hour of TGF-. beta.action, cells were washed with PBS and replaced with DMEM-ITS, and then cultured for various periods of time. Specifically, confluent human skin fibroblasts were incubated in DMEM-ITS containing 5. mu.g/ml insulin, 5. mu.g/ml transferrin and 5. mu.g/ml selenium for 24 hours, followed by addition of TGF-. beta.s. After 1 hour of treatment with 10ng/ml TGF-. beta.the cells were washed with PBS and incubated in DMEM-ITS for the indicated time. Northern blot analysis showed that CTGF mRNA was still strongly induced 4-30 hours after TGF- β removal (FIG. 2A).

This example also examined the ability of TGF- β to induce CTGF transcription in the presence of several protein synthesis inhibitors. Fig. 2B shows the effect of cycloheximide on CTGF mRNA induction. Lanes a and H are untreated control cells at 4 hours and 24 hours, respectively. Lane B: 4 hours, Cycloheximide (CHX) (10. mu.g/ml); lane C: 4 hours, enabling the cycloheximide to act for 2 hours, and adding TGF-beta to act for 1 hour at the 1 st hour; lane E: RNA was prepared as in B, but 24 hours after cycloheximide addition; lane f: 24 h TGF-. beta.s (10. mu.g/ml); lane G: RNA was prepared as in D, but 24 hours after cycloheximide addition and 22 hours after TGF-. beta.removal.

As shown in FIG. 2B, TGF- β stimulation in the presence of cycloheximide for 1 hour was sufficient to induce CTGF mRNA after 4 hours and after 24 hours. The ability of cycloheximide alone to increase CTGF mRNA after 4 hours suggests the possibility of stabilizing the mRNA as has been reported for cycloheximide induced transcription of other genes such as c-fos and c-myc (Greenberg et al, Nature (London)), Vol.311, pages 433-438, 1984; Kruijer et al, Nature (Nature), Vol.312, pages 711-716, 1984). However, reports by Edwards and Mahadevan (Edwards, D.R., and L.C. Mahadevan, EMBO J. (EMBO J.). 11: pp. 2415-2424, 1992) indicate that protein synthesis inhibitors cycloheximide and anisomycin, but not puromycin, can act to stimulate transcription of the c-fos and c-jun genes, and thus messenger stabilization is not the only possible mechanism of action for these compounds.

The ability of anisomycin and puromycin to inhibit TGF- β induction of CTGF transcription was compared to the ability of cycloheximide to enhance CTGF transcription. FIG. 2C shows the effect of protein synthesis inhibitors on CTGF mRNA induction. Cells were treated with puromycin or anisomycin for 4 hours. TGF-. beta.was added 1 hour after the addition of the protein synthesis inhibitor, the cells were incubated for 3 hours, and then total RNA was isolated. CTGF transcription was analyzed by Northern blot.

Puromycin also failed to induce CTGF mRNA at the concentrations tested up to 100 μ g/ml, which were 10-fold higher than those required to completely block protein synthesis in these cells. Even at such high concentrations, it had no effect on the ability of TGF-beta to induce CTGF mRNA. In contrast, anisomycin did increase CTGF transcription (FIG. 2C), as seen with cycloheximide treatment, although TGF-. beta.treatment increased CTGF mRNA levels in the presence of anisomycin.

These findings are similar to those reported by Edwards and Mahadevan (Edwards, D.R., and L.C.Mahadevan, EMBO J. (EMBO J.), volume 11: pages 2415-2424, 1992), which found that anisomycin or cycloheximide alone induced c-fos and c-jun, but not puromycin alone. These data strongly suggest that TGF- β directly regulates CTGF gene expression, the mechanism of which is independent of protein synthesis, and may act primarily at the transcriptional level.

Example 9

Isolation of human CTGF Gene

To elucidate the structure of the CTGF gene, a fragment of the CTGF gene was first obtained by PCR. 4 micrograms of genomic DNA prepared from human skin fibroblasts as template and oligonucleotides HO2 and HO3 as primers. After 30 reaction cycles, a 900bp fragment was recovered which was 390bp longer than expected from the cDNA sequence, and nucleotide sequence analysis of this fragment (HO 900) revealed the presence of a 387bp intron in the central part of the fragment. By HO900For the probe, 3 phage clones were obtained from a human genomic library, which contained a 4.3kb XbaI fragment representing the entire coding sequence of the CTGF gene and a large portion of the putative promoter region. As shown in fig. 1A, the CTGF gene has 5 exons and 4 introns. Oligonucleotide primer extension identified a TATA sequence that appeared 24 nucleotides upstream of the mRNA cap. Between nucleotides-380 to-390, there is a consensus sequence of the CArG box, which is CC (A/T)₆GG is the inner core of a seroreactive element (SRE) of the feature. Other potential regulatory elements also exist including a CAT box, two Spl sites and two AP-1 sites. Furthermore, the CTGF promoter has an NF-1-like site (TGGN) between-194 and-182 sites₆GCCAA) (6# sequence), and a TGF-beta inhibitory element-like sequence (GNNTTGGTGA) (7# sequence) between-119 and-128. These two elements differ from The reported consensus sequence by only a single base (Edwards, D.R., and J.K.Heath, hormone control regulation of gene transcription, Vol.16: pages 333-347). DNA sequence comparison showed 80% sequence identity between the human CTGF promoter and the murine fsp-12 promoter in the 5' 300 nucleotide region from the transcription start point (FIG. 1B). The similarity of the two DNA sequences is much lower in the more upstream region.

Example 10

Investigation of CTGF promoter

To examine whether the 5' -untranslated region of the CTGF gene can be used as a promoter inducible by TGF-. beta.A fusion gene comprising the CTGF promoter (nucleotides-823 to +74) and the coding region of the firefly luciferase gene was constructed and inserted into the vector pGL 2-basic. Luciferase activity was detected in a transient transfection assay with NIH/3T3 cells. This construct resulted in a 15-30 fold increase in luciferase activity induced after 24 hours of TGF- β stimulation compared to control cultured cells. For CTGF mRNA levels, other growth factors such as PDGF, EGF and FEF were also able to stimulate induction of luciferase activity only 2-3 fold increase under equivalent conditions (table 1).

TABLE 1

Regulation of the CTGF promoter by cell types and growth factors

Luciferase activity relative fold induction cell type TGF-beta PDGF FGF EGF after growth factor treatment

NIH/3T3	25.7	2.9	3.3	1.4
					HSF	9.2	2.4	3.1	2.2
VSMC	9.8	ND	ND	ND
					HBL100	1.1	ND	1.3	1.4
HEP G2	1.3	ND	1.4	1.8

ND does not detect GSF-human foreskin fibroblast (primary) VSMC-fetal bovine aortic smooth muscle cell (primary) HBL100 human mammary epithelial cell line (non-tumorigenic) HEP G2 human liver epithelial cell line (non-tumorigenic) (CTGF gene fragments from nucleotide-823 to +74 were inserted into pGL2-basic vector plasmid was transfected with lipofectin for 6 hours, cells were incubated for 16 hours in DMEM-ITS with additional growth factors, cell extracts were prepared after 24 hours incubation and luciferase activity was detected.

When the promoter fragment was cloned in the reverse direction (+ 74-823), only basal levels of luciferase activity were detected, and cells were not affected by treatment with TGF-. beta.or other growth factors. The same growth factor induction pattern was also observed when human skin fibroblasts were used in the experiment instead of NIH/3T3 cells (Table 1). TGF- β does not induce luciferase activity in several epithelial cell lines (table 1), indicating that TGF- β is cell type specific for the regulation of the CTGF gene. The lack of any response by epithelial cells is not due to the lack of TGF-beta response, as the growth of these cells can be inhibited by TGF-beta (10 ng/ml). Luciferase activity induction under the control of the CTGF promoter requires only a short effect of TGF- β on the cells, since the fold induction of 1 hour of TGF- β treatment on cells was almost identical to that of sustained TGF- β action on cells at 4 hours and 24 hours (table 2). These results confirm the data obtained by the Northern blot described above and indicate that transcriptional regulation plays a major role in the control of CTGF gene expression by TGF-. beta.s.

TABLE 2 short-term TGF-B treatment stimulates long-term CTGF promoter activity¹Luciferase Activity test time

Duration of TGF-beta treatment	4 hours	24 hours
			(Continuous)	3.8	21
1 hour	3.5	19

¹Fold induction of luciferase activity was determined as illustrated in table 1 and example 7. NIH/3T3 cells were used for these experiments.

Example 11

Identification of promoter elements required for TGF-beta Induction

To determine which region of the promoter sequence is responsible for TGF- β induction, deletion variants of the CTGF promoter were constructed by PCR using primers designed to delete known transcription factor consensus elements. The promoter region was deleted in sequence starting from the most distal end of the 5' region and gradually moving toward the transcription initiation point (FIG. 3A). Removal of the promoter region preceding base-363, which includes an AP1 site and the CArG box, had no significant effect on TGF- β induced luciferase activity. When the second AP-1 was deleted (-363 to-276), luciferase activity was reduced by about 30%, although the fold induction by TGF-. beta.was still high (20-fold). Removal of the NF-1-like sites (-276 to-128) in the P4 construct abolished the TGF- β inducibility of the promoter, suggesting that this region contains TGF-B response elements. By using two BsmI sites, nucleotides from-162 to-110 are deleted, and other partial promoters are kept intact. This construct showed complete loss of TGF- β inducibility, suggesting that the sequence between-162 and-128 is essential for TGF- β to induce luciferase activity. This region contains a TGF- β inhibitory element (TIC) -like site, contiguous with an NF-1-like site, while NF-1-like sites have been reported to play a role in TGF- β regulation of α 2(1) collagen gene expression (Oikarinen, J., A. Hatamochi, and B. De Crombghe., J. biol. chem., 262: 11064-11070, 1987) and in TGF- β regulation of type 1 plaminogen activator inhibitor (PAI-1) gene expression (Riccio, et al, molecular cell biology (mol. cell. biol.), 12: 1846-1855, 1992).

The nucleotide sequence from-275 to-106 of the CTGF promoter was placed upstream of the enhancer-free SV40 promoter, and the SV40 promoter regulated luciferase gene, thus constructing a fusion gene for determining whether the region of the CTGF promoter was inducible by the TGF-. beta.gene (FIG. 3B). The SV40 promoter without enhancer is not regulated by TGF-. beta.s. However, the promoters containing the CTGF sequences-275 to-106 showed nearly 9-fold induction after TGF-beta treatment. Insertion of this fragment in the opposite direction slightly stimulates luciferase activity upon treatment with TGF-. beta.s. These data demonstrate that the region between nucleotides-275 to-106 of the CTGF promoter can act as a TGF-beta regulator element.

A series of competitive gel change and methylation interference experiments were used to elucidate the binding of this region of the CTGF promoter to nucleoprotein. Competitive gel changes were originally used to elucidate which region of the sequence between positions-205 to-109 was the target for protein binding. The probes and various competitor fragments are illustrated graphically (FIG. 4). The results of these studies showed that any fragment containing the NH3 region (-169 to-149) can be used as a specific competitor for the labeled promoter fragment containing bases-204 to-109 (FIG. 4). This region is located between NF-1-like and TIE-like sites. In the gel change assay, oligonucleotide fragments containing only the TIE-like region or the NF-1-like region but not the NH3 region were not competitive.

To further elucidate the regulatory elements, methylation interference assays were performed. The promoter used was originally a fragment from-275 th to-106 th. The results of these studies suggest that neither NF-1-like sites nor TIE elements appear to interact with any of the nucleoproteins present in control or TGF- β treated cells, further confirming the results of the gel-shift competition assay. However, the region from-157 to-145 located between these sites contains several G residues that are not methylated in the altered electrophoretic band, suggesting that this region is a nuclear protein binding site (FIG. 5A). Analysis of a smaller fragment (nt-169-139) in this region further identified some important G residues (dispute 5B). The data from this analysis confirm the results obtained with the larger fragment analysis and the positioning of the G residues within the sequence determined by the competitive gel changes.

To better identify the exact site of TGF- β response, the ability of the complete sequence and several of the missing sequences to competitively bind to the protein was compared in a gel change assay (FIG. 6). These data confirm the results of methylation interference experiments and suggest that the promoter contains at least a portion of cis-regulatory elements from the-159 to-143 region involved in the regulation of CTGF gene expression by TGF-. beta.

The regions of the sequence believed to be involved in TGF-. beta.induction were point mutated and these promoters were detected in the luciferase reporter gene construct that we constructed (FIG. 7). Two point mutations were detected, both of which reduced the inducibility of the TGF-. beta.to the gene. One mutation reduced induction by 25% and the other by 80% compared to the control. The two mutated sequences had no effect on the basal expression level compared to the control non-mutated native sequence.

The point mutation was constructed by synthesizing oligonucleotides containing the desired altered base and using two BsmI sites in the CTGF promoter. All constructs were confirmed by nucleotide sequence analysis to contain only the desired base changes, while all other bases in the nucleotide sequence were identical to the normal promoter. Assays were performed as described above for other CTGF promoter-luciferase constructs using NIH/3T3 cells as target cells. The data listed in the table in fig. 7 were obtained from a single experiment, with one trial repeated for each experimental condition. This experiment was repeated several times to confirm the results obtained. These data indicate that a single mutation in this region of the promoter can reduce TGF-. beta.induction by 85%, which is only less than 15% of the normal gene. These data indicate that the identified sequences are essential for TGF-. beta.induction of the CTGF gene.

Example 12

TGF-B inhibition of TGF-beta induced CTGF gene expression by stimulation of increased levels of anchoring independent growth a.cAMP via CTGF-dependent pathways

Both herbimycin and phorbol ester were used to determine whether tyrosine kinase or protein kinase C had any role in TGF-beta induced regulation of CTGF gene expression. These studies were performed by transfecting NIH/3T3 cells with the CTGF promoter (-803 to +74) luciferase reporter gene construct.

NIH/3T3 cells grew to 50% confluence in DMEM/10% FCS and LIPOFECTIN was used as described in example 7^The PO CTGF promoter (nucleotide-823- +74) was transfected into all cells and drives the expression of pGL2 basic vector firefly luciferase. Inhibitors were added after 24 hours of culture in DMEM/ITS medium. After all formulations were added to the culture system for 2 hours, 10ng/ml TGF-. beta.was added. Cells were incubated for 24 hours and then trypsinized with Tropix luciferaseThe activity of luciferase is measured by a measuring kit and a Beckman scintillation counter equipped with a single-photon probe. In a related experiment, PMA was added 24 hours prior to the addition of TGF-. beta.to deplete protein kinase C in the cells. This treatment also did not affect the ability of TGF-. beta.to induce luciferase activity under the control of the CTGF promoter. Furthermore, as a control experiment for herbimycin studies, the activity of the formulation in inhibiting PDGF-induced cell division was also investigated. NIH/3T3 cells that stopped proliferating due to overgrowth were treated with herbimycin at the indicated concentration for 2 hours, followed by addition of recombinant PDGF BB. After addition of PDGF24 hours, cells were trypsinized and counted. The division index represents the percentage of cells that are dividing.

None of these compounds has any effect on the ability of TGF-beta to induce CTGF gene expression, nor do they alter the basal level of CTGF gene expression in the target cell. However, both cholera toxin and 8 bromo-cAMP are potent inhibitors of TGF- β induced CTGF gene (fig. 8A).

These data indicate that neither tyrosine kinase nor protein kinase C are part of the signaling pathways regulated by TGF- β that lead to CTGF gene induction. Furthermore, cyclic nucleotide regulatory proteins do not appear to be part of the TGF- β regulatory pathway for CTGF gene expression. However, elevated cAMP levels in cells abrogate the induction of CTGF gene expression by TGF- β. In a related experiment we found that addition of cAMP or cholera toxin 8 hours after addition of TGF- β still effectively blocked the expression of the CTGF gene. This suggests that the role of cAMP is not through the receptor, but may affect the transcription factor that binds to the CTGF promoter. cAMP does not block all effects of TGF-beta on fibroblasts

FIG. 8B shows 4 micrographs of NIH/3T3 cells from the experiment described above: control) no agent was added; TGF-. beta.) TGF-. beta.10 ng/ml; cAMP)8 bromocAMP (1000. mu.M); cAMP + TGF-. beta.) 8 bromocAMP (1000. mu.M) and TGF-. beta.s (10ng/ml), followed by luciferase activity assay. These data indicate that although cAMP caused a significant change in the morphology of NIH/3T3 cells, the addition of TGF- β to these cells resulted in morphology of these cells that appeared similar, if not identical, to control cells treated with TGF- β. Thus, although cAMP blocks TGF- β induction of CTGF gene expression, it has no effect on biochemical events that modulate morphological changes seen in these monolayer cell cultures. These results suggest that there are various components involved in the action of TGF-. beta.s that can be regionally blocked by cAMP on fibroblasts. No significant difference was detected in cultured cells in terms of total cellular protein content or SV40/β -galactosidase control reporter gene expression, indicating that these changes were not due to the toxic effect of cAMP. The morphological changes resulting from cholera toxin treatment were similar to those seen with 8-bromocAMP treated cells, and these changes were reversed by the addition of TGF-. beta.s. Inhibition of TGF-beta induced anchorage-independent growth by cAMP and reversal of inhibition by rCTGF

In view of the results of the previous studies, the following experiments were performed in order to determine whether cAMP would block TGF-. beta.induced anchorage-independent growth. First, the effects of 8 bromocAMP, 8 bromocGMP and cholera toxin on TGF- β induced anchorage-independent growth of NRK cells. Anchorage-independent growth tests were carried out essentially as described by Guadagna and Assoian (J.cell.biol., Vol.115: pp.1419-1425, 1991). Briefly, NRK cells normally maintained as a monolayer culture were seeded on agar layers in DMEM/10% FCS containing 5ng/ml EGF. TGF-. beta.or CTGF was added and the cells were incubated for 72 hours. Then performing a DNA synthesis assay by³H-thymidine (2. mu. Ci/ml) was labeled for 24 hours, and the cells were harvested and treated with TCA precipitation or the like. Inhibitors were added simultaneously with growth factors and remained present throughout the experiment (fig. 8C). (abbreviation: Cholera Toxin (CTX)).

As seen in FIG. 8C, both 8-bromocAMP and cholera toxin are potent growth inhibitors in this assay, while 8-bromocGMP concentrations up to 10mM remain ineffective. Since elevated cAMP levels in cells block CTGF gene expression, an experiment was conducted to determine whether rCTGF could overcome this inhibition. As shown in the left panel of fig. 8B, addition of rCTGF to NRK cells failed to stimulate anchorage-independent growth and therefore failed to replace TGF- β. However, for cells treated with TGF- β and inhibited with 8-bromocAMP or cholera toxin, the same dose of rCTGF was added to overcome the inhibition and allow the cells to grow at a rate comparable to cells treated with TGF- β but without cAMP or cholera toxin (right-most panel). These studies suggest that there is a direct link between CTGF production and NRK cell growth in suspension. These studies also show that although TGF- β induces some effect on fibroblasts with elevated cAMP levels, these effects are not sufficient to anchor cell independent growth. Moreover, since CTGF alone is also insufficient to stimulate this biological response, it does not represent all of the effects of TGF- β on fibroblasts. These results suggest that TGF- β induces two effects in target cells (NRK) -CTGF-dependent and CTGF-independent effects, which act synergistically to produce a specific cellular response (anchorage-independent growth).

Example 13

Formulations for increasing cAMP levels inhibit TGF-beta induced granulation tissue formation

TGF- β has been shown to induce fibrosis in several animal model studies. For example, one group injected 400-800 ng TGF-. beta.s into the subcutaneous space of the back of newborn mice. When injected once daily for three consecutive days, a wide range of fibrotic tissue forms (Roberts, et al, Advance in American national academy of sciences (Proc. Natl. Acad. Sci. USA), Vol.83: page 4167, 1986). This example shows that comparative studies of TGF- β and CTGF indicate that CTGF induces connective tissue formation very similar, if not identical, to that induced by TGF- β. Other growth factors such as PDGF or EGF induce tissues that differ from those induced by TGF- β, suggesting that CTGF may play a role in tissue formation induced by TGF- β injection.

Whereas the results of example 12 show that cAMP levels block the induction of CTGF in cultured cells, it is important in animals to determine whether elevated cAMP levels in cells block the in vivo effects of TGF- β. The following experiments were performed using the injection model described above and by Roberts et al. Newborn mice were divided into 4 groups, and the following agents were injected, one daily for three consecutive days:

TGF-. beta.400 ng; cholera toxin (100 ng); TGF-. beta.400 ng and cholera toxin 100 ng; or saline. Each group had 3 mice. After preparing the injected tissue by a conventional histological method, the injected area was examined under an optical microscope by hematoxylin and eosin staining.

As expected, saline injection had no effect on the type of tissue present in the skin of mice, whereas TGF- β injection induced a large amount of new connective tissue similar to granulation tissue. Such tissues contain an increased number of fibroblasts and an increased amount of collagen and other matrix components. Injection of cholera toxin alone did not stimulate granulation tissue formation. Simultaneous injection of TGF- β and cholera toxin also did not show granulation tissue formation, indicating that cholera toxin blocks the induction of granulation tissue formation by TGF- β. These results suggest the therapeutic use of agents that block CTGF production or action as anti-fibrotic drugs.

Sequence listing (1) general data (i) applicants: subject of the invention of University of South Florida (ii): number of connective tissue growth factor (iii) sequences: 2(iv) communication address:

(A) the receiver: FISH & RICHARDSON

(B) Street: 4225 Executive Square, Suite 1400

(C) City: la Jolla

(D) State: CA

(E) The state is as follows: US

(F) And (3) post code: 92037(v) computer readable form:

(A) media type: flexible disk

(B) A computer: IBM PC compatible machine

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: patent in Release #1.0, Version #1.25(vi) current application data:

(A) application No.: PCT/US 96-

(B) Application date: 30-05-1996

(C) And (4) classification: (viii) lawyer/attorney profile:

(A) name: haile, ph.d., Lisa a.

(B) Registration number: 38,347

(C) Query/tag number: 07414/003WO1(ix) Telecommunications data:

(A) telephone: 619-678-5070

(B) Electric transmission: 619-678-5099(2)1# sequence data: (i) the sequence characteristics are as follows: length (A): 2075 base pairs (type B): nucleic acid (C) strand type: single chain (D) topology: linear (ii) molecular type: (vii) direct source of cDNA: (B) cloning: ctgf (ix) characteristics: (A) name/keyword: cds (b) position: sequence representation of 1177 (xi): 1# sequence: CCCGGCCGAC AGCCCCGAGA CGACAGCCCG GCGCGTCCCG GTCCCCACCT CCGACCACCG60CCAGCGCTCC AGGCCCCGCG CTCCCCGCTC GCCGCCACCG CGCCCTCCGC TCCGCCCGCA 120GTGCCAACC ATG ACC GCC GCC AGT ATG GGC CCC GTC CGC GTC GCC TTC 168

Met Thr Ala Ala Ser Met Gly Pro Val Arg Val Ala Phe

1 5 10GTG GTC CTC CTC GCC CTC TGC AGC CGG CCG GCC GTC GGC CAG AAC TGC 216Val Val Leu Leu Ala Leu Cys Ser Arg Pro Ala Val Gly Gln Asn Cys

15 20 25AGC GGG CCG TGC CGG TGC CCG GAC GAG CCG GCG CCG CGC TGC CCG GCG 264Ser Gly Pro Cys Arg Cys Pro Asp Glu Pro Ala Pro Arg Cys Pro Ala30 35 40 45GGC GTG AGC CTC GTG CTG GAC GGC TGC GGC TGC TGC CGC GTC TGC GCC 312Gly Val Ser Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala

50 55 60AAG CAG CTG GGC GAG CTG TGC ACC GAG CGC GAC CCC TGC GAC CCG CAC 360Lys Gln Leu Gly Glu Leu Cys Thr Glu Arg Asp Pro Cys Asp Pro His

65 70 75AAG GGC CTC TTC TGT GAC TTC GGC TCC CCG GCC AAC CGC AAG ATC GGC 408Lys Gly Leu Phe Cys Asp Phe Gly Ser Pro Ala Asn Arg Lys Ile Gly

80 85 90GTG TGC ACC GCC AAA GAT GGT GCT CCC TGC ATC TTC GGT GGT ACG GTG 456Val Cys Thr Ala Lys Asp Gly Ala Pro Cys Ile Phe Gly Gly Thr Val

95 100 105TAC CGC AGC GGA GAG TCC TTC CAG AGC AGC TGC AAG TAC CAG TGC ACG 504Tyr Arg Ser Gly Glu Ser Phe Gln Ser Ser Cys Lys Tyr Gln Cys Thr110 115 120 125TGC CTG GAC GGG GCG GTG GGC TGC ATG CCC CTG TGC AGC ATG GAC GTT 552Cys Leu Asp Gly Ala Val Gly Cys Met Pro Leu Cys Ser Met Asp Val

130 135 140CGT CTG CCC AGC CCT GAC TGC CCC TTC CCG AGG AGG GTC AAG CTG CCC 600Arg Leu Pro Ser Pro Asp Cys Pro Phe Pro Arg Arg Val Lys Leu Pro

145 150 155GGG AAA TGC TGC GAG GAG TGG GTG TGT GAC GAG CCC AAG GAC CAA ACC 648Gly Lys Cys Cys Glu Glu Trp Val Cys Asp Glu Pro Lys Asp Gln Thr

160 165 170GTG GTT GGG CCT GCC CTC GCG GCT TAC CGA CTG GAA GAC ACG TTT GGC 696Val Val Gly Pro Ala Leu Ala Ala Tyr Arg Leu Glu Asp Thr Phe Gly

175 180 185CCA GAC CCA ACT ATG ATT AGA GCC AAC TGC CTG GTC CAG ACC ACA GAG 744Pro Asp Pro Thr Met Ile Arg Ala Asn Cys Leu Val Gln Thr Thr Glu190 195 200 205TGG AGC GCC TGT TCC AAG ACC TGT GGG ATG GGC ATC TCC ACC CGG GTT 792Trp Ser Ala Cys Ser Lys Thr Cys Gly Met Gly Ile Ser Thr Arg Val

210 215 220ACC AAT GAC AAC GCC TCC TGC AGG CTA GAG AAG CAG AGC CGC CTG TGC 840Thr Asn Asp Asn Ala Ser Cys Arg Leu Glu Lys Gln Ser Arg Leu Cys

225 230 235ATG GTC AGG CCT TGC GAA GCT GAC CTG GAA GAG AAC ATT AAG AAG GGC 888Met Val Arg Pro Cys Glu Ala Asp Leu Glu Glu Asn Ile Lys Lys Gly

240 245 250AAA AAG TGC ATC CGT ACT CCC AAA ATC TCC AAG CCT ATC AAG TTT GAG 936Lys Lys Cys Ile Arg Thr Pro Lys Ile Ser Lys Pro Ile Lys Phe Glu

255 260 265CTT TCT GGC TGC ACC AGC ATG AAG ACA TAC CGA GCT AAA TTC TGT GGA 984Leu Ser Gly Cys Thr Ser Met Lys Thr Tyr Arg Ala Lys Phe Cys Gly270 275 280 285GTA TGT ACC GAC GGC CGA TGC TGC ACC CCC CAC AGA ACC ACC ACC CTG 1032Val Cys Thr Asp Gly Arg Cys Cys Thr Pro His Arg Thr Thr Thr Leu

290 295 300CCG GTG GAG TTC AAG TGC CCT GAC GGC GAG GTC ATG AAG AAG AAC ATG 1080Pro Val Glu Phe Lys Cys Pro Asp Gly Glu Val Met Lys Lys Asn Met

305 310 315ATG TTC ATC AAG ACC TGT GCC TGC CAT TAC AAC TGT CCC GGA GAC AAT 1128Met Phe Ile Lys Thr Cys Ala Cys His Tyr Asn Cys Pro Gly Asp Asn

320 325 330GAC ATC TTT GAA TCG CTG TAC TAC AGG AAG ATG TAC GGA GAC ATG GCA T 1177Asp Ile Phe Glu Ser Leu Tyr Tyr Arg Lys Met Tyr Gly Asp Met Ala

335340345 GAAGCCAGAG AGTGAGAGAC ATTAACTCAT TAGACTGGAA CTTGAACTGA TTCACATCTC 1237ATTTTTCCGT AAAAATGATT TCAGTAGCAC AAGTTATTTA AATCTGTTTT TCTAACTGGG 1297GGAAAAGATT CCCACCCAAT TCAAAACATT GTGCCATGTC AAACAAATAG TCTATCTTCC 1357CCAGACACTG GTTTGAAGAA TGTTAAGACT TGACAGTGGA ACTACATTAG TACACAGCAC 1417CAGAATGTAT ATTAAGGTGT GGCTTTAGGA GCAGTGGGAG GGTACCGGCC CGGTTAGTAT 1477CATCAGATCG ACTCTTATAC GAGTAATATG CCTGCTATTT GAAGTGTAAT TGAGAAGGAA 1537AATTTTAGCG TGCTCACTGA CCTGCCTGTA GCCCCAGTGA CAGCTAGGAT GTGCATTCTC 1597CAGCCATCAA GAGACTGAGT CAAGTTGTTC CTTAAGTCAG AACAGCAGAC TCAGCTCTGA 1657CATTCTGATT CGAATGACAC TGTTCAGGAA TCGGAATCCT GTCGATTAGA CTGGACAGCT 1717TGTGGCAAGT GAATTTGCCT GTAACAAGCC AGATTTTTTA AAATTTATAT TGTAAATATT 1777GTGTGTGTGT GTGTGTGTGT ATATATATAT ATATATGTAC AGTTATCTAA GTTAATTTAA 1837AGTTGTTTGT GCCTTTTTAT TTTTGTTTTT AATGCTTTGA TATTTCAATG TTAGCCTCAA 1897TTTCTGAACA CCATAGGTAG AATGTAAAGC TTGTCTGATC GTTCAAAGCA TGAAATGGAT 1957ACTTATATGG AAATTCTGCT CAGATAGAAT GACAGTCCGT CAAAACAGAT TGTTTGCAAA 2017GGGGAGGCAT CAGTGTCTTG GCAGGCTGAT TTCTAGGTAG GAAATGTGGT AGCTCACG 2075(2) sequence data # 2: (i) the sequence characteristics are as follows:

(A) length: 349 amino acid

(B) Type (2): amino acids

(D) Topological structure: linear (ii) molecular type: protein (xi) sequence representation: 2# sequence: met Thr Ala Ala Ser Met Gly Pro Val Arg Val Ala Phe Val Val Leu 151015 Leu Ala Leu Cys Ser Arg Pro Ala Val Gly Gln Asn Cys Ser Gly Pro

20 25 30Cys Arg Cys Pro Asp Glu Pro Ala Pro Arg Cys Pro Ala Gly Val Ser

35 40 45Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala Lys Gln Leu

50 55 60Gly Glu Leu Cys Thr Glu Arg Asp Pro Cys Asp Pro His Lys Gly Leu65 70 75 80Phe Cys Asp Phe Gly Ser Pro Ala Asn Arg Lys Ile Gly Val Cys Thr

85 90 95Ala Lys Asp Gly Ala Pro Cys Ile Phe Gly Gly Thr Val Tyr Arg Ser

100 105 110Gly Glu Ser Phe Gln Ser Ser Cys Lys Tyr Gln Cys Thr Cys Leu Asp

115 120 125Gly Ala Val Gly Cys Met Pro Leu Cys Ser Met Asp Val Arg Leu Pro

130 135 140Ser Pro Asp Cys Pro Phe Pro Arg Arg Val Lys Leu Pro Gly Lys Cys145 150 155 160Cys Glu Glu Trp Val Cys Asp Glu Pro Lys Asp Gln Thr Val Val Gly

165 170 175Pro Ala Leu Ala Ala Tyr Arg Leu Glu Asp Thr Phe Gly Pro Asp Pro

180 185 190Thr Met Ile Arg Ala Asn Cys Leu Val Gln Thr Thr Glu Trp Ser Ala

195 200 205Cys Ser Lys Thr Cys Gly Met Gly Ile Ser Thr Arg Val Thr Asn Asp

210 215 220Asn Ala Ser Cys Arg Leu Glu Lys Gln Ser Arg Leu Cys Met Val Arg225 230 235 240Pro Cys Glu Ala Asp Leu Glu Glu Asn Ile Lys Lys Gly Lys Lys Cys

245 250 255Ile Arg Thr Pro Lys Ile Ser Lys Pro Ile Lys Phe Glu Leu Ser Gly

260 265 270Cys Thr Ser Met Lys Thr Tyr Arg Ala Lys Phe Cys Gly Val Cys Thr

275 280 285Asp Gly Arg Cys Cys Thr Pro His Arg Thr Thr Thr Leu Pro Val Glu

290 295 300Phe Lys Cys Pro Asp Gly Glu Val Met Lys Lys Asn Met Met Phe Ile305 310 315 320Lys Thr Cys Ala Cys His Tyr Asn Cys Pro Gly Asp Asn Asp Ile Phe

325 330 335Glu Ser Leu Tyr Tyr Arg Lys Met Tyr Gly Asp Met Ala

340 345

While the invention has been described with respect to presently preferred embodiments, it will be understood that various modifications may be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims (33)

1. A method for accelerating wound healing in a subject in need thereof, the method comprising contacting the wound site with a therapeutically effective amount of a composition comprising a CTGF polypeptide.

2. The method of claim 1, wherein the composition further comprises an agent that stimulates the production of CTGF.

3. The method of claim 2, wherein the agent is transforming growth factor beta (TGF- β).

4. A method of diagnosing a pathological state in a patient suspected of having a disease characterized by a CTGF-associated cell proliferation disorder, the method comprising:

obtaining a sample suspected of containing CTGF from a patient;

determining the level of CTGF in the sample; and

the CTGF levels of the specimen and normal standard samples were compared.

5. The method of claim 4, wherein the disease is selected from the group consisting of fibrotic diseases and atherosclerosis.

6. A method for ameliorating a CTGF-associated cell proliferation disorder comprising treating a subject having a CTGF-reactive agent at a site of the disorder.

7. The method of claim 6, wherein the cell proliferative disorder is due to cell overgrowth.

8. The method of claim 6, wherein the cell proliferative disorder is due to connective tissue cell overgrowth.

9. The method of claim 6, wherein the CTGF-reactive agent is an antagonist of CTGF.

10. The method of claim 9, wherein the antagonist is an antibody that specifically binds to a CTGF polypeptide.

11. The method of claim 6, wherein the cell proliferation disorder is due to low cell growth.

12. The method of claim 6, wherein the CTGF reactive agent is transforming growth factor beta (TGF- β).

13. A method of identifying a composition that affects CTGF expression, the method comprising:

a) incubating ingredients comprising the composition with a TGF-beta regulatory element (T beta RE), which

Under conditions and for a time sufficient for the components to interact; and

b) determining the effect of the composition on CTGF expression.

14. The method of claim 13, further comprising adding TGF- β in step (a).

15. The method of claim 13, wherein the effect is inhibition of CTGF expression.

16. The method of claim 13, wherein the effect is stimulation of CTGF expression.

17. The method of claim 13, wherein the reporter gene is operably linked.

18. The method of claim 17, wherein the reporter gene is selected from the group consisting of: beta-lactamase, Chloramphenicol Acetyltransferase (CAT), Adenosine Deaminase (ADA), aminoglycoside phosphotransferase (neo, G418), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), Thymidine Kinase (TK), beta-galactosidase (beta-gal), and Xanthine Guanine Phosphoribosyltransferase (XGPRT).

19. An isolated polynucleotide encoding the CTGF polypeptide of figure 2 (sequence # 2).

20. The polynucleotide of claim 19, wherein the polynucleotide is sequence #1.

21. An isolated polynucleotide comprising 5 'and 3' untranslated nucleotide sequences flanking a polynucleotide sequence encoding CTGF.

22. The polynucleotide of claim 21, wherein the polynucleotide is the polynucleotide of figure 2.

23. An isolated polynucleotide having 5'-GTGTCAAGGGGTC-3' (sequence # 8) and a sequence substantially complementary thereto.

24. A method of treating a patient having a cell proliferative disorder associated with CTGF gene expression, the method comprising administering to a patient having the disorder a therapeutically effective amount of an agent that modulates CTGF gene expression, thereby treating the disorder.

25. The method of claim 24, wherein the agent is a polynucleotide that modulates expression of CTGF.

26. The method of claim 25, wherein the polynucleotide is selected from the group consisting of an antisense nucleotide, a ribozyme, and a triplex formulation.

27. The method of claim 24, wherein the agent induces one cyclic nucleotide.

28. The method of claim 27, wherein the agent is selected from the group consisting of cholera mycin and 8 bromo-cAMP.

29. The method of claim 27, wherein the cyclic nucleotide is cAMP.

30. The method of claim 24, wherein the agent comprises a TGF- β responsive element (T β RE) polynucleotide.

31. The method of claim 30, wherein the polynucleotide comprises nucleotides from about-162 to about-128 of the CTGF modulating polynucleotide of figure 1.

32. The method of claim 31, wherein the polynucleotide comprises nucleotides at about-154 to-145 of the CTGF modulating polynucleotide of figure 1.

33. A pharmaceutical composition comprising a therapeutically effective amount of CTGF formulated in a pharmaceutically acceptable carrier.