CTGF - Journal of Endocrinology

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RAPID COMMUNICATION The heparin-binding 10 kDa fragment of connective tissue growth factor (CTGF) containing module 4 alone stimulates cell adhesion D K Ball1,4, A W Rachfal1,3,4, S A Kemper4 and D R Brigstock1,2,3,4 1

Department of Surgery, The Ohio State University, Columbus, Ohio 43212, USA

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Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43212, USA

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Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio 43212, USA

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Center for Cellular and Vascular Biology, Children’s Research Institute, Columbus, Ohio 43205, USA

(Requests for offprints should be addressed to D R Brigstock, Children’s Research Institute, Room W309, 700 Children’s Drive, Columbus Ohio 43205, USA; Email: [email protected])

Abstract Connective tissue growth factor (CTGF) is a 349-residue mosaic protein that contains four structural modules implicated in protein-protein interactions. To address the functionality of residues 247–349 (containing module 4 alone), this region of CTGF was produced as a maltose binding protein (MBP) fusion protein in E. coli. After removal of MBP, recombinant CTGF commenced at Glu247, was of Mr 10 000, was immunoreactive with anti-CTGF[247–260], bound strongly to heparin, and promoted dose-dependent adhesion of fibroblasts, myofibroblasts, endothelial cells, and epithelial cells. An 8 kDa

presumptive C-terminally truncated form of CTGF commencing at Glu247 also promoted cell adhesion. CTGFmediated cell adhesion was abolished by heparin or EDTA. These data demonstrate the presence of heparinbinding and cell-adhesion motifs within the C-terminal 103 residues of CTGF and show that CTGF-mediated cell adhesion is heparin-and divalent cation-dependent. Thus, CTGF isoforms comprising essentially module 4 are intrinsically functional in the absence of the other constituent modules of CTGF.

Introduction

which is structurally related to CTGF (Sampath, Zhu et al. 2001, Xie, Miller et al. 2001, Sampath et al. 2002). Both CYR61 and CTGF are expressed in steroid-dependent tumors of the breast or uterus (Frazier & Grotendorst 1997, Tsai et al. 2000, Uzumcu et al. 2000, Sampath, Winneker et al. 2001, Sampath, Zhu et al. 2001, Xie, Miller et al. 2001, Xie, Nakachi et al. 2001, Sampath et al. 2002, Tsai et al. 2002). The CTGF protein is organized into four modules which structurally resemble an insulin-like growth factor binding motif (module 1), a von Willebrand factor type C repeat (module 2), a thrombospondin-1 domain (module 3), and a cysteine knot (module 4) (Bork 1993). This modular structure is conserved in other CTGF-related proteins (CYR61, NOV, WISP-1, -2, -3) that collectively comprise the CTGF/CYR61/NOV (CCN) family (Bork 1993, Brigstock 1999, Lau & Lam 1999, Perbal 2001). Data from several laboratories support an important role for module 4 in the activity of several CCN proteins since it is involved in regulation of mitosis by CTGF, heparin-binding by CTGF or CYR61, regulation of the

Connective tissue growth factor (CTGF) is a matricellular protein that regulates a diverse variety of functions in several cell types (Brigstock 1999, Lau & Lam 1999, Moussad & Brigstock 2000, Perbal 2001). CTGF is involved in pathways of hormone action, particularly in organs that are targets of sex steroids. Ovarian CTGF appears to be involved in recruitment and mitosis of theca cells and maintenance of the corpus luteum (Wandji et al. 2000, Slee et al. 2001, Harlow et al. 2002, Harlow & Hillier 2002, Liu et al. 2002) while uterine CTGF has been implicated in angiogenesis and extracellular matrix remodeling during placentation and decidualization. (Surveyor et al. 1998, Moussad & Brigstock 2000, Uzumcu et al. 2000, Moussad et al. 2002). Expression of CTGF in the ovary is regulated by TGF- and gonadotrophins (Harlow et al. 2002, Liu et al. 2002) while in the uterus it is regulated by TGF-, estrogen and progesterone (Rageh et al. 2001). Estrogen and progesterone also regulate the production of cysteine-rich 61 (CYR61)

Journal of Endocrinology (2003) 176, R1–R7

Journal of Endocrinology (2003) 176, R1–R7 Accepted 9 January 2003 0022–0795/03/0176–R1 2003 Society for Endocrinology Printed in Great Britain

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· Recombinant production and action of CTGF module 4

Figure 1 Induction and Factor Xa cleavage of MBP-CTGF[247–349] in E. coli clone DB-10K. A 1 liter culture of E. coli DB-10K cells was induced with IPTG for 1 h and subjected to amylose affinity purification. The eluted fusion protein was then digested in the presence or absence of Factor Xa for 72 h at room temperature, during which time aliquots of the reaction mixture were removed, mixed with SDS-PAGE sample buffer and boiled. The figure shows SDS-PAGE and Western blot analysis of (left panel) 10 µl of DB-10K cell cultures before (Lane 1) and after (Lane 2) IPTG induction and (right panel) 3 µl of the cell lysate prior to amylose column chromatography (Lane 1), 5 µl of mock-treated reaction mixture after 72 h (Lane 2), or 5 µl of Factor Xa-treated reaction mixture at 2, 4, 8, 24, 32, 48, 56, and 72 h (Lanes 3–10 respectively). CTGFimmunoreactive proteins were detected using anti-CTGF[247–260] peptide antiserum.

angiogenic activity of VEGF by CTGF, or the binding of Notch or fibulin 1C by NOV (Brigstock et al. 1997, Perbal et al. 1999, Chen N. et al. 2000, Sakamoto et al. 2002). Here we report that a recombinant form of CTGF comprising essentially module 4 alone is a heparin-binding protein that supports heparin- and divalent cationdependent adhesion of fibroblasts, epithelial cells, endothelial cells, and myofibroblasts. These data emphasize the intrinsic functionality of module 4. Materials and Methods Generation of 10 kDa CTGF fusion protein 10 kDa human CTGF, corresponding to residues 247– 349, was produced in E. coli using the maltose-binding protein fusion system. Briefly, cDNA encoding 10 kDa CTGF was generated by RT-PCR of human fibroblast mRNA. The amplified product was blunt end ligated into the Xmn I site of pMAL-c2 (New England BioLabs, Beverly, MA, USA) using T4 DNA ligase (GIBCO/ BRL, Grand Island, NY, USA) to generate pMAL-c2/10 KCTGF. E. coli strain BL21 (F- ompT rB- mB-) (Novagen, Madison, WI, USA) was transformed with pMAL-c2/10 KCTGF, plated onto LB agar plates with 100 µg/ml ampicillin, and a CTGF-positive clone, termed DB-10K, was selected. Production and purification of recombinant CTGF[247–349] in E. coli DB-10K cells were grown in 1 liter of LB broth containing 100 µg/ml ampicillin and 2 g glucose until the optical Journal of Endocrinology (2003) 176, R1–R7

density at 600 nm was 4·5. Recombinant protein was induced with 0·3 mM IPTG for 1 h and the bacterial cells were lysed at 16 000 psi in 40 ml amylose column buffer (10 mM Tris–HCl, 200 mM NaCl, 1 mM EDTA) using a French Press. The homogenate was clarified by centrifugation and the supernatant was diluted 1:10 with amylose column buffer and loaded on to an amylose column (5 cm x 10 cm; BioRad, Hercules, CA) at 4 C. Bound proteins were eluted using 10 mM maltose in amylose column buffer and fractions containing peak protein levels were combined (approx 40 ml) and digested for up to three days at room temperature with 250 µg of Factor Xa. The sample was then clarified using a 0·45 µm filter and subjected to sequential steps of heparin affinity FPLC and C8 reverse-phase HPLC, essentially as previously described (Brigstock et al. 1997). Protein analysis Aliquots of column fractions were subjected to SDSPAGE and Western blotting using rabbit antiCTGF[247–260] peptide antiserum (Brigstock et al. 1997). Automated N-terminal amino acid sequence analysis of HPLC-purified CTGF was performed using an ABI Procise 491 protein sequencer (Applied Biosystems, Foster City, CA, USA). Cell adhesion 50 µl PBS containing CTGF samples were incubated overnight at 4 C in 96-well round-bottom ELISA plates (Corning Inc, Reynoldsburg, OH, USA). Wells were www.endocrinology.org

Recombinant production and action of CTGF module 4 ·

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Figure 2 Purification of recombinant human CTGF[247–349]. The figure shows sequential steps of (A) amylose-affinity chromatography followed by Factor Xa cleavage of the eluted fusion protein, (B) heparin-affinity chromatography of the cleavage reaction; and (C) C8 reverse-phase HPLC of the 0·8 M NaCl heparin eluate. The insets show CTGF immunoreactivity in 10 µl (A,C) or 5 µl (B) of selected fractions that were analyzed by SDS-PAGE on 15% (A) or 18% (B,C) gels followed by Western blotting using anti-CTGF[247–260] peptide antiserum. 3 µl (B) or 20 µl (C) of selected fractions were diluted to a total volume of 100 µl in PBS and tested for their ability to promote adhesion of Balb/c 3T3 cells to U-bottom wells of non-tissue culture 96-well plates. www.endocrinology.org


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Figure 3 N-terminal sequence analysis of recombinant CTGF proteins and their relationship to full length CTGF. The figure shows the modular structure of the 349-residue primary translational product of CTGF as well as the structure of native and recombinant N-terminally truncated CTGF. (1) Native 10 kDa CTGF from ref (Brigstock et al. 1997); (2) Recombinant 10 kDa CTGF present in HPLC fraction #43 (see Figure 2C): Initial yield 144 pmol, repetitive yield 63%; (3) Recombinant 8 kDa CTGF protein in HPLC fraction #39 with possible C-terminal truncation (see Figure 2C): Initial yield 314 pmol, repetitive yield 64%.

Figure 4 Dose-dependent stimulation of Balb/c 3T3 cell adhesion by10 kDa CTGF

blocked with 200 µl PBS containing 3% BSA and then incubated for 1 h at 37 C with 100µl PBS containing approximately 5 x 104 Balbc/3T3 cells, intestinal epithelial cells (IEC-6), bovine aortic endothelial cells (BAEC), or myofibroblastic hepatic stellate cells (HSC-T6; kindly provided by Scott Friedman M.D., Mount Sinai Hospital, New York, NY, USA). Some incubations were done in the presence of 5 ug/ml heparin or 10 mM EDTA. Adherent cells were then fixed for 15 min with 5% formaldehyde and non-adherent cells were removed by washing each well three times with PBS. The remaining cells were measured by fluorescent emission from the wells at 520 nm following addition of 100 µl Cytoquant reagent (Molecular Probes, Eugene, OR, USA) in lysis buffer. Journal of Endocrinology (2003) 176, R1–R7

Results A 52 kDa CTGF-immunoreactive protein doublet, representing the MBP-10 kDa CTGF fusion protein, was produced in DB-10K cells following treatment with IPTG (Figure 1). In the presence of Factor Xa, a 10 kDa cleavage product was generated in a time-dependent manner, with complete production by 48–72 h (Figure 1). The fusion protein was isolated by amylose-affinity purification (Figure 2A) after which it was cleaved with Factor Xa and subjected to heparin-affinity FPLC. Uncleaved fusion protein and 10 kDa CTGF were eluted from the heparin column by, respectively, 0·5 M NaCl (fractions #7–9) and 0·8 M NaCl (fractions #16–19) (Figure 2B), the www.endocrinology.org

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Figure 5 Heparin- and EDTA-dependence of 10 kDa CTGF-mediated adhesion in multiple cell types. The indicated cell lines were incubated alone (open bars) or in the presence of either 5 µg/ml heparin (grey bars) or 10 mM EDTA (black bars) in U-bottom non-tissue culture wells that had been pre-coated with 3 ng or 9 ng 10 kDa CTGF. Values shown are mean S.D. of triplicate determinations of the number of adherent cells.

latter of which is an identical elution position as that of the native 10 kDa CTGF protein (Brigstock et al. 1997). Both the fusion protein and 10 kDa cleavage product supported adhesion of 3T3 cells to non-tissue culture plastic (Figure 2B). C8 reverse-phase HPLC of the 0·8 M NaCl eluate resulted in the separation of several CTGF proteins in the 8–10 kDa range, all of which promoted 3T3 cell adhesion (Figure 2C). N-terminal sequence analysis of the most abundant 8 kDa and 10 kDa proteins demonstrated that they both commenced at Glu247 (Figure 3) suggesting that the 8 kDa protein was C-terminally truncated. HPLC-purified 10 kDa CTGF promoted 3T3 cell adhesion in a dose-dependent manner, with maximal binding at 8 ng/well (Figure 4). 10 kDa CTGF also promoted the dose-dependent adhesion of several additional cell types including endothelial, epithelial, and myofibroblastic cells (Figure 5). The binding of each cell type to 10 kDa CTGF was completely inhibited by 5 µg/ml heparin (Figure 5), suggesting that cell surface heparan sulfate proteoglycans (HSPGs) act as adhesion receptors for 10 kDa CTGF. In view of the recent findings that full-length CTGF or CYR61 bind to cell-surface integrins and that this process is dependent on divalent cations (Lau & Lam 1999), we next tested the effect of EDTA on 10 kDa CTGF-mediated cell adhesion. Binding of all cell types to 10 kDa CTGF was substantially reduced by EDTA treatment, though 3T3 cells were less affected as compared with the other cell types (Figure 5). www.endocrinology.org

Discussion Although CTGF and other CCN proteins were recognized as modular proteins almost a decade ago (Bork 1993), there has been very little progress in understanding the functional significance of their constituent modules, either collectively or individually. This aspect of CCN biology has been confounded by the discovery that the ‘4-module’ structure is not rigorously conserved in all CCN proteins. For example, WISP-2 completely lacks module 4 (Zhang et al. 1998, Kumar et al. 1999) while module 2 is absent in a short form of WISP-1 called WISP1 v (Tanaka et al. 2001). Additionally, N-terminal truncations have been described that result in the absence of module 1 or modules 1 and 2 from NOV (Joliot et al. 1992, Perbal 1999) or in the absence of modules 1 and 2 or modules 1, 2, and 3 from CTGF (Brigstock et al. 1997, Ball et al. 1998). Although the biological significance of this structural heterogeneity remains largely unexplored, it may be a means of generating variants that exhibit a diverse range of agonistic and antagonistic activities, as well as bioavailabilites. Based on the structure of their constituent modules, CCN proteins are predicted to bind to a diverse variety of other molecules (Bork 1993). Among the interactions that have been identified are partnering of integrins with CTGF or CYR61 (Lau & Lam 1999), low density lipoprotein-related protein with CTGF (Segarini et al. 2001), heparin with CTGF or Journal of Endocrinology (2003) 176, R1–R7

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CYR61 (Kireeva et al. 1996, Brigstock et al. 1997, Kireeva et al. 1997, Chen N. et al. 2000, Grzeszkiewicz et al. 2002), insulin-like growth factors with CTGF or NOV (Kim et al. 1997, Burren et al. 1999) and fibulin 1C, RNA polymerase II, Notch, or S100A4 (mts1) calcium binding protein with NOV (Perbal 1999, Perbal et al. 1999, Li et al. 2002, Sakamoto et al. 2002). Our data show that a 10 kDa recombinant form of CTGF produced in E. coli exhibits comparable physicochemical and biochemical properties as its native counterpart that arises from proteolysis of the intact molecule in utero (Brigstock et al. 1997, Ball et al. 1998). This protein, corresponding essentially to module 4, contained sufficient structural information to support adhesion of four cell types, a phenomenon that was shown to be heparindependent and one which likely reflects the heparinbinding properties of the 10 kDa CTGF molecule. The EDTA-sensitive cell adhesion data are consistent with recent data showing that full length CTGF or CYR61 utilize integrin V3 to promote endothelial cell adhesion or integrin 61 to promote adhesion of fibroblasts or vascular smooth muscle cells (Kireeva et al. 1996, Kireeva et al. 1997, Kireeva et al. 1998, Babic et al. 1999, Shimo et al. 1999, Chen N. et al. 2000, Chen C. C. et al. 2001, Grzeszkiewicz et al. 2002). In the latter case, cell surface HSPGs act as co-receptors that are essential for CYR61-integrin 61 interactions (Chen N. et al. 2000, Grzeszkiewicz et al. 2002). Although the precise location and inter-dependence of the heparin-binding and integrin-binding sites in CTGF will require detailed studies of mutant proteins, our data suggest that module 4 alone contains both types of binding site. While this does not rule out the possibility that, in full length CTGF, the repertoire of protein-protein interactions is modified through the presence of additional binding sites in one or more of the other modules, module 4 appears to be a fundamentally important functional unit in the CTGF protein. The somewhat lower sensitivity of 3T3 cells to EDTA treatment may relate to the specific integrin subunits involved or the presence of additional nonintegrin binding sites for module 4 on the surface of this cell type. Additional structural and functional studies of the modules, both individually and collectively, will provide important insight into the mechanisms by which CTGF regulates cell function. Acknowledgements We thank Laurey Steinke Ph.D. (Protein Structure Core Facility, University of Nebraska Medical Center, Omaha, NE, USA) for assistance with protein sequencing. This work was supported by USDA grant 98–35206–6430 awarded to DRB and by a research award from FibroGen Inc in which DRB has an equity interest. DKB was supported in part by NIH grant 2 T32 CA09498–11 awarded to FM Robertson. Journal of Endocrinology (2003) 176, R1–R7

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Recombinant production and action of CTGF module 4 · Kireeva M, Lam S & Lau L 1998 Adhesion of human umbilical vein endothelial cells to the immediate- early gene product Cyr61 is mediated through integrin alphavbeta3. Journal of Biological Chemistry 273 3090–3096. Kumar S, Hand A, Connor J, Dodds R, Ryan P, Trill J, Fisher S, Nuttall M, Lipshutz D, Zou C, Hwang S, Votta B, James I, Rieman D, Gowen M & Lee JC 1999 Identification and cloning of a connective tissue growth factor-like cDNA from human osteoblasts encoding a novel regulator of osteoblast functions. Journal of Biological Chemistry 274 17123–17131. Lau L & Lam S 1999 The CCN family of angiogenic regulators: the integrin connection. Experimental Cell Research 248 44–57. Li C, Martinez V, He B, Lombet A & Perbal B 2002 A role for CCN3 (NOV) in calcium signalling. Molecular Pathology 55 250–261. Liu J, Kosma V, Vanttinen T, Hyden-Granskog C & Voutilainen R 2002 Gonadotrophins inhibit the expression of insulin-like growth factor binding protein-related protein-2 mRNA in cultured human granulosa- luteal cells. Molecular Human Reproduction 8 136–141. Moussad E & Brigstock D 2000 Connective tissue growth factor: what’s in a name? Molecular Genetics and Metabolism 71 276–292. Moussad E, Rageh M, Wilson A, Geisert R & Brigstock D 2002 Temporal and spatial expression of connective tissue growth factor (CCN2; CTGF) and transforming growth factor beta type 1 (TGF-beta1) at the utero-placental interface during early pregnancy in the pig. Molecular Pathology 55 186–192. Perbal B 1999 Nuclear localisation of NOVH protein: a potential role for NOV in the regulation of gene expression. Molecular Pathology 52 84–91. Perbal B, Martinerie C, Sainson R, Werner M, He B & Roizman B 1999 The C-terminal domain of the regulatory protein NOVH is sufficient to promote interaction with fibulin 1C: a clue for a role of NOVH in cell- adhesion signaling. PNAS 96 869–874. Perbal B 2001 NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues. Molecular Pathology 54 57–79. Rageh M, Moussad E, Wilson A & Brigstock D 2001 Steroidal regulation of connective tissue growth factor (CCN2;CTGF) synthesis in the mouse uterus. Molecular Pathology 54 338–346 Sakamoto K, Yamaguchi S, Ando R, Miyawaki A, Kabasawa Y, Takagi M, Li C, Perbal B & Katsube K 2002 The Nephroblastoma Overexpressed gene (NOV/ccn3) protein associates with Notch1 extracellular domain and inhibits myoblast differentiation via Notch signaling pathway. Journal of Biological Chemistry 277 29399–29405. Sampath D, Winneker RC & Zhang Z 2001 Cyr61, a member of the CCN family, is required for MCF-7 cell proliferation: regulation by 17 beta-estradiol and overexpression in human breast cancer. Endocrinology 142 2540–2548. Sampath D, Zhu Y, Winneker R & Zhang Z 2001 Aberrant expression of Cyr61, a member of the CCN (CTGF/Cyr61/ Cef10/NOVH) family, and dysregulation by 17 beta-estradiol and

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