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Identification and Cloning of a Connective Tissue Growth Factor-like cDNA from Human Osteoblasts Encoding a Novel Regulator of Osteoblast Functions

Identification and Cloning of a Connective Tissue Growth Factor-like cDNA from Human Osteoblasts... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 24, Issue of June 11, pp. 17123–17131, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Identification and Cloning of a Connective Tissue Growth Factor-like cDNA from Human Osteoblasts Encoding a Novel Regulator of Osteoblast Functions* (Received for publication, February 26, 1999) Sanjay Kumar‡§, Annalisa T. Hand‡, Janice R. Connor‡, Robert A. Dodds‡, Paul J. Ryan¶, John J. Trill¶, Seth M. Fisheri, Mark E. Nuttall‡, David B. Lipshutz‡, Cheng Zou‡, Shing M. Hwang‡, Bartholomew J. Votta‡, Ian E. James‡, David J. Rieman‡, Maxine Gowen‡, and John C. Lee‡ From the Departments of ‡Bone and Cartilage Biology, ¶Gene Expression Sciences, and iProtein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406 We have identified and cloned a novel connective tis- tified as a major chemotactic and mitogenic factor from endo- sue growth factor-like (CTGF-L) cDNA from primary hu- thelial cells (1). CTGF is distinct from, but immunologically man osteoblast cells encoding a 250-amino acid single related to, platelet-derived growth factor and competes for chain polypeptide. Murine CTGF-L cDNA, encoding a binding to the platelet-derived growth factor receptor (1). In polypeptide of 251 amino acids, was obtained from a fibroblasts, transforming growth factor b induces the produc- murine lung cDNA library. CTGF-L protein bears signif- tion of CTGF mRNA and protein, which in turn induces type I icant identity (;60%) to the CCN (CTGF, Cef10/Cyr61, collagen gene expression (2). Other members of this family Nov) family of proteins. CTGF-L is composed of three include Fisp12 (3), which is the murine orthologue of human distinct domains, an insulin-like growth factor binding CTGF; human and murine Cyr61 (4, 5); the chicken orthologue domain, a von Willebrand Factor type C motif, and a of Cyr61, Cef10 (6); and human and Xenopus Nov (7, 8). These thrombospondin type I repeat. However, unlike CTGF, genes, with the exception of nov, are immediate-early genes CTGF-L lacks the C-terminal domain implicated in that are induced by serum, growth factors, or certain oncogenes dimerization and heparin binding. CTGF-L mRNA (;1.3 and are collectively referred to as the CCN (CTGF, cef10/cyr61, kilobases) is expressed in primary human osteoblasts, nov) family (9). The nov gene was identified as a gene that was fibroblasts, ovary, testes, and heart, and a ;26-kDa pro- induced as a result of proviral rearrangement due to insertion tein is secreted from primary human osteoblasts and of nephroblastosis associated virus (7). fibroblasts. In situ hybridization indicates high expres- Members of CCN family are cysteine-rich proteins that are sion in osteoblasts forming bone, discrete alkaline phos- organized into four distinct motifs (9). The first motif contains phatase positive bone marrow cells, and chondrocytes. I-labeled insulin-like growth fac- an insulin-like growth factor (IGF) binding domain (GCGC- Specific binding of CXXC) common to all seven known IGF-binding proteins tors to CTGF-L was demonstrated by ligand Western blotting and cross-linking experiments. Recombinant (IGFBPs). The second domain contains a von Willebrand factor human CTGF-L promotes the adhesion of osteoblast type C (VWC) module that is suspected to be involved in oli- cells and inhibits the binding of fibrinogen to integrin gomerization. The third is a thrombospondin type I repeat receptors. In addition, recombinant human CTGF-L in- (TSP1) that is thought to play a role in cell attachment and hibits osteocalcin production in rat osteoblast-like Ros binding to matrix proteins and sulfated glycoconjugates (9). 17/2.8 cells. Taken together, these results suggest that The fourth, a C-terminal (CT) domain, has been implicated in CTGF-L may play an important role in modulating bone heparin binding and dimerization (10). The CT domain (;10 turnover. kDa) of CTGF is present in biological fluid and is sufficient for some biological activities (10). Osteoblasts are specialized mesenchymal cells that are re- Connective tissue growth factor (CTGF) was originally iden- sponsible for synthesizing and secreting the complex mixture of collagenous and noncollagenous proteins that make up bone matrix. These cells are also responsible for subsequent miner- * The costs of publication of this article were defrayed in part by the alization of this matrix (11). During the process of bone forma- payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to tion and remodeling, there is an integrated process of oste- indicate this fact. oclast-mediated bone resorption and osteoblast-derived bone The nucleotide sequence(s) reported in this paper has been submitted formation (11, 12). The process of bone formation and remod- TM to the GenBank /EBI Data Bank with accession number(s) AF083500 eling is under tight regulation by numerous factors, including and AF126063. endocrine hormones, cytokines, growth factors, adhesion mol- § To whom correspondence should be addressed: SmithKline Beecham, Bone and Cartilage Biology, UW2109, 709 Swedeland Rd., ecules, and extracellular matrix components. King of Prussia, PA 19406. Tel.: 610-270-7245; Fax: 610-270-5598; E- In the present report, we describe the identification, cloning, mail: Sanjay_Kumar@sbphrd.com. expression, and functional characterization of a novel CTGF- The abbreviations used are: CTGF, connective tissue growth factor; like (CTGF-L) cDNA from primary human osteoblast cells. CTGF-L, CTGF-like; rhCTGF-L, recombinant human CTGF-L; IGF, insulin-like growth factor; IGFBP, IGF-binding protein; TSP1, throm- CTGF-L contains the first three domains present in CCN fam- bospondin type I domain; VWC, von Willebrand factor type C domain; ily members but lacks the fourth CT domain. We show that CT, caboxyl terminal; TGF, transforming growth factor; GAPDH, glyc- CTGF-L is expressed at high levels in human bone tissue. eraldehyde-3-phosphate dehydrogenase; PAGE, polyacrylamide gel Recombinant hCTGF-L protein binds to IGFs and promotes electrophoresis; BSA, bovine serum albumin; EST, expressed sequenced tag; PBS, phosphate-buffered saline. adhesion of osteoblast cells. In addition, rhCTGF-L inhibits the This paper is available on line at http://www.jbc.org 17123 This is an Open Access article under the CC BY license. 17124 Identification, Cloning, and Characterization of CTGF-L oxide and sodium azide. The slides were washed in PBS and blocked binding of fibrinogen to integrin receptors and inhibits osteo- with PBS containing 10% goat and 1% human AB serum. The sections calcin production from rat osteoblast-like cells. were reacted with anti-CTGF-L or preimmune serum (1:250 in PBS), washed and reacted with horseradish peroxidase-conjugated secondary MATERIALS AND METHODS antibody, and visualized by labeled streptavidine-biotin 2 peroxidase Cells and Cell Culture—Primary osteoblasts were grown from ex- kit and the liquid diaminobenzidine substrate/chromogen system, ac- plants of human trabecular bone fragments from knee joints taken at cording to the manufacturer’s protocols (Dako). A brown stain indicated surgery (kindly provided by the Rothman Institute, Pennsylvania Hos- positive reactivity. pital, Philadelphia, PA). The osteoblasts were cultured in Eagle’s mod- Immunoprecipitation of CTGF-L from Fibroblast and Osteoblast ified minimum essential medium supplemented with 10% fetal calf Cells—For metabolic labeling, exponentially growing primary human serum (Hyclone, Logan, UT), 2 mML-glutamine, and antibiotics for 2–3 osteoblasts and fibroblasts were incubated for4hin cysteine-free weeks as described previously (13, 14). Cells from up to three passages medium containing 5% dialyzed fetal bovine serum with 100 –150 were used for all experiments. Primary human fibroblasts, human mCi/ml of S-labeled cysteine (;1000 Ci/mmol, ICN Biomedicals, Costa osteosarcoma MG 63 and SaOS-2, HeLa, and human mesenglial cells Mesa, CA). For immunoprecipitation, 5.0 ml of preimmune or anti- were obtained from ATCC (Manassas, VA). Human stromal TF274 cells CTGF-L immune-serum was mixed with precleared S-labeled condi- have been described previously (15). tioned medium and 20 ml of protein A-agarose (Life Technologies, Inc.) RNA Isolation and Northern Blot Analysis—Total RNA was isolated and incubated overnight at 4 °C. Immune complex beads were collected from primary human osteoblasts and different cell lines using Trizol by centrifugation and washed three times with PBST (PBS containing reagent (Life Technologies, Inc.) according to the manufacturer’s rec- 0.1% Tween 20) buffer. The beads were solubilized in sample buffer and ommendation. RNA was fractionated by electrophoresis on 1.2% aga- resolved through SDS-PAGE, fixed, dried, and processed for fluorogra- rose-formaldehyde gels, transferred to Genscreen plus membranes, and phy and autoradiography. cross-linked using an UV Stratalinker-180 (Stratagene, La Jolla, CA). Ligand Western Blotting and Cross-linking—Recombinant hCTGF-L The blots were probed with P-labeled CTGF-L cDNA and the house- (10 –300 pmol) was separated by SDS-PAGE under nonreducing condi- keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ac- tions. The protein was transferred to nitrocellulose membrane, blocked cording to standard procedures (16). A commercial human multiple with 5% nonfat dry milk in PBST, and probed sequentially with 1 3 10 tissue Northern blot (CLONTECH, Palo Alto, CA) containing 2 mgof cpm of I-labeled IGF-I or -II (;2000 Ci/mmol, Amersham Pharmacia poly(A) RNA from various tissues was processed according to the Biotech) or anti-CTGF-L antibody (1:1000) in PBST containing 0.1% manufacturer’s instructions. BSA for 18 h. The membrane was then washed 4 times for 15 min each Cloning of CTGF-L cDNAs and Expression of Recombinant Protein— with PBST and either dried and exposed to autoradiographic film for Human CTGF-L cDNA was identified by expressed sequenced tag ligand blots or developed with ECL for Western blot (Amersham Phar- (EST) analysis (17) of a cDNA library derived from primary human macia Biotech). For cross-linking, 300 pmol of rhCTGF-L was mixed 5 125 osteoblasts. After sequencing, the clone HOEBG39 was found to contain with 1 3 10 cpm of I-labeled IGF-I or -II for2hat4 °C. Water the entire open reading frame of hCTGF-L. An EST (AA754979) from a soluble homobifunctional cross-linker BS (Sigma) was added to the murine lung library, with high homology to the human CTGF-L cDNA, reaction mixture at a final concentration of 5 mM for 1 h. The reaction TM was identified from the GenBank data base. This EST contained the was stopped by addition of SDS-PAGE sample buffer, and the products 39-untranslated region and encoded about 35 amino acids from the C were separated by 12.5% SDS-PAGE. In some cases, the cross-linking terminus of CTGF-L protein. Based on the most 59 sequence of this EST, was performed in the presence of excess unlabeled IGF-I or IGF-II (Life primers were designed, and the full-length cDNA was isolated using the Technologies, Inc.). 59 rapid amplification of cDNA ends from a marathon cDNA library Cell Adhesion Studies—Corning 96-well enzyme-linked immunosor- prepared from murine lung (CLONTECH). For recombinant protein bent assay plates (Corning, NY) were precoated overnight at 4 °C with expression, the coding region of hCTGF-L was subcloned into CDN various concentrations of rhCTGF-L, 0.1 ml of human vitronectin (0.2 vector, where the expression of hCTGF-L was driven by the cytomega- mg/ml in PBS), or BSA (3 mg/ml). The plates were washed once with lovirus promoter (18). Two epitope tags, an N-terminal human immu- PBS and blocked with 3% BSA in PBS for1hat room temperature. nodeficiency virus GP120 V3 (SKSIRIQRGPGR) and His , were in- Cells were trypsinized and resuspended in RPMI medium and supple- serted after the signal sequence, and an enterokinase cleavage site was mented with 20 mM Hepes, pH 7.4, and 0.1% BSA at a density of 5 3 10 engineered between the epitope tags and CTGF-L protein. The plasmid cells/ml, and 0.1 ml of cell suspension was added to each well. Following was transfected into Chinese hamster ovary cells by electroporation, 1 h of incubation at 37 °C, the cells were fixed by the addition of 25 ml and cells expressing hCTGF-L were bulk-selected in nuceloside-free of a 10% formaldehyde solution, pH 7.4, at room temperature for 10 medium. Conditioned medium from a large-scale culture of Chinese min. The plates were washed three times with 0.2 ml of PBS, and the hamster ovary-hCTGF-L cells was used to purify rhCTGF-L using a adherent cells were stained with 0.1 ml of 0.5% toluidine blue for 20 min TALON metal affinity column (CLONTECH). Poor expression of soluble at room temperature. Excess stain was removed by extensive washing secreted CTGF-L was observed possibly due to the high number of with deionized water. The toluidine blue incorporated into cells was cysteine residues (11% of total protein), and hence only small amounts eluted by the addition of 0.1 ml of 50% ethanol containing 50 mM HCl of purified recombinant protein were obtained. The expression and and quantitated by measuring absorbance at 630 nm on a microtiter authenticity of rhCTGF-L was confirmed by immunoblotting with anti- plate reader (Titertek Multiskan MC, Sterling, VA). CTGF-L antibodies and N-terminal sequencing of purified protein. Integrin Binding—Vitronectin receptor, a b (0.12 mg), and fibrino- v 3 Anti-CTGF-L antibodies were prepared by injecting KLH-conjugated gen receptor, aII b (1 mg), purified from human placenta and blood, b 3 135 151 peptides derived from hCTGF-L ( SEDVRLPSWDCPHPRRV and respectively (22) were added to 96-well microtiter plates and incubated 234 248 SRPCPPSRGRSPQNS ) into rabbits according to standard overnight at 4 °C. At the time of experiment, the protein solutions were procedures. aspirated and the wells were incubated in 0.1 ml of Buffer A (50 mM Tissue Processing, in Situ Hybridization, and Immunocytochemis- Tris, 100 mM NaCl, 1 mM MgCl ,1mM MnCl , pH 7.4) containing 3% 2 2 try—Human tissues were obtained through the Anatomical Gift Foun- BSAfor1hat room temperature to block the nonspecific binding. After dation of Arizona (Phoenix, AZ). Human osteophytes were dissected aspirating the blocking solution, various concentrations of CTGF-L was from osteoarthritic femoral heads. Osteoclastoma tissue was prepared added to the wells followed by the addition of 8 nM biotinylated fibrin- from freshly dissected giant cell tumors. All tissues were processed for ogen in 100 ml of Buffer A containing 0.1% BSA. The plates were cryosectioning as described (19). The hCTGF-L cDNA cloned into incubated for1hat room temperature and washed twice with 100 mlof pBluescript was linearized and transcribed using T3- and T7-promoter binding buffer. Anti-biotin antibody conjugated to alkaline phosphatase sites to generate the antisense and sense strand riboprobes using the (1:2000 dilution, Sigma) was then added for 10 min followed by two Promega (Madison, WI) In Vitro transcription kit with [ S]thio-CTP washes with binding buffer containing 0.1% Tween 20. The reaction (Amersham Pharmacia Biotech). Riboprobes with a specific activity in was quantitated with alkaline phosphate substrate kit (Bio-Rad) by excess of 10 cpm/mg were used for in situ hybridization as described measuring absorbance at 405 nm using a microtiter plate reader. previously (20, 21). For immunocytochemistry, cryostat sections (8 mm) Osteocalcin Measurement—ROS 17/2.8 cells were seeded in 24-well were placed onto 3-aminopropyltriethoxy silane-coated glass slides, plates at a concentration of 3 3 10 /0.5 ml/well in assay medium (Ham’s fixed in 10% formalin, and washed in phosphate-buffered saline (PBS). F-12 medium supplemented with 1% fetal calf serum, 2 mML-gluta- Antigen retrieval was performed by heating the sections at 95 °C for 20 mine, and antibiotics) and incubated for 3– 4 h. Fresh medium, with or min in a Coplin jar containing appropriately diluted target retrieval without 10 nM 1,25(OH) D and various concentrations of CTGF-L, was 2 3 buffer (Dako, Carpenteria, CA). Endogenous peroxidase activity was then added in duplicate, and the cultures were incubated for an addi- blocked by incubating the sections for 10 min in 0.03% hydrogen per- tional 48 h. Osteocalcin in the culture supernatants was measured by Identification, Cloning, and Characterization of CTGF-L 17125 radioimmunoassay using reagents purchased from Biomedical Technol- noted at sites of secondary remodeling (secondary spongiosa), ogies (Stoughton, MA). chondrocytes, and osteoclasts. These data are summarized in Table I. RESULTS Polyclonal antibodies were generated to peptides derived Identification and Cloning of CTGF-L cDNA—By analyzing from CTGF-L and used to immunolocalize CTGF-L protein in a human osteoblast cDNA library using expressed sequence tag bone. Intense CTGF-L staining was associated with osteoblasts analysis, we identified an EST (HOEBG39) that contained an lining trabecular and periosteal bone surfaces from human IGF binding domain (GCGCCXXC). Complete sequencing of fetal bone tissue (Fig. 3, C and D). We also examined the HOEBG39 revealed that it contained an open reading frame expression of CTGF-L protein in primary human osteoblasts encoding a polypeptide of 250 amino acids. Based on sequence and fibroblasts that show high expression of CTGF-L mRNA. analysis, the HOEBG39-encoded protein exhibited significant In both cell types, a ;26-kDa protein was specifically immu- identity to the CCN family of proteins of which CTGF and 35 noprecipitated by anti-CTGF-L antibodies from S-labeled Cyr61 are the most fully characterized members. HOEBG39 conditioned medium (Fig. 4, A, lanes 2 and 4) and cell lysate encoded protein is 30 – 60% identical to CCN proteins (Fig. 1A) (not shown). The apparent molecular mass (;26 kDa) of im- and is most closely related to CTGF (;60% identity). Therefore, munoprecipitated protein is consistent with the expected size of we termed it CTGF-L protein. The first 23 amino acids of the protein encoded by the open reading frame of CTGF-L CTGF-L encode a putative signal sequence followed by three of mRNA after cleavage of the signal peptide. A similar sized the four distinct domains that are found in the CCN family of protein was also immunoprecipitated from HeLa and TF274 proteins (9). An IGF binding domain is present from residue cells that were transfected with an expression vector encoding 24 93 98 Gln to Leu . A VWC repeat is found from residue Ser to hCTGF-L (data not shown). Gly . A third TSP1 domain is contained within residues Recombinant hCTGF-L was expressed as an N terminus 194 237 Cys to Cys (Fig. 1, A and B). A fourth CT domain (;100 human immunodeficiency virus GP120 V3 and His epitope tag residues) that has been implicated in the heparin binding and in Chinese hamster ovary cells and was purified by TALON mitogenic activity of CTGF is missing from CTGF-L. An align- metal affinity chromatography as described under “Materials ment of CTGF-L to various members of the CCN family is and Methods.” The purified tagged protein of ;30 kDa (expect- shown in Fig. 1A. ed size due to the tag) was detected by Coomassie Blue staining An EST data base search with hCTGF-L sequence identified (Fig. 4B, lane 1) and immunostaining (Fig. 4B, lane 2). a murine EST (AA754979) that exhibited significant identity Binding of CTGF-L to IGF-I and IGF-II—The presence of an (;74% at nucleotide level) to hCTGF-L. We cloned the full- IGF binding domain within CTGF-L prompted us to investigate length mCTGF-L cDNA from a murine lung cDNA library the binding of CTGF-L to IGFs using ligand Western blotting using marathon 59 rapid amplification of cDNA ends as de- and cross-linking. Increasing amounts of rhCTGF-L were sep- scribed under “Materials and Methods.” The protein encoded arated by nonreducing SDS-PAGE and transferred onto a ni- by mCTGF-L cDNA is ;70% identical to hCTGF-L protein (Fig. trocellulose membrane. The blot was probed sequentially with 1A). Similar to hCTGF-L, the mCTGF-L also encodes a protein 125 125 I-labeled IGF-I, I-labeled IGF-II, and anti-CTGF-L anti- of 251 amino acids and contains all but the fourth CT domain body. As little as 10 pmol of CTGF-L bound to labeled IGFs (Fig. 1, A and B). Phylogentic analysis with all available mem- (Fig. 5A). rhCTGF-L showed a dose-dependent binding to I- bers of the CCN family grouped both human and murine labeled IGFs, with IGF-II exhibiting relatively higher binding CTGF-L together in a separate group (Fig. 1C). It appears that than IGF-I. Immunostaining confirmed the increasing elm is the oldest gene from which both CTGF-L and other CCN amounts of rhCTGF-L loaded in each lane (Fig. 5A, bottom members originated (Fig. 1C). Given the similar arrangement, panel). As a control, an unrelated His-tagged protein, p38 MAP composition, and length of various domains between the mu- kinase, did not bind either IGF-I or IGF-II (Fig. 5A, lane 5). In rine and human CTGF-L protein (Fig. 1, A and B), it is clear a cross-linking experiment using a homobifunctional cross- that the murine cDNA that we isolated is the murine ortho- linker BS , both I-labeled IGF-I and -II bound to rhCTGF-L logue of human CTGF-L. (Fig. 5B, lane 1). The size of cross-linked product (;37–38 kDa) Expression of CTGF-L mRNA and Protein—Northern blots is consistent with the cross-linking of rhCTGF-L (;30 kDa) to containing RNAs from various human tissues and bone-derived IGFs (;7– 8 kDa). We also performed a competitive cross- cells were hybridized to a P-labeled CTGF-L cDNA probe. A linking experiment in which the cross-linking was competed ;1.3-kilobase CTGF-L mRNA was highly expressed in primary with increasing amounts of unlabeled IGF-I (Fig. 5B, lanes 2–5) human osteoblasts, fibroblasts, ovary and testes (Fig. 2, A, or IGF-II (lanes 6 –9). The signal obtained by cross-linking of lanes 1 and 9, and B, lanes 12 and 13). A lower level of expres- rhCTGF-L to I-labeled IGF-II was much higher than with sion of CTGF-L was also observed in heart, lung, skeletal I-labeled IGF I. In addition, only 30 ng of IGF-II was needed muscle, prostate, and colon (Fig. 2B, lanes 1, 4, 6, 11, and 15). to compete rhCTGF-L cross-linking to IGF-I (Fig. 5B, lane 7, However, CTGF-L was not expressed in human osteosarcoma upper panel), whereas even 300 ng of IGF-I could not compete SaOS-2 or MG 63 cells, stromal TF274 cells, osteoclastoma the cross-linking of rhCTGF-L to IGF-II (lane 5, lower panel). tissue, or HeLa or mesenglial cells (Fig. 2). The expression of These data suggest that both IGF I and IGF II bind to CTGF-L in primary osteoblast or fibroblast cultures appears to rhCTGF-L and that IGF-II has at least 10-fold higher affinity be constitutive as treatment with a variety of osteotropic for CTGF-L compared with IGF-I. It should be noted that the agents, including parathyroid hormone, transforming growth specific activity of both I-labeled IGF-I and IGF-II was com- factor-b, 1,25(OH) D , and estrogen, failed to modulate its ex- 2 3 pression (data not shown). parable (;2000 Ci/mmol). Adhesion of Osteoblast Cells to CTGF-L—The TSP1 domain In situ hybridization indicated strong expression of CTGF-L mRNA in bone-forming osteoblasts on calcified cartilage spi- has been implicated in the promotion of cell attachment (9). Therefore, we tested whether rhCTGF-L was able to promote cules (primary spongiosa) in fetal bone (Fig. 3, A and B) and human osteophytic tissue (Table I). Discrete alkaline phospha- the adhesion of osteoblastic cells. Primary human osteoblast, tase positive cells in bone marrow of osteophyte and discrete osteosarcoma MG63, and rat osteoblast-like osteosarcoma Ros 17/2.8 cells attached to immobilized rhCTGF-L in a dose-de- macrophage-like cells from giant cell tumor also exhibited strong expression of CTGF-L mRNA. Weaker expression was pendent manner (Fig. 6A). Although maximal adhesion was 17126 Identification, Cloning, and Characterization of CTGF-L FIG.1. Alignment of CTGF-L with various members of the CCN family of proteins. A, protein sequence alignment was performed using the MEGALIGN program of Lasergene (DNASTAR Inc., Madison, WI) according to the clustal algorithm. The amino acids identical to CTGF are hidden and indicated by dots, whereas dashes indicate gaps. The broken, dashed, solid and dotted lines with arrows indicate the IGF binding domain, the VWC domain, the TSP1 domain, and the CT domain, respectively. B, schematic representation of different domains, including signal sequence (SS) of CCN proteins. IGF BD, IGF binding domain. The amino acid number flanking each domain of hCTGF-L is also shown. Note that the CT domain is missing in CTGF-L. C, phylogenetic relationship between various members of the CCN family. observed with Ros 17/2.8 cells, the extent of cell adhesion to at a coating concentration of ;300 ng/ml (;10 nM), whereas no rhCTGF-L for all cells was comparable to that observed with cell adhesion was observed using a protein preparation in vitronectin. Half-maximal adhesion to rhCTGF-L was observed which CTGF-L was depleted using anti-CTGF-L antibodies or Identification, Cloning, and Characterization of CTGF-L 17127 FIG.2. Expression of human CTGF-L mRNA in various human cells and tissue preparations. A, total RNA from primary human osteoblasts (lane 1), human osteosarcoma SaOS2 (lane 2), human osteoclastoma tissue (lane 3), human osteosarcoma HOS (lane 4), human osteosarcoma MG63 (lane 5), human stromal TF274 (lane 6), HeLa (lane 7), human mesenglial (lane 8), and synovial fibroblasts (lane 9) was hybridized with P-labeled hCTGF-L and GAPDH cDNA probe. The location of 28 S and 18 S ribosomal RNA, CTGF-L, and GAPDH are indicated. B, a multiple tissue Northern blot containing poly(A) RNA from heart (lane 1), brain (lane 2), placenta (lane 3), lung (lane 4), liver (lane 5), skeletal muscle (lane 6), kidney (lane 7), pancreas (lane 8), spleen (lane 9), thymus (lane 10), prostate (lane 11), testes (lane 12), ovary (lane 13), small intestine (lane 14), colon (lane 15), and peripheral blood leukocytes (lane 16) was hybridized with P-labeled CTGF-L and GAPDH cDNA probe. The locations of RNA markers (in kilobases), CTGF-L, and GAPDH are indicated. report that Cyr61, another CCN family member, binds a b v 3 (23) prompted us to examine the effect of CTGF-L on the binding of purified integrins to matrix protein. The purified integrin receptors, a b and aII b , demonstrated high affinity v 3 b 3 binding to biotinylated fibrinogen. As shown in Fig. 7, rh- CTGF-L inhibited the binding of fibrinogen to both integrins in a dose-dependent manner. The IC for a b (Fig. 7, filled 50 v 3 triangles) was ;100 nM, whereas weaker inhibition was ob- served for aII b (IC ;1 mM)(open circles), suggesting some b 3 50 degree of selectivity. Effect of rhCTGF-L on Osteocalcin Production—To deter- mine whether rhCTGF-L could modulate osteoblast function, we examined its effect on osteocalcin production in rat osteo- blast-like osteosarcoma, ROS 17/2.8 cells. Osteocalcin is a marker of mineralizing osteoblasts, and its expression is used routinely as a measure of osteoblast function. ROS 17/2.8 cells endogenously secrete low levels of osteocalcin, which can be up-regulated by treatment with 1,25(OH) D . As shown in Fig. 2 3 8, rhCTGF-L inhibited both basal and 1,25(OH) D -stimulated 2 3 osteocalcin production in a dose dependent manner with an IC of ;300 ng/ml (;10 nM). Treatment with a CTGF-L de- pleted protein preparation or with an unrelated His -tagged protein, p38, had no effect on osteocalcin production (data not shown). The inhibition of osteocalcin expression was at the mRNA level because rhCTGF-L also inhibited the osteocalcin mRNA induction by 1,25(OH) D both in Ros 17/2.8 and in 2 3 primary human osteoblasts (data not shown). DISCUSSION During the past few years, several proteins belonging to the expanding CCN family have been described (9). Most members of this family are immediate-early genes that are induced by FIG.3. CTGF-L mRNA and protein expression during human treatment with serum or growth factors (1, 3, 5, 24, 25). All endochondral ossification. A, a section of human fetal femoral members of this family contain four distinct domains, including growth plate was hybridized with antisense CTGF-L riboprobe. Osteo- the IGF binding domain, the VWC domain, the TSP1 domain, blasts (Obs)(arrows) forming osteoid on calcified cartilage spicules demonstrated intense mRNA expression; adjacent formation sites and the CT domain. showed reduced expression (arrowheads). Magnification, 3 50; counter- We have identified human (and murine) CTGF-L as a novel stained with methylene blue. B, high power view of the area shown in member of the CCN family. There are two striking features A demonstrates CTGF-L mRNA expression in osteoblasts (Obs)(ar- that distinguish CTGF-L from the other CCN family members. rows) and osteoprogenitor cells (Opg) within the marrow (magnifica- tion, 3 100). C, a section of fetal bone stained with the anti-CTGF-L First, CTGF-L lacks the fourth CT domain found in all other polyclonal antibody. Osteoblasts lining the trabecular (arrows) and family members. Second, CTGF-L is not induced by serum or periosteal (arrowheads) bone surface express high levels of CTGF-L growth factors. Although an alternatively spliced form may (magnification, 3 25). D, high power view of the area shown in C shows exist, we have been unable to identify a CTGF-L variant that expression of CTGF-L in osteoblasts (Obs)(arrows) and discrete cells within the marrow (arrowheads) (magnification, 3 100). encodes a protein containing the CT domain in libraries de- rived from cells and tissues expressing CTGF-L. to an unrelated His -tagged protein, p38 (Fig. 6B). In addition, immunoprecipitation of CTGF-L by anti- Binding of rhCTGF-L to Purified Integrin Receptors—The CTGF-L antibodies detected only a single ;26-kDa protein in attachment of cells to CTGF-L-coated plates and a previous both osteoblasts and fibroblasts. We did not detect a protein of 17128 Identification, Cloning, and Characterization of CTGF-L TABLE I Expression of CTGF-L mRNA in human bone by in situ hybridization Cryostat sections of various tissues were hybridized to S-labeled antisense or sense CTGF-L riboprobe. Tissue type Cell type mRNA expression Osteophyte, including synovium and granulation tissue Osteoblasts Primary spongiosa 111 Secondary spongiosa 1 Chondrocytes 1/2 Synovial macrophages 11 Marrow cells 1 to 111 Osteoclasts 1/2 Fetal bone Osteoblasts Primary spongiosa 11 Secondary spongiosa 1 Chondrocytes 11 Myocytes 1 Giant cell tumor, including areas of bone formation and alkaline phosphatase-positive stroma Stromal cells 1 Osteoblasts 1 Osteocytes 1 Marrow cells 1/2 Osteoclasts 2 Blood vessels 2 Macrophages (discrete) 111 2 indicates negative signal; 1 to 111 indicates moderate to very strong signal obtained with antisense probe. No signal was obtained with sense probe. non-bone-forming giant cell tumors were negative. including osteosarcoma, and it appears that its expression is restricted to primary cells, such as osteoblasts and fibrolasts. Human tissues expressing the highest levels of CTGF-L mRNA are bone, ovary, and testes. We have shown that both CTGF-L mRNA and protein are expressed at high levels in osteoblasts forming bone in the periosteum and primary spon- giosa of human fetal and osteophytic bone. These bone forma- tion areas are zones of appositional and longitudinal bone growth. Lower levels of expression were observed in osteoblasts lining the relatively slower remodeling trabeculae of the sec- ondary spongiosa. Thus, CTGF-L mRNA was highly expressed by osteoblasts at sites of high bone turnover (Table I) associ- ated with net gain in bone mass. This highly selective expres- sion is suggestive of a specific role for CTGF-L in the control of bone formation. We have examined the activity of rhCTGF-L in several as- FIG.4. Expression of endogenous and recombinant CTGF-L says to elucidate potential functions for various domains of this protein. A, primary human fibroblasts (Fib.) and osteoblasts (Obs.) were labeled with [ S]cysteine, and the conditioned medium was im- protein, including cell adhesion, IGFs and integrin binding, munoprecipitated with preimmune (PI) or immune (IM) anti-CTGF-L and osteocalcin production. IGFs have growth promoting activ- antibody. The location of ;26-kDa CTGF-L is indicated. B, recombinant ity and play an important role in bone formation and remodel- CTGF-L was expressed as N terminus GP120/His -tagged protein in ing (26). Several IGF-binding proteins regulate the activity and Chinese hamster ovary cells and purified by TALON metal affinity chromatography. Molecular weight markers (in thousands) and the availability of IGFs in bone and other tissues (26). Although we location of purified rhCTGF-L as detected by Coomassie Blue staining have unequivocally shown that rhCTGF-L binds to both IGF-I (lane 1) and Western blot (lane 2) are shown. and IGF-II, to date, no significant change in IGF action has been observed in the presence of CTGF-L. This suggests that CTGF-L may serve not to modulate IGFs activity, but rather to higher molecular mass in the anti-CTGF-L immunoprecipi- increase the local concentrations of IGFs. Although cross-link- tates that might contain the fourth CT domain. Furthermore, ing and ligand Western blotting studies did not allow a precise murine CTGF-L cDNA encodes an open reading frame that determination of binding affinities, it appears that the relative terminates at the same amino acid residue as human CTGF-L. affinity of rhCTGF-L for IGF-II is at least 10-fold higher than CTGF-L without the CT domain, therefore, is the functional for IGF-I. In this regard, it has been reported that human protein being produced and secreted by osteoblast and fibro- blast cells. osteoblasts produce and respond better to IGF-II, whereas ro- dent osteoblasts produce and are more responsive to IGF-I (26). Another feature that distinguishes CTGF-L from all other members of the CCN family is the inability of serum or various In a recent report, rhCTGF was also shown to bind both IGF-I and IGF-II (27) but with much lower affinity than the binding growth factors to induce its expression. The expression of CTGF-L in primary osteoblast or fibroblast cultures appears to of IGFBP3 to IGFs. Unlike IGFBPs, no reports on modulation be constitutive, and to date, we have not been able to modulate of IGFs activity by CCN family of proteins have been described. its expression by treatment with a variety of osteotropic agents, Whereas the binding of CTGF-L to IGFs is most likely me- including parathyroid hormone, transforming growth factor-b, diated via the IGF binding domain similar to the IGFBPs, cell 1,25(OH) D , and estrogen. An additional feature is the ab- adhesion is most likely mediated via the TSP1 domain, perhaps 2 3 sence of CTGF-L mRNA expression in various tumor cell lines, through binding to integrin receptors. Indeed, we have demon- Identification, Cloning, and Characterization of CTGF-L 17129 FIG.5. Binding of rhCTGF-L to IGFs. A, various amounts of CTGF-L (10 –300 pmol) and p38 (300 pmol) were separated by SDS-PAGE under nonreducing conditions and probed sequentially with I-labeled IGF-I or IGF-II or with anti-CTGF-L antibody. Between experiments, the blot was stripped and extensively washed. The final immunoblot was developed with ECL. The locations of the ;30-kDa marker and CTGF-L as 125 125 detected with each probe are indicated. B, rhCTGF-L was cross-linked to I-labeled IGF-I (top)or I-labeled IGF-II (bottom) with a homobifunctional cross-linking agent, BS . The cross-linking was competed with increasing amounts of unlabeled IGF-I (lanes 2–5) or IGF-II (lanes 6–9) as indicated. FIG.7. Inhibition of fibrinogen binding to purified integrins by rhCTGF-L. Biotinylated fibrinogen (8 nM) was added to microtiter wells coated with integrin receptors in the presence or absence of various concentration of CTGF-L and incubated for1hat room tem- perature to measure the effect of CTGF-L on the binding of biotinylated fibrinogen to a b (filled triangles) and aII b (open circles). Inhibition v 3 b 3 of fibrinogen binding by CTGF-L was quantitated by staining with alkaline phosphatase conjugated anti-biotin antibody and by measuring absorbance at 405 nM. Total binding (control 5 100%) was determined in absence of CTGF-L. The inhibition is expressed as a percentage of control. The data are representative of three independent experiments. Values shown are the means 6 S. E. of triplicate determinations. FIG.6. Promotion of cell adhesion by rhCTGF-L. A, 96-well CTGF-L or Cyr61, which has also been shown to bind a b v 3 microtiter plates were coated with or 3 mg/ml BSA, 200 ng/ml vitronec- integrin (23). Therefore, CTGF-L binding must occur through tin (Vn), or various amounts of rhCTGF-L as indicated. After blocking an RGD-independent mechanism. Consistent with this notion, with BSA, MG 63 (black columns) primary human osteoblasts (gray columns), and Ros 17/2.8 (white columns) cells were allowed to adhere the adhesion of osteoblasts to vitronectin, but not to CTGF-L, for 30 min, washed extensively, fixed, and stained to quantitate the was inhibited by the RGD-containing snake venom protein adhesion. B, adhesion of MG 63 (black columns) and Ros 17/2.8 (white echistatin (data not shown). Therefore, CTGF-L may inhibit columns) cells to microtiter plates coated with equal amounts (1 mg/ml) matrix/integrin interaction by binding close to the RGD recog- of CTGF-L, antibody depleted CTGF-L (depleted) or unrelated protein, p38. The data are representative of three independent experiments. nition site due to steric hindrance or by inducing a conforma- Values shown are the means 6 S. E. of triplicate determinations. tional change. Similarly, the effect of CTGF-L on osteocalcin release may occur through the TSP1 domain and integrin bind- strated that rhCTGF-L directly inhibits fibrinogen binding to ing because it is known that integrin binding to extracellular purified integrin receptors. Fibrinogen, like many extracellular matrix proteins can result in signal transduction leading to matrix proteins, contains an RGD motif that is crucial for changes in cell proliferation, gene expression, and cellular dif- integrin binding. However, no RGD sequence is present in ferentiation (28). It is possible that these changes may be 17130 Identification, Cloning, and Characterization of CTGF-L role in wound repair and/or fibrosis (1, 10, 31–33). Cyr61 has been shown to be an extracellular matrix-associated molecule that has mitogenic activity when used in conjunction with basic fibroblast growth factor and can also promote adhesion and migration of fibroblasts and vascular endothelial cells (34, 35). Recently, it has been determined that Cyr61 mediates the adhesion of cells via a b integrin binding and has been sug- v 3 gested to promote tumor growth and angiogenesis (36, 37). Elm, another family member, is expressed at low levels in metastatic cancer and suppresses metastasis (25). While this report was in preparation, two laboratories re- ported the cloning of murine and rat CTGF-L. WISP-2, the murine orthologue of hCTGF-L, was cloned by subtractive hy- bridization as a Wnt-1 inducible gene, the expression of which was reduced in some tumors (38). The rat orthologue of CTGF-L, rCOP-1, was cloned by differential display analysis as a gene of which the expression was lost as a result of cell FIG.8. Inhibition of osteocalcin production by rhCTGF-L. Ros transformation (39). An identical sequence has also been sub- cells were treated with different concentrations of rhCTGF-L in the TM mitted to the GenBank data base as a CTGF-related protein, absence (open circles) or presence (filled triangles)of10nM 1,25(OH) D . Osteocalcin present in 48-h culture supernatants was 2 3 CT 58, which was found in a yeast two-hybrid screen from measured by radioimmunoassay. The data are representative of three epithelial cell library using mucin, Muc1, as a bait (Gen- independent experiments. Values shown are the means 6 S. E. of TM Bank accession number AF074604). Mucin is a family of triplicate determinations. highly glycosylated secreted proteins that are aberrantly ex- pressed in epithelial tumors, including breast carcinomas (40). modulated directly or indirectly via interactions of CTGF-L Although these studies suggest a role for CTGF-L in tumori- with matrix molecules and integrin receptors. Indeed, the genesis possibly as a tumor suppressive gene, they are yet to be TSP1 domain has been implicated in binding to both soluble confirmed using purified protein. Given the range of activities and matrix molecules and to sulfated glycoconjugates (9, 29). It associated with this class of proteins, the exact function(s) of was recently reported that binding of integrin antagonist CTGF-L is not readily predictable. echistatin inhibits IGF-I signaling in vascular smooth muscle We have shown that rhCTGF-L is active at nanomolar con- cells (30). This led to the proposal that ligand-induced activa- centrations in assays measuring the inhibition of osteocalcin tion of a b integrin receptor results in its interaction with the v 3 production, the promotion of cell adhesion, and integrin bind- IGF-I signal transduction pathway. It is possible that CTGF-L ing, suggesting that these could be among the major functions by its ability to bind both integrin and IGFs may modulate the of this protein. We do not yet know whether CTGF-L is depos- activity of IGFs. However, we did not observe any significant ited in bone matrix, where it might facilitate osteoblast adher- modulation of IGF activity by CTGF-L, at least in osteoblastic ence leading to osteoid formation. By virtue of its ability to cells (data not shown). It is also possible that CTGF-L may bind promote cell adhesion and binding to IGFs, CTGF-L may in- to some as yet unidentified receptor. crease the local concentration of IGFs to augment osteoblast Although the VWC domain has been implicated in oligomer- activity. In this regard, it is tempting to speculate that CTGF-L ization, we have not observed either dimers or any other higher may also function to maintain or extend the osteoblast matrix order species, nor have oligomers been reported for other CCN maturation phase by inhibiting the production of osteocalcin, a proteins. CTGF-L lacks the CT domain, which alone is suffi- standard marker of mineralizing osteoblasts. The high expres- cient for heparin binding and mitogenic activity of CTGF (10). sion of CTGF-L in bone and suggested tumor suppressive ac- This suggests an interesting possibility that CTGF-L may an- tivity of some CCN protein suggests that it may also play a role tagonize the effect of CT domain in one or more CCN proteins, in preventing tumor metastasis to bone and other tissues. The as osteoblasts are capable of expressing various CCN members, identification of CTGF-L adds to the diversity of the CCN including CTGF, Cyr61 (24), and CTGF-L. family and suggests their potential role in bone metabolism. Due to the multidomain structure of CTGF-L, antisera gen- Further studies with CTGF-L will help elucidate its role in erated against peptides derived from the VWC domain and the bone and other tissues. C terminus of CTGF-L were nonneutralizing and, therefore, were not useful for blocking experiments. Alternatively, we Acknowledgments—We thank Drs. Michael Lark, Larry Suva, and used a similarly tagged but unrelated protein, p38, or a prep- Peter Young for critical reading of the manuscript; Dr. Rothman of the Rothman institute, Pennsylvania Hospital, for providing bone samples; aration in which rhCTGF-L was depleted with anti-CTGF-L Dr. Kyung Johanson for purified integrin receptors; Heather McClung antibodies. Neither protein preparations had any effect in any for technical assistance; DNA synthesis group for oligonucleotide syn- of the assays tested, suggesting that the functional results thesis and DNA sequencing; and Wendy Crowell for artwork. obtained were due to CTGF-L protein. Similarly, we have also confirmed various activities reported here using a rhCTGF-L REFERENCES preparation in which the epitope tags were removed by en- 1. Bradham, D. M., Igarashi, A., Potter, R. L., and Grotendorst, G. R. (1991) J. Cell Biol. 114, 1285–1294 terokinase digestion, suggesting that the presence of epitope 2. Grotendorst, G. R. (1997) Cytokine Growth Factor Rev. 8, 171–179 tags did not have any effect on the rhCTGF-L protein activity 3. Ryseck, R.-P., Macdonald-Bravo, H., Mattei, M.-G., and Bravo, R. (1991) Cell (data not shown). Growth Differ. 2, 225–233 4. Jay, P., Berge-Lefrac, J. L., Marsollier, C., C., M., Taviaux, S., and Berta, P. 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Identification and Cloning of a Connective Tissue Growth Factor-like cDNA from Human Osteoblasts Encoding a Novel Regulator of Osteoblast Functions

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 24, Issue of June 11, pp. 17123–17131, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Identification and Cloning of a Connective Tissue Growth Factor-like cDNA from Human Osteoblasts Encoding a Novel Regulator of Osteoblast Functions* (Received for publication, February 26, 1999) Sanjay Kumar‡§, Annalisa T. Hand‡, Janice R. Connor‡, Robert A. Dodds‡, Paul J. Ryan¶, John J. Trill¶, Seth M. Fisheri, Mark E. Nuttall‡, David B. Lipshutz‡, Cheng Zou‡, Shing M. Hwang‡, Bartholomew J. Votta‡, Ian E. James‡, David J. Rieman‡, Maxine Gowen‡, and John C. Lee‡ From the Departments of ‡Bone and Cartilage Biology, ¶Gene Expression Sciences, and iProtein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406 We have identified and cloned a novel connective tis- tified as a major chemotactic and mitogenic factor from endo- sue growth factor-like (CTGF-L) cDNA from primary hu- thelial cells (1). CTGF is distinct from, but immunologically man osteoblast cells encoding a 250-amino acid single related to, platelet-derived growth factor and competes for chain polypeptide. Murine CTGF-L cDNA, encoding a binding to the platelet-derived growth factor receptor (1). In polypeptide of 251 amino acids, was obtained from a fibroblasts, transforming growth factor b induces the produc- murine lung cDNA library. CTGF-L protein bears signif- tion of CTGF mRNA and protein, which in turn induces type I icant identity (;60%) to the CCN (CTGF, Cef10/Cyr61, collagen gene expression (2). Other members of this family Nov) family of proteins. CTGF-L is composed of three include Fisp12 (3), which is the murine orthologue of human distinct domains, an insulin-like growth factor binding CTGF; human and murine Cyr61 (4, 5); the chicken orthologue domain, a von Willebrand Factor type C motif, and a of Cyr61, Cef10 (6); and human and Xenopus Nov (7, 8). These thrombospondin type I repeat. However, unlike CTGF, genes, with the exception of nov, are immediate-early genes CTGF-L lacks the C-terminal domain implicated in that are induced by serum, growth factors, or certain oncogenes dimerization and heparin binding. CTGF-L mRNA (;1.3 and are collectively referred to as the CCN (CTGF, cef10/cyr61, kilobases) is expressed in primary human osteoblasts, nov) family (9). The nov gene was identified as a gene that was fibroblasts, ovary, testes, and heart, and a ;26-kDa pro- induced as a result of proviral rearrangement due to insertion tein is secreted from primary human osteoblasts and of nephroblastosis associated virus (7). fibroblasts. In situ hybridization indicates high expres- Members of CCN family are cysteine-rich proteins that are sion in osteoblasts forming bone, discrete alkaline phos- organized into four distinct motifs (9). The first motif contains phatase positive bone marrow cells, and chondrocytes. I-labeled insulin-like growth fac- an insulin-like growth factor (IGF) binding domain (GCGC- Specific binding of CXXC) common to all seven known IGF-binding proteins tors to CTGF-L was demonstrated by ligand Western blotting and cross-linking experiments. Recombinant (IGFBPs). The second domain contains a von Willebrand factor human CTGF-L promotes the adhesion of osteoblast type C (VWC) module that is suspected to be involved in oli- cells and inhibits the binding of fibrinogen to integrin gomerization. The third is a thrombospondin type I repeat receptors. In addition, recombinant human CTGF-L in- (TSP1) that is thought to play a role in cell attachment and hibits osteocalcin production in rat osteoblast-like Ros binding to matrix proteins and sulfated glycoconjugates (9). 17/2.8 cells. Taken together, these results suggest that The fourth, a C-terminal (CT) domain, has been implicated in CTGF-L may play an important role in modulating bone heparin binding and dimerization (10). The CT domain (;10 turnover. kDa) of CTGF is present in biological fluid and is sufficient for some biological activities (10). Osteoblasts are specialized mesenchymal cells that are re- Connective tissue growth factor (CTGF) was originally iden- sponsible for synthesizing and secreting the complex mixture of collagenous and noncollagenous proteins that make up bone matrix. These cells are also responsible for subsequent miner- * The costs of publication of this article were defrayed in part by the alization of this matrix (11). During the process of bone forma- payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to tion and remodeling, there is an integrated process of oste- indicate this fact. oclast-mediated bone resorption and osteoblast-derived bone The nucleotide sequence(s) reported in this paper has been submitted formation (11, 12). The process of bone formation and remod- TM to the GenBank /EBI Data Bank with accession number(s) AF083500 eling is under tight regulation by numerous factors, including and AF126063. endocrine hormones, cytokines, growth factors, adhesion mol- § To whom correspondence should be addressed: SmithKline Beecham, Bone and Cartilage Biology, UW2109, 709 Swedeland Rd., ecules, and extracellular matrix components. King of Prussia, PA 19406. Tel.: 610-270-7245; Fax: 610-270-5598; E- In the present report, we describe the identification, cloning, mail: Sanjay_Kumar@sbphrd.com. expression, and functional characterization of a novel CTGF- The abbreviations used are: CTGF, connective tissue growth factor; like (CTGF-L) cDNA from primary human osteoblast cells. CTGF-L, CTGF-like; rhCTGF-L, recombinant human CTGF-L; IGF, insulin-like growth factor; IGFBP, IGF-binding protein; TSP1, throm- CTGF-L contains the first three domains present in CCN fam- bospondin type I domain; VWC, von Willebrand factor type C domain; ily members but lacks the fourth CT domain. We show that CT, caboxyl terminal; TGF, transforming growth factor; GAPDH, glyc- CTGF-L is expressed at high levels in human bone tissue. eraldehyde-3-phosphate dehydrogenase; PAGE, polyacrylamide gel Recombinant hCTGF-L protein binds to IGFs and promotes electrophoresis; BSA, bovine serum albumin; EST, expressed sequenced tag; PBS, phosphate-buffered saline. adhesion of osteoblast cells. In addition, rhCTGF-L inhibits the This paper is available on line at http://www.jbc.org 17123 This is an Open Access article under the CC BY license. 17124 Identification, Cloning, and Characterization of CTGF-L oxide and sodium azide. The slides were washed in PBS and blocked binding of fibrinogen to integrin receptors and inhibits osteo- with PBS containing 10% goat and 1% human AB serum. The sections calcin production from rat osteoblast-like cells. were reacted with anti-CTGF-L or preimmune serum (1:250 in PBS), washed and reacted with horseradish peroxidase-conjugated secondary MATERIALS AND METHODS antibody, and visualized by labeled streptavidine-biotin 2 peroxidase Cells and Cell Culture—Primary osteoblasts were grown from ex- kit and the liquid diaminobenzidine substrate/chromogen system, ac- plants of human trabecular bone fragments from knee joints taken at cording to the manufacturer’s protocols (Dako). A brown stain indicated surgery (kindly provided by the Rothman Institute, Pennsylvania Hos- positive reactivity. pital, Philadelphia, PA). The osteoblasts were cultured in Eagle’s mod- Immunoprecipitation of CTGF-L from Fibroblast and Osteoblast ified minimum essential medium supplemented with 10% fetal calf Cells—For metabolic labeling, exponentially growing primary human serum (Hyclone, Logan, UT), 2 mML-glutamine, and antibiotics for 2–3 osteoblasts and fibroblasts were incubated for4hin cysteine-free weeks as described previously (13, 14). Cells from up to three passages medium containing 5% dialyzed fetal bovine serum with 100 –150 were used for all experiments. Primary human fibroblasts, human mCi/ml of S-labeled cysteine (;1000 Ci/mmol, ICN Biomedicals, Costa osteosarcoma MG 63 and SaOS-2, HeLa, and human mesenglial cells Mesa, CA). For immunoprecipitation, 5.0 ml of preimmune or anti- were obtained from ATCC (Manassas, VA). Human stromal TF274 cells CTGF-L immune-serum was mixed with precleared S-labeled condi- have been described previously (15). tioned medium and 20 ml of protein A-agarose (Life Technologies, Inc.) RNA Isolation and Northern Blot Analysis—Total RNA was isolated and incubated overnight at 4 °C. Immune complex beads were collected from primary human osteoblasts and different cell lines using Trizol by centrifugation and washed three times with PBST (PBS containing reagent (Life Technologies, Inc.) according to the manufacturer’s rec- 0.1% Tween 20) buffer. The beads were solubilized in sample buffer and ommendation. RNA was fractionated by electrophoresis on 1.2% aga- resolved through SDS-PAGE, fixed, dried, and processed for fluorogra- rose-formaldehyde gels, transferred to Genscreen plus membranes, and phy and autoradiography. cross-linked using an UV Stratalinker-180 (Stratagene, La Jolla, CA). Ligand Western Blotting and Cross-linking—Recombinant hCTGF-L The blots were probed with P-labeled CTGF-L cDNA and the house- (10 –300 pmol) was separated by SDS-PAGE under nonreducing condi- keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ac- tions. The protein was transferred to nitrocellulose membrane, blocked cording to standard procedures (16). A commercial human multiple with 5% nonfat dry milk in PBST, and probed sequentially with 1 3 10 tissue Northern blot (CLONTECH, Palo Alto, CA) containing 2 mgof cpm of I-labeled IGF-I or -II (;2000 Ci/mmol, Amersham Pharmacia poly(A) RNA from various tissues was processed according to the Biotech) or anti-CTGF-L antibody (1:1000) in PBST containing 0.1% manufacturer’s instructions. BSA for 18 h. The membrane was then washed 4 times for 15 min each Cloning of CTGF-L cDNAs and Expression of Recombinant Protein— with PBST and either dried and exposed to autoradiographic film for Human CTGF-L cDNA was identified by expressed sequenced tag ligand blots or developed with ECL for Western blot (Amersham Phar- (EST) analysis (17) of a cDNA library derived from primary human macia Biotech). For cross-linking, 300 pmol of rhCTGF-L was mixed 5 125 osteoblasts. After sequencing, the clone HOEBG39 was found to contain with 1 3 10 cpm of I-labeled IGF-I or -II for2hat4 °C. Water the entire open reading frame of hCTGF-L. An EST (AA754979) from a soluble homobifunctional cross-linker BS (Sigma) was added to the murine lung library, with high homology to the human CTGF-L cDNA, reaction mixture at a final concentration of 5 mM for 1 h. The reaction TM was identified from the GenBank data base. This EST contained the was stopped by addition of SDS-PAGE sample buffer, and the products 39-untranslated region and encoded about 35 amino acids from the C were separated by 12.5% SDS-PAGE. In some cases, the cross-linking terminus of CTGF-L protein. Based on the most 59 sequence of this EST, was performed in the presence of excess unlabeled IGF-I or IGF-II (Life primers were designed, and the full-length cDNA was isolated using the Technologies, Inc.). 59 rapid amplification of cDNA ends from a marathon cDNA library Cell Adhesion Studies—Corning 96-well enzyme-linked immunosor- prepared from murine lung (CLONTECH). For recombinant protein bent assay plates (Corning, NY) were precoated overnight at 4 °C with expression, the coding region of hCTGF-L was subcloned into CDN various concentrations of rhCTGF-L, 0.1 ml of human vitronectin (0.2 vector, where the expression of hCTGF-L was driven by the cytomega- mg/ml in PBS), or BSA (3 mg/ml). The plates were washed once with lovirus promoter (18). Two epitope tags, an N-terminal human immu- PBS and blocked with 3% BSA in PBS for1hat room temperature. nodeficiency virus GP120 V3 (SKSIRIQRGPGR) and His , were in- Cells were trypsinized and resuspended in RPMI medium and supple- serted after the signal sequence, and an enterokinase cleavage site was mented with 20 mM Hepes, pH 7.4, and 0.1% BSA at a density of 5 3 10 engineered between the epitope tags and CTGF-L protein. The plasmid cells/ml, and 0.1 ml of cell suspension was added to each well. Following was transfected into Chinese hamster ovary cells by electroporation, 1 h of incubation at 37 °C, the cells were fixed by the addition of 25 ml and cells expressing hCTGF-L were bulk-selected in nuceloside-free of a 10% formaldehyde solution, pH 7.4, at room temperature for 10 medium. Conditioned medium from a large-scale culture of Chinese min. The plates were washed three times with 0.2 ml of PBS, and the hamster ovary-hCTGF-L cells was used to purify rhCTGF-L using a adherent cells were stained with 0.1 ml of 0.5% toluidine blue for 20 min TALON metal affinity column (CLONTECH). Poor expression of soluble at room temperature. Excess stain was removed by extensive washing secreted CTGF-L was observed possibly due to the high number of with deionized water. The toluidine blue incorporated into cells was cysteine residues (11% of total protein), and hence only small amounts eluted by the addition of 0.1 ml of 50% ethanol containing 50 mM HCl of purified recombinant protein were obtained. The expression and and quantitated by measuring absorbance at 630 nm on a microtiter authenticity of rhCTGF-L was confirmed by immunoblotting with anti- plate reader (Titertek Multiskan MC, Sterling, VA). CTGF-L antibodies and N-terminal sequencing of purified protein. Integrin Binding—Vitronectin receptor, a b (0.12 mg), and fibrino- v 3 Anti-CTGF-L antibodies were prepared by injecting KLH-conjugated gen receptor, aII b (1 mg), purified from human placenta and blood, b 3 135 151 peptides derived from hCTGF-L ( SEDVRLPSWDCPHPRRV and respectively (22) were added to 96-well microtiter plates and incubated 234 248 SRPCPPSRGRSPQNS ) into rabbits according to standard overnight at 4 °C. At the time of experiment, the protein solutions were procedures. aspirated and the wells were incubated in 0.1 ml of Buffer A (50 mM Tissue Processing, in Situ Hybridization, and Immunocytochemis- Tris, 100 mM NaCl, 1 mM MgCl ,1mM MnCl , pH 7.4) containing 3% 2 2 try—Human tissues were obtained through the Anatomical Gift Foun- BSAfor1hat room temperature to block the nonspecific binding. After dation of Arizona (Phoenix, AZ). Human osteophytes were dissected aspirating the blocking solution, various concentrations of CTGF-L was from osteoarthritic femoral heads. Osteoclastoma tissue was prepared added to the wells followed by the addition of 8 nM biotinylated fibrin- from freshly dissected giant cell tumors. All tissues were processed for ogen in 100 ml of Buffer A containing 0.1% BSA. The plates were cryosectioning as described (19). The hCTGF-L cDNA cloned into incubated for1hat room temperature and washed twice with 100 mlof pBluescript was linearized and transcribed using T3- and T7-promoter binding buffer. Anti-biotin antibody conjugated to alkaline phosphatase sites to generate the antisense and sense strand riboprobes using the (1:2000 dilution, Sigma) was then added for 10 min followed by two Promega (Madison, WI) In Vitro transcription kit with [ S]thio-CTP washes with binding buffer containing 0.1% Tween 20. The reaction (Amersham Pharmacia Biotech). Riboprobes with a specific activity in was quantitated with alkaline phosphate substrate kit (Bio-Rad) by excess of 10 cpm/mg were used for in situ hybridization as described measuring absorbance at 405 nm using a microtiter plate reader. previously (20, 21). For immunocytochemistry, cryostat sections (8 mm) Osteocalcin Measurement—ROS 17/2.8 cells were seeded in 24-well were placed onto 3-aminopropyltriethoxy silane-coated glass slides, plates at a concentration of 3 3 10 /0.5 ml/well in assay medium (Ham’s fixed in 10% formalin, and washed in phosphate-buffered saline (PBS). F-12 medium supplemented with 1% fetal calf serum, 2 mML-gluta- Antigen retrieval was performed by heating the sections at 95 °C for 20 mine, and antibiotics) and incubated for 3– 4 h. Fresh medium, with or min in a Coplin jar containing appropriately diluted target retrieval without 10 nM 1,25(OH) D and various concentrations of CTGF-L, was 2 3 buffer (Dako, Carpenteria, CA). Endogenous peroxidase activity was then added in duplicate, and the cultures were incubated for an addi- blocked by incubating the sections for 10 min in 0.03% hydrogen per- tional 48 h. Osteocalcin in the culture supernatants was measured by Identification, Cloning, and Characterization of CTGF-L 17125 radioimmunoassay using reagents purchased from Biomedical Technol- noted at sites of secondary remodeling (secondary spongiosa), ogies (Stoughton, MA). chondrocytes, and osteoclasts. These data are summarized in Table I. RESULTS Polyclonal antibodies were generated to peptides derived Identification and Cloning of CTGF-L cDNA—By analyzing from CTGF-L and used to immunolocalize CTGF-L protein in a human osteoblast cDNA library using expressed sequence tag bone. Intense CTGF-L staining was associated with osteoblasts analysis, we identified an EST (HOEBG39) that contained an lining trabecular and periosteal bone surfaces from human IGF binding domain (GCGCCXXC). Complete sequencing of fetal bone tissue (Fig. 3, C and D). We also examined the HOEBG39 revealed that it contained an open reading frame expression of CTGF-L protein in primary human osteoblasts encoding a polypeptide of 250 amino acids. Based on sequence and fibroblasts that show high expression of CTGF-L mRNA. analysis, the HOEBG39-encoded protein exhibited significant In both cell types, a ;26-kDa protein was specifically immu- identity to the CCN family of proteins of which CTGF and 35 noprecipitated by anti-CTGF-L antibodies from S-labeled Cyr61 are the most fully characterized members. HOEBG39 conditioned medium (Fig. 4, A, lanes 2 and 4) and cell lysate encoded protein is 30 – 60% identical to CCN proteins (Fig. 1A) (not shown). The apparent molecular mass (;26 kDa) of im- and is most closely related to CTGF (;60% identity). Therefore, munoprecipitated protein is consistent with the expected size of we termed it CTGF-L protein. The first 23 amino acids of the protein encoded by the open reading frame of CTGF-L CTGF-L encode a putative signal sequence followed by three of mRNA after cleavage of the signal peptide. A similar sized the four distinct domains that are found in the CCN family of protein was also immunoprecipitated from HeLa and TF274 proteins (9). An IGF binding domain is present from residue cells that were transfected with an expression vector encoding 24 93 98 Gln to Leu . A VWC repeat is found from residue Ser to hCTGF-L (data not shown). Gly . A third TSP1 domain is contained within residues Recombinant hCTGF-L was expressed as an N terminus 194 237 Cys to Cys (Fig. 1, A and B). A fourth CT domain (;100 human immunodeficiency virus GP120 V3 and His epitope tag residues) that has been implicated in the heparin binding and in Chinese hamster ovary cells and was purified by TALON mitogenic activity of CTGF is missing from CTGF-L. An align- metal affinity chromatography as described under “Materials ment of CTGF-L to various members of the CCN family is and Methods.” The purified tagged protein of ;30 kDa (expect- shown in Fig. 1A. ed size due to the tag) was detected by Coomassie Blue staining An EST data base search with hCTGF-L sequence identified (Fig. 4B, lane 1) and immunostaining (Fig. 4B, lane 2). a murine EST (AA754979) that exhibited significant identity Binding of CTGF-L to IGF-I and IGF-II—The presence of an (;74% at nucleotide level) to hCTGF-L. We cloned the full- IGF binding domain within CTGF-L prompted us to investigate length mCTGF-L cDNA from a murine lung cDNA library the binding of CTGF-L to IGFs using ligand Western blotting using marathon 59 rapid amplification of cDNA ends as de- and cross-linking. Increasing amounts of rhCTGF-L were sep- scribed under “Materials and Methods.” The protein encoded arated by nonreducing SDS-PAGE and transferred onto a ni- by mCTGF-L cDNA is ;70% identical to hCTGF-L protein (Fig. trocellulose membrane. The blot was probed sequentially with 1A). Similar to hCTGF-L, the mCTGF-L also encodes a protein 125 125 I-labeled IGF-I, I-labeled IGF-II, and anti-CTGF-L anti- of 251 amino acids and contains all but the fourth CT domain body. As little as 10 pmol of CTGF-L bound to labeled IGFs (Fig. 1, A and B). Phylogentic analysis with all available mem- (Fig. 5A). rhCTGF-L showed a dose-dependent binding to I- bers of the CCN family grouped both human and murine labeled IGFs, with IGF-II exhibiting relatively higher binding CTGF-L together in a separate group (Fig. 1C). It appears that than IGF-I. Immunostaining confirmed the increasing elm is the oldest gene from which both CTGF-L and other CCN amounts of rhCTGF-L loaded in each lane (Fig. 5A, bottom members originated (Fig. 1C). Given the similar arrangement, panel). As a control, an unrelated His-tagged protein, p38 MAP composition, and length of various domains between the mu- kinase, did not bind either IGF-I or IGF-II (Fig. 5A, lane 5). In rine and human CTGF-L protein (Fig. 1, A and B), it is clear a cross-linking experiment using a homobifunctional cross- that the murine cDNA that we isolated is the murine ortho- linker BS , both I-labeled IGF-I and -II bound to rhCTGF-L logue of human CTGF-L. (Fig. 5B, lane 1). The size of cross-linked product (;37–38 kDa) Expression of CTGF-L mRNA and Protein—Northern blots is consistent with the cross-linking of rhCTGF-L (;30 kDa) to containing RNAs from various human tissues and bone-derived IGFs (;7– 8 kDa). We also performed a competitive cross- cells were hybridized to a P-labeled CTGF-L cDNA probe. A linking experiment in which the cross-linking was competed ;1.3-kilobase CTGF-L mRNA was highly expressed in primary with increasing amounts of unlabeled IGF-I (Fig. 5B, lanes 2–5) human osteoblasts, fibroblasts, ovary and testes (Fig. 2, A, or IGF-II (lanes 6 –9). The signal obtained by cross-linking of lanes 1 and 9, and B, lanes 12 and 13). A lower level of expres- rhCTGF-L to I-labeled IGF-II was much higher than with sion of CTGF-L was also observed in heart, lung, skeletal I-labeled IGF I. In addition, only 30 ng of IGF-II was needed muscle, prostate, and colon (Fig. 2B, lanes 1, 4, 6, 11, and 15). to compete rhCTGF-L cross-linking to IGF-I (Fig. 5B, lane 7, However, CTGF-L was not expressed in human osteosarcoma upper panel), whereas even 300 ng of IGF-I could not compete SaOS-2 or MG 63 cells, stromal TF274 cells, osteoclastoma the cross-linking of rhCTGF-L to IGF-II (lane 5, lower panel). tissue, or HeLa or mesenglial cells (Fig. 2). The expression of These data suggest that both IGF I and IGF II bind to CTGF-L in primary osteoblast or fibroblast cultures appears to rhCTGF-L and that IGF-II has at least 10-fold higher affinity be constitutive as treatment with a variety of osteotropic for CTGF-L compared with IGF-I. It should be noted that the agents, including parathyroid hormone, transforming growth specific activity of both I-labeled IGF-I and IGF-II was com- factor-b, 1,25(OH) D , and estrogen, failed to modulate its ex- 2 3 pression (data not shown). parable (;2000 Ci/mmol). Adhesion of Osteoblast Cells to CTGF-L—The TSP1 domain In situ hybridization indicated strong expression of CTGF-L mRNA in bone-forming osteoblasts on calcified cartilage spi- has been implicated in the promotion of cell attachment (9). Therefore, we tested whether rhCTGF-L was able to promote cules (primary spongiosa) in fetal bone (Fig. 3, A and B) and human osteophytic tissue (Table I). Discrete alkaline phospha- the adhesion of osteoblastic cells. Primary human osteoblast, tase positive cells in bone marrow of osteophyte and discrete osteosarcoma MG63, and rat osteoblast-like osteosarcoma Ros 17/2.8 cells attached to immobilized rhCTGF-L in a dose-de- macrophage-like cells from giant cell tumor also exhibited strong expression of CTGF-L mRNA. Weaker expression was pendent manner (Fig. 6A). Although maximal adhesion was 17126 Identification, Cloning, and Characterization of CTGF-L FIG.1. Alignment of CTGF-L with various members of the CCN family of proteins. A, protein sequence alignment was performed using the MEGALIGN program of Lasergene (DNASTAR Inc., Madison, WI) according to the clustal algorithm. The amino acids identical to CTGF are hidden and indicated by dots, whereas dashes indicate gaps. The broken, dashed, solid and dotted lines with arrows indicate the IGF binding domain, the VWC domain, the TSP1 domain, and the CT domain, respectively. B, schematic representation of different domains, including signal sequence (SS) of CCN proteins. IGF BD, IGF binding domain. The amino acid number flanking each domain of hCTGF-L is also shown. Note that the CT domain is missing in CTGF-L. C, phylogenetic relationship between various members of the CCN family. observed with Ros 17/2.8 cells, the extent of cell adhesion to at a coating concentration of ;300 ng/ml (;10 nM), whereas no rhCTGF-L for all cells was comparable to that observed with cell adhesion was observed using a protein preparation in vitronectin. Half-maximal adhesion to rhCTGF-L was observed which CTGF-L was depleted using anti-CTGF-L antibodies or Identification, Cloning, and Characterization of CTGF-L 17127 FIG.2. Expression of human CTGF-L mRNA in various human cells and tissue preparations. A, total RNA from primary human osteoblasts (lane 1), human osteosarcoma SaOS2 (lane 2), human osteoclastoma tissue (lane 3), human osteosarcoma HOS (lane 4), human osteosarcoma MG63 (lane 5), human stromal TF274 (lane 6), HeLa (lane 7), human mesenglial (lane 8), and synovial fibroblasts (lane 9) was hybridized with P-labeled hCTGF-L and GAPDH cDNA probe. The location of 28 S and 18 S ribosomal RNA, CTGF-L, and GAPDH are indicated. B, a multiple tissue Northern blot containing poly(A) RNA from heart (lane 1), brain (lane 2), placenta (lane 3), lung (lane 4), liver (lane 5), skeletal muscle (lane 6), kidney (lane 7), pancreas (lane 8), spleen (lane 9), thymus (lane 10), prostate (lane 11), testes (lane 12), ovary (lane 13), small intestine (lane 14), colon (lane 15), and peripheral blood leukocytes (lane 16) was hybridized with P-labeled CTGF-L and GAPDH cDNA probe. The locations of RNA markers (in kilobases), CTGF-L, and GAPDH are indicated. report that Cyr61, another CCN family member, binds a b v 3 (23) prompted us to examine the effect of CTGF-L on the binding of purified integrins to matrix protein. The purified integrin receptors, a b and aII b , demonstrated high affinity v 3 b 3 binding to biotinylated fibrinogen. As shown in Fig. 7, rh- CTGF-L inhibited the binding of fibrinogen to both integrins in a dose-dependent manner. The IC for a b (Fig. 7, filled 50 v 3 triangles) was ;100 nM, whereas weaker inhibition was ob- served for aII b (IC ;1 mM)(open circles), suggesting some b 3 50 degree of selectivity. Effect of rhCTGF-L on Osteocalcin Production—To deter- mine whether rhCTGF-L could modulate osteoblast function, we examined its effect on osteocalcin production in rat osteo- blast-like osteosarcoma, ROS 17/2.8 cells. Osteocalcin is a marker of mineralizing osteoblasts, and its expression is used routinely as a measure of osteoblast function. ROS 17/2.8 cells endogenously secrete low levels of osteocalcin, which can be up-regulated by treatment with 1,25(OH) D . As shown in Fig. 2 3 8, rhCTGF-L inhibited both basal and 1,25(OH) D -stimulated 2 3 osteocalcin production in a dose dependent manner with an IC of ;300 ng/ml (;10 nM). Treatment with a CTGF-L de- pleted protein preparation or with an unrelated His -tagged protein, p38, had no effect on osteocalcin production (data not shown). The inhibition of osteocalcin expression was at the mRNA level because rhCTGF-L also inhibited the osteocalcin mRNA induction by 1,25(OH) D both in Ros 17/2.8 and in 2 3 primary human osteoblasts (data not shown). DISCUSSION During the past few years, several proteins belonging to the expanding CCN family have been described (9). Most members of this family are immediate-early genes that are induced by FIG.3. CTGF-L mRNA and protein expression during human treatment with serum or growth factors (1, 3, 5, 24, 25). All endochondral ossification. A, a section of human fetal femoral members of this family contain four distinct domains, including growth plate was hybridized with antisense CTGF-L riboprobe. Osteo- the IGF binding domain, the VWC domain, the TSP1 domain, blasts (Obs)(arrows) forming osteoid on calcified cartilage spicules demonstrated intense mRNA expression; adjacent formation sites and the CT domain. showed reduced expression (arrowheads). Magnification, 3 50; counter- We have identified human (and murine) CTGF-L as a novel stained with methylene blue. B, high power view of the area shown in member of the CCN family. There are two striking features A demonstrates CTGF-L mRNA expression in osteoblasts (Obs)(ar- that distinguish CTGF-L from the other CCN family members. rows) and osteoprogenitor cells (Opg) within the marrow (magnifica- tion, 3 100). C, a section of fetal bone stained with the anti-CTGF-L First, CTGF-L lacks the fourth CT domain found in all other polyclonal antibody. Osteoblasts lining the trabecular (arrows) and family members. Second, CTGF-L is not induced by serum or periosteal (arrowheads) bone surface express high levels of CTGF-L growth factors. Although an alternatively spliced form may (magnification, 3 25). D, high power view of the area shown in C shows exist, we have been unable to identify a CTGF-L variant that expression of CTGF-L in osteoblasts (Obs)(arrows) and discrete cells within the marrow (arrowheads) (magnification, 3 100). encodes a protein containing the CT domain in libraries de- rived from cells and tissues expressing CTGF-L. to an unrelated His -tagged protein, p38 (Fig. 6B). In addition, immunoprecipitation of CTGF-L by anti- Binding of rhCTGF-L to Purified Integrin Receptors—The CTGF-L antibodies detected only a single ;26-kDa protein in attachment of cells to CTGF-L-coated plates and a previous both osteoblasts and fibroblasts. We did not detect a protein of 17128 Identification, Cloning, and Characterization of CTGF-L TABLE I Expression of CTGF-L mRNA in human bone by in situ hybridization Cryostat sections of various tissues were hybridized to S-labeled antisense or sense CTGF-L riboprobe. Tissue type Cell type mRNA expression Osteophyte, including synovium and granulation tissue Osteoblasts Primary spongiosa 111 Secondary spongiosa 1 Chondrocytes 1/2 Synovial macrophages 11 Marrow cells 1 to 111 Osteoclasts 1/2 Fetal bone Osteoblasts Primary spongiosa 11 Secondary spongiosa 1 Chondrocytes 11 Myocytes 1 Giant cell tumor, including areas of bone formation and alkaline phosphatase-positive stroma Stromal cells 1 Osteoblasts 1 Osteocytes 1 Marrow cells 1/2 Osteoclasts 2 Blood vessels 2 Macrophages (discrete) 111 2 indicates negative signal; 1 to 111 indicates moderate to very strong signal obtained with antisense probe. No signal was obtained with sense probe. non-bone-forming giant cell tumors were negative. including osteosarcoma, and it appears that its expression is restricted to primary cells, such as osteoblasts and fibrolasts. Human tissues expressing the highest levels of CTGF-L mRNA are bone, ovary, and testes. We have shown that both CTGF-L mRNA and protein are expressed at high levels in osteoblasts forming bone in the periosteum and primary spon- giosa of human fetal and osteophytic bone. These bone forma- tion areas are zones of appositional and longitudinal bone growth. Lower levels of expression were observed in osteoblasts lining the relatively slower remodeling trabeculae of the sec- ondary spongiosa. Thus, CTGF-L mRNA was highly expressed by osteoblasts at sites of high bone turnover (Table I) associ- ated with net gain in bone mass. This highly selective expres- sion is suggestive of a specific role for CTGF-L in the control of bone formation. We have examined the activity of rhCTGF-L in several as- FIG.4. Expression of endogenous and recombinant CTGF-L says to elucidate potential functions for various domains of this protein. A, primary human fibroblasts (Fib.) and osteoblasts (Obs.) were labeled with [ S]cysteine, and the conditioned medium was im- protein, including cell adhesion, IGFs and integrin binding, munoprecipitated with preimmune (PI) or immune (IM) anti-CTGF-L and osteocalcin production. IGFs have growth promoting activ- antibody. The location of ;26-kDa CTGF-L is indicated. B, recombinant ity and play an important role in bone formation and remodel- CTGF-L was expressed as N terminus GP120/His -tagged protein in ing (26). Several IGF-binding proteins regulate the activity and Chinese hamster ovary cells and purified by TALON metal affinity chromatography. Molecular weight markers (in thousands) and the availability of IGFs in bone and other tissues (26). Although we location of purified rhCTGF-L as detected by Coomassie Blue staining have unequivocally shown that rhCTGF-L binds to both IGF-I (lane 1) and Western blot (lane 2) are shown. and IGF-II, to date, no significant change in IGF action has been observed in the presence of CTGF-L. This suggests that CTGF-L may serve not to modulate IGFs activity, but rather to higher molecular mass in the anti-CTGF-L immunoprecipi- increase the local concentrations of IGFs. Although cross-link- tates that might contain the fourth CT domain. Furthermore, ing and ligand Western blotting studies did not allow a precise murine CTGF-L cDNA encodes an open reading frame that determination of binding affinities, it appears that the relative terminates at the same amino acid residue as human CTGF-L. affinity of rhCTGF-L for IGF-II is at least 10-fold higher than CTGF-L without the CT domain, therefore, is the functional for IGF-I. In this regard, it has been reported that human protein being produced and secreted by osteoblast and fibro- blast cells. osteoblasts produce and respond better to IGF-II, whereas ro- dent osteoblasts produce and are more responsive to IGF-I (26). Another feature that distinguishes CTGF-L from all other members of the CCN family is the inability of serum or various In a recent report, rhCTGF was also shown to bind both IGF-I and IGF-II (27) but with much lower affinity than the binding growth factors to induce its expression. The expression of CTGF-L in primary osteoblast or fibroblast cultures appears to of IGFBP3 to IGFs. Unlike IGFBPs, no reports on modulation be constitutive, and to date, we have not been able to modulate of IGFs activity by CCN family of proteins have been described. its expression by treatment with a variety of osteotropic agents, Whereas the binding of CTGF-L to IGFs is most likely me- including parathyroid hormone, transforming growth factor-b, diated via the IGF binding domain similar to the IGFBPs, cell 1,25(OH) D , and estrogen. An additional feature is the ab- adhesion is most likely mediated via the TSP1 domain, perhaps 2 3 sence of CTGF-L mRNA expression in various tumor cell lines, through binding to integrin receptors. Indeed, we have demon- Identification, Cloning, and Characterization of CTGF-L 17129 FIG.5. Binding of rhCTGF-L to IGFs. A, various amounts of CTGF-L (10 –300 pmol) and p38 (300 pmol) were separated by SDS-PAGE under nonreducing conditions and probed sequentially with I-labeled IGF-I or IGF-II or with anti-CTGF-L antibody. Between experiments, the blot was stripped and extensively washed. The final immunoblot was developed with ECL. The locations of the ;30-kDa marker and CTGF-L as 125 125 detected with each probe are indicated. B, rhCTGF-L was cross-linked to I-labeled IGF-I (top)or I-labeled IGF-II (bottom) with a homobifunctional cross-linking agent, BS . The cross-linking was competed with increasing amounts of unlabeled IGF-I (lanes 2–5) or IGF-II (lanes 6–9) as indicated. FIG.7. Inhibition of fibrinogen binding to purified integrins by rhCTGF-L. Biotinylated fibrinogen (8 nM) was added to microtiter wells coated with integrin receptors in the presence or absence of various concentration of CTGF-L and incubated for1hat room tem- perature to measure the effect of CTGF-L on the binding of biotinylated fibrinogen to a b (filled triangles) and aII b (open circles). Inhibition v 3 b 3 of fibrinogen binding by CTGF-L was quantitated by staining with alkaline phosphatase conjugated anti-biotin antibody and by measuring absorbance at 405 nM. Total binding (control 5 100%) was determined in absence of CTGF-L. The inhibition is expressed as a percentage of control. The data are representative of three independent experiments. Values shown are the means 6 S. E. of triplicate determinations. FIG.6. Promotion of cell adhesion by rhCTGF-L. A, 96-well CTGF-L or Cyr61, which has also been shown to bind a b v 3 microtiter plates were coated with or 3 mg/ml BSA, 200 ng/ml vitronec- integrin (23). Therefore, CTGF-L binding must occur through tin (Vn), or various amounts of rhCTGF-L as indicated. After blocking an RGD-independent mechanism. Consistent with this notion, with BSA, MG 63 (black columns) primary human osteoblasts (gray columns), and Ros 17/2.8 (white columns) cells were allowed to adhere the adhesion of osteoblasts to vitronectin, but not to CTGF-L, for 30 min, washed extensively, fixed, and stained to quantitate the was inhibited by the RGD-containing snake venom protein adhesion. B, adhesion of MG 63 (black columns) and Ros 17/2.8 (white echistatin (data not shown). Therefore, CTGF-L may inhibit columns) cells to microtiter plates coated with equal amounts (1 mg/ml) matrix/integrin interaction by binding close to the RGD recog- of CTGF-L, antibody depleted CTGF-L (depleted) or unrelated protein, p38. The data are representative of three independent experiments. nition site due to steric hindrance or by inducing a conforma- Values shown are the means 6 S. E. of triplicate determinations. tional change. Similarly, the effect of CTGF-L on osteocalcin release may occur through the TSP1 domain and integrin bind- strated that rhCTGF-L directly inhibits fibrinogen binding to ing because it is known that integrin binding to extracellular purified integrin receptors. Fibrinogen, like many extracellular matrix proteins can result in signal transduction leading to matrix proteins, contains an RGD motif that is crucial for changes in cell proliferation, gene expression, and cellular dif- integrin binding. However, no RGD sequence is present in ferentiation (28). It is possible that these changes may be 17130 Identification, Cloning, and Characterization of CTGF-L role in wound repair and/or fibrosis (1, 10, 31–33). Cyr61 has been shown to be an extracellular matrix-associated molecule that has mitogenic activity when used in conjunction with basic fibroblast growth factor and can also promote adhesion and migration of fibroblasts and vascular endothelial cells (34, 35). Recently, it has been determined that Cyr61 mediates the adhesion of cells via a b integrin binding and has been sug- v 3 gested to promote tumor growth and angiogenesis (36, 37). Elm, another family member, is expressed at low levels in metastatic cancer and suppresses metastasis (25). While this report was in preparation, two laboratories re- ported the cloning of murine and rat CTGF-L. WISP-2, the murine orthologue of hCTGF-L, was cloned by subtractive hy- bridization as a Wnt-1 inducible gene, the expression of which was reduced in some tumors (38). The rat orthologue of CTGF-L, rCOP-1, was cloned by differential display analysis as a gene of which the expression was lost as a result of cell FIG.8. Inhibition of osteocalcin production by rhCTGF-L. Ros transformation (39). An identical sequence has also been sub- cells were treated with different concentrations of rhCTGF-L in the TM mitted to the GenBank data base as a CTGF-related protein, absence (open circles) or presence (filled triangles)of10nM 1,25(OH) D . Osteocalcin present in 48-h culture supernatants was 2 3 CT 58, which was found in a yeast two-hybrid screen from measured by radioimmunoassay. The data are representative of three epithelial cell library using mucin, Muc1, as a bait (Gen- independent experiments. Values shown are the means 6 S. E. of TM Bank accession number AF074604). Mucin is a family of triplicate determinations. highly glycosylated secreted proteins that are aberrantly ex- pressed in epithelial tumors, including breast carcinomas (40). modulated directly or indirectly via interactions of CTGF-L Although these studies suggest a role for CTGF-L in tumori- with matrix molecules and integrin receptors. Indeed, the genesis possibly as a tumor suppressive gene, they are yet to be TSP1 domain has been implicated in binding to both soluble confirmed using purified protein. Given the range of activities and matrix molecules and to sulfated glycoconjugates (9, 29). It associated with this class of proteins, the exact function(s) of was recently reported that binding of integrin antagonist CTGF-L is not readily predictable. echistatin inhibits IGF-I signaling in vascular smooth muscle We have shown that rhCTGF-L is active at nanomolar con- cells (30). This led to the proposal that ligand-induced activa- centrations in assays measuring the inhibition of osteocalcin tion of a b integrin receptor results in its interaction with the v 3 production, the promotion of cell adhesion, and integrin bind- IGF-I signal transduction pathway. It is possible that CTGF-L ing, suggesting that these could be among the major functions by its ability to bind both integrin and IGFs may modulate the of this protein. We do not yet know whether CTGF-L is depos- activity of IGFs. However, we did not observe any significant ited in bone matrix, where it might facilitate osteoblast adher- modulation of IGF activity by CTGF-L, at least in osteoblastic ence leading to osteoid formation. By virtue of its ability to cells (data not shown). It is also possible that CTGF-L may bind promote cell adhesion and binding to IGFs, CTGF-L may in- to some as yet unidentified receptor. crease the local concentration of IGFs to augment osteoblast Although the VWC domain has been implicated in oligomer- activity. In this regard, it is tempting to speculate that CTGF-L ization, we have not observed either dimers or any other higher may also function to maintain or extend the osteoblast matrix order species, nor have oligomers been reported for other CCN maturation phase by inhibiting the production of osteocalcin, a proteins. CTGF-L lacks the CT domain, which alone is suffi- standard marker of mineralizing osteoblasts. The high expres- cient for heparin binding and mitogenic activity of CTGF (10). sion of CTGF-L in bone and suggested tumor suppressive ac- This suggests an interesting possibility that CTGF-L may an- tivity of some CCN protein suggests that it may also play a role tagonize the effect of CT domain in one or more CCN proteins, in preventing tumor metastasis to bone and other tissues. The as osteoblasts are capable of expressing various CCN members, identification of CTGF-L adds to the diversity of the CCN including CTGF, Cyr61 (24), and CTGF-L. family and suggests their potential role in bone metabolism. Due to the multidomain structure of CTGF-L, antisera gen- Further studies with CTGF-L will help elucidate its role in erated against peptides derived from the VWC domain and the bone and other tissues. C terminus of CTGF-L were nonneutralizing and, therefore, were not useful for blocking experiments. Alternatively, we Acknowledgments—We thank Drs. Michael Lark, Larry Suva, and used a similarly tagged but unrelated protein, p38, or a prep- Peter Young for critical reading of the manuscript; Dr. Rothman of the Rothman institute, Pennsylvania Hospital, for providing bone samples; aration in which rhCTGF-L was depleted with anti-CTGF-L Dr. Kyung Johanson for purified integrin receptors; Heather McClung antibodies. 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Published: Jun 1, 1999

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