Stimulation of Fibroblast Cell Growth, Matrix Production, and Granulation Tissue Fortnation by Connective Tissue Growth Factor

Ken Frazier, Shawn Williams, Devashish Kothapalli, Helene Klapper, and Gary R. Grotendorst
Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida, U.S.A.

Connective tissue growth factor (CTGF) is a 36- to 38-kDa peptide that is selectively induced by transfornUng growth factor-fJ (TGF-fJ)in fibroblastic cell types. We compared the biologic activities of CTGF with TGF-J3 on fibroblasts in culture and in animal models of fibroplasia. CTGF was active as a mitogen in monolayer cultures of normal rat kidney fibroblasts. CTGF did not stimulate anchorage-independent growth ofNRI{ fibroblasts, however, or inhibit the growth of mink lung epithelial cells, distinguishing CTGF's growth-regulatory activities from those ofTGF-J3. In NRK fibroblasts, both TGF-J3 and CTGF significantly increased the transcripts encoding 0:1 type I collagen, as integrin, and fibronectin. Stimulation of type I collagen and fibronectin protein synthesis by TGF-J3 and CTGF was confirmed by

pulse labeling of cells with [35S]methionine. Subcutaneous injection of TGF-J3and CTGF into neonatal NIH Swiss resulted in a large stimulation of granulation tissue and fibrosis at the site of injection. Itt siuc hybridization studies revealed that TGF-J3 injection induced high levels of CTGF mRNA in the dermal fibroblasts at the injection site, demonstrating that TGF-{3 can induce the expression of CTGFin connective tissue cells ill vivo. No CTGF transcripts were detected in the epidermal cells in either control or TGF-{3-injected skin or in fibroblasts in control (saline-injected) skin. These results demonstrate that, like TGF-I3, CTGF can induce connective tissue cell proliferation arrd extracellular rrratzix synthesis. Key words: TGF-J3lcollagenlwomld repairlfihrosis. J Invest Dermatol. 107:404-411, 1996

issue regeneration and repair proceed in a cascade fashion beginning with a coagulation and inflammatOI), phase, folJowed by granulation tissue formation and finally extracellulnr matrix deposition and termination of the response. Peptide growth factors play a central role in this process. It is likely that factors released by platelets and inflammatory cells serve as initiators of the regeneration/repait response. Similarly, wound repair disorders, as well as organ-specific fibrosis, may be caused by dysfunctional cascades. One of the principal regulatory factors that appears to function as an initiator in these processes is rransforming growth factor-{3 (TGF-f3) (Arnento and Beck, 1991; Rnghow, 1991; Wahl, 1992; Roberts and Sporn, 1993). TGF-,B has been shown to act as a potent stimulatory signal for connective tissue formation during wound repair and in fibrotic conditions. Elevated TGF-f3 mRNA or protein levels have been documented in tissue during normal wound repair (Igarashi et al, 1993; Levine et al, 1993), and fibrotic disorders of the skin (Kulozik et ill, 1990; Peltonen et ai, 1990; Smith and Lek.oy, 1990) and internal organs and tissues (Nagy et ill, 1991; Kagami et al, 1993; Bahadori et ill, 1995). The increased fibrotic tissue has been attributed to several actions of TGF-,B, including: (i) increased Manuscript received February 23, 1996; revised April 29, 1996; accepted for publication May 21, 1996. . Reprint requests to: Dr. Gary R.. Grotendorst, Department of Cell Biology and Anatomy, University of Miami School of Medicine (R-124), 1600 NW 10th Avenue, Miami, FL 33136. Abbreviations: CTGF, connective tissue growth factor; rCTGF, recombinant connective tissue growth factor.


fibroblast proliferation (Del.arco and Todaro, 1978; e( ill, 1984; Leof el ill, 1986; Soma and Grorcndorst, 1989; Ishikawa dill, 1990; Battegay ct ill, 1990); (ii) elevated synthesis of extracellular matrix components including fibronectin, type I collagen, inregrins, laminin, and glycosarninoglycans production (Ignotz and Massague, :1986; Roberts ct al, 1986; Raghow dill, 1987; Varga et. III, 1987; Penttinen ct ai, 1988), and (iii) decreased degradation of extracellular matrix due to direct inhibition of protease activity and stimulation of the synthesis of protease inhibitors (Laiho et al, 1986; Lnnd et al, 1987; Kerr ct ill, 1990). Previous studies have demonstnated that '.1 large portion of the TG F-{3 induction of matrix protein synthesis is not shared by other growth factors such as fibroblast growth factor (FGF) or platelet derived growth factors (PDGF) (Ignotz and Massague, 1986; Roberts e/ ill, 1986; Penttinen et ai, 1988). Connective tissue growth factor (CTGF) is a cysteine-rich mitogenic peptide that was originally identified as a growth factor secreted by vascular endothelial cells in culture (Bradham et ill, 1991). CTGF is selectively induced in fibroblasts after activation with TGF-{3 (Soma and Grotendorst, 1989; Igarashi e( ill, 1993). CTGF is a member of a family of peptides that include seruminduced gene products ceflO (Simmons et ai, 1989), cyro l (O'Brien et al, 1990), flsp12/ {3IG Ml (Brunner et ai, 1991; Ryscck et Ill, 1991) and 11 chicken transforming gene, nov (joliet et al, 1992). CTGF also shares significant sequence homology with a Drosopliiln gene product, twisted gastrulation (twg) (Mason et ill, 1994), that determines cell fates during dorsal/ventral pattern formation in the embryo. Previous studies have demonstrated coordinate expression of TGF-/31 and CTGF in granulation tissue beds during wound


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© 1996 by The Society for Investigative




MD). based ct nt.800 ng ofEGF (n = 8) or ng of recombiuanr CTGF (n = 38). Om studies indicate that recomb-inant CTGF is mitogenic for Nl~K fibroblasts in monolayer culture.9-kb EwIU/Hi"dlII fragment derived 6'0111 a 2. and northern blot analysis was performed as described previously (Igarashi et al. 1994). 1. Slides were then developed and sections counter stained in Mayer's hematoxylin and eosin. .A260/2S0 ratios. 100. Growth Inhibition Assays The mink lung epithelial cell line. Assoian. indicating that the CTGF gene is directly regulated by TGF-/3. For large scale production of recombinant CTGF (rCTGF).0% paraforrnaldehyde for 1.fixation.) [II situ hybridization for CTGF mRNA was performed using previously described methods (Pava et al. . CAl. Purified murine EGF was purchased from Biomedical Technologies. Medical Insritutc. S'v" fetal bovine serum. 1984. Mitogenic and Anchorage-Independent Growth Assays Mitogenic assays were performed in monolayer cultures using 48-well plates and NRK Stfmularlon of Matrix Protein Synthesis Cell cultures were grown to . cultures were 35 pulse labeled 'with 50 {LCi [ S]med1ionille per 1111 in methionine-free DMEM for 2 h in the presence of the indicated growth factor. CollaborutivcP . but not by other growth factors. Bedford. 1996). csearch.5 X :J 0" ce:llsper ml and infected with rCTGF baculovirus at an MOl = 7. The t1'5 intcgrin probe was produced from a cDNA insert containing a portion of the human eDNA containing the open reading frame (R. total cellular RNA was extracted after 24 . The TGF-{31 probe was a Lfl-kb IVm:! fragment derived 6'0111 <J 2. '1. 107.h of NIU( fibroblasts (DeLarco and Todaro. Double-stranded cDNA fragments used for probes were labeled with 2 p]dCTP using a random prime labeling kit (Boehringer Mannhcim. Type I collagen was analyzed by overnight pepsin digestion (5 JLgper ml in 'I M acetic acid) in the presence oflO JLgof carrier bovine type I co llagen. described by Laernmli ~1970). basic FGF) (Igarashi et al. Protein synthesis is not required for TGF-J! stimulation of CTGF gene expression (Igarashi et al.MD) with 3. Cultures were then placed in DMEM containing 100 JLgbovine serum albumin pcr 1111 and either 10 ug TGF-{3 per ml. MD) and maintained in Dulbeccos modified Eagle's medium (DMEM) with 5'){.400. NO. 1993). transferrin. Cell proteins were extracted from the cell pellet with 0.ction ofCTGF by TGF-f3 in fibroblasts. blots were reprobed with an actin cDNA probe. Subcutaneous Administration N1J-l Swiss mice were injected in JLIof total volume of either saline (II = 15).The Tf3RE sequence present in the human and mouse CTGF genes is not present in the promoters of other TGF-/3-regulafed gene that has been described to date. H.1 % Triton Xl 00. Lenexa. such as epidermal growth factor (EGF). MATERIALS AND METHODS 49F fibroblasts as target cells as described previously (Soma and Grotendorst. 1991) are not shared by CTGF. 61323). Gaithersburg. The CTG!' probe was derived from a ·. CTGF does not share TGF-f3's ability to stimulate anchorage-independent growth of normal fibroblasts or act as a growth inhibitor for epithelial cells.1-kb human eDNA fragment that encompassed the open reading frame of the CTGF transcript. polyacrylamide gels containing SDS as. and of Growth Factors to Mice Neonatal the nape of the neck daily for 3 d with 20 control (n = 12). We have now identified a novel TGF-{:l response element (T/31lE) in the CTGI' promoter (Grotendorst et al. Growth factors were added to the cell cultures. High Five cells (Invirrogcu.fetal bovine serum and 10 JLg gentamicin per 1111. Inc. 1990). Samples used for the. Immunobloccing was performed by electroblotting the proteins in the acrylarnide gel to a nitrocellulose membrane as described previously (Soma and Grotendorst. which begins to define the molecular basis for the selective regulation of tins gene by TGF-{3. and selenium (ITS. High Five cells were grown in shaker cultures to a density 0(3. cotrausfcctcd with linearized wild-type AcMNPV into Sf') cells according to prescribed techniques (Ausubel "I nl. Extracellular fibroblasts Matrix Protein mRNA Induction Assays NRK rat 'were grown to conAucncc in DMEM with 50/0 feral bovine serum and chenserum starved in DM.VOL. Recombinant CTGF was produced in the laboratory using a bncculoviral expression system employing the plslucbac II transfer plasmid (Invitrogen) containing the CTGF open rending frame. 19-89).h.i1l1.L Bell.200. MA). 20 ng CTGF per rnl. Indianapolis.1. Grotcridorst et al . confluence. San Diego. As an additional control. MA) for 18 h. was used as targer for growrh-inhibitory assays with TGF-/3 and CTGF. NR:I. 5 'v" Nuscrum (Collaborative Biomedical. MvlLu (ATTC No. or FGFs (acidic FGF. To ensure that equivalent amounts of total RNA were added to each lane on a gel. MD. We have produced recombinant CTGF to address this question. (GIBCO.l11assie staining of sodium dodecyl sulfate (SDS)-polyacrylamidc gel electrophoresis gels. and samples were autoradiographed. CAl were grown in shaker and monolayer culture and maintained in ExCel1405 serum-free insect cell media (JRH Bioscience. 3. Sections were prepared for histopathologic examination after routine formalin.33 g lactalbumin hydrolysate per 1111. KS). (Stoughton. 1995). RNA was quantitated by .% Pluronic F68 (spinner cultures only)]. Gel Electrophoresis and Imrrruuoblorrtng Electrophoresis was performed using 12'X. and leupeptin for 10 min. processing. or nothing (negative control) and incubated for 22 h. St. 50. Bedford. CCL 64).EM with 1% bovine serum albumin for 24 h.samples of dennis and subcutis were taken 6:01l1 the site of injection 8 h after the second injection. 10 JLg gentamicin per rnl. III Siru Hybridization NIH Swiss neonatal mice were injected as described aboveexcept that. Because of the pleiotropic actions of TGF-f3 on cells and the selective iudu. C Cell Cultures Sf9 cells were grown in spinner and monolayer culture and maintained in TNM-FH media JGracc's Insect Culture Media. and equivalent transfer was assured by comparing ribosomal 28S and 18S RNA bands in each lane after staining with ethidiurn bromide. Assays were performed as described by Ogawa and Scycdin (1991).33 g yeastolatc per rnl.C49F and Mv1Lu cells were obtained from American Type Culture Collection (Rockville. Until now. Ogawa and Seyedin.5 h and then flashfrozen and embedded. 400 or 800 ng ofTGF-/3 BB (n = 12). RESULTS Production of Recomb ill ant CTGF Recombinant CTGF was produced using a baculoviral vector system that contained the open reading frame of the human CTGI' transcript under the regulation . Polysciencc) and incubated for 8 d at 4°C. Animals were sacrificed 24 h biopsies of the area containing and large surrounding the injection site were removed. The human actin (control RNA) probe was purchased from Oncor Co. or 800 on a previous protocol (Roberts after the last injection.3 SEPTEMBER 1996 CTCF STIMULATION OF FlBROPLASIA 405 repair (Igarashi et til. 1989). SDS-polyacrylamide gel electrophoresis was performed. we wanted to compare the biologic activities of CTGF with TGF-f3. MO). Ma). Samples were assayed for endotoxin using an endotoxin enzymeIinkcd immunosorbent assay kit (Sigma. 1993. (Gaithersburg. the limited availability of natural CTGF made it difficult to perform studies on the biologic actions of the molecule.ionine were used in each sample.H. These results demonstrate that CTGF stimulates fibroblast proliferation and extracellular matrix synthesis in a manner similar to TGF-{3. University of Miami). IN). Fibronecrin was immunoprecipitared using Protein A-SephaJ"ose using prescribed methods (Roberts et al.5 111M phenylmethylsulfonyl fluoride. The CTGF mRNA is selectively induced in fibroblasts by TGF:"'{3. 1993). PDGF. the growth-inhibitory actions ofTGF-f3 on epithelial cells (Tucker et al . After this time. Sources of Growrh Factors TGF-J3 isolated from platelets was a gift from Richard Assoian (University of Miami). University of Chicago). and staining with Mayer's hcmatoxvlin and eosin. Peak fracrions containing rCTGF were determined by immunoblotting and COO. The recombinant CTGF was purified using heparin Sepharose affinity chromatography. Louis.2-kb eDNA clone containing the 3' region of the open reading frame provided by F. 0. 1990). CTGF induces elevated expression and synthesis of both collagen and fibronectill in fibroblasts in culture and induces fibroplasia in the skin of newborn mice. and O. 1988). Recombinant platelet-derived growth factor EE was obtained fi:0111 Chiron (Emeryville. Woessner (University of Miami).. Gaithersburg.0-kb human TGF-J31 cDNA present (G. TGF-f3 in the presence of EGF and serum is capable of stimulating anchorage-independentgl'owt. then serum-starved in DMEM and insulin. 1996). 1978.. Sections were cur at 5 JLI11 and placed on TESPAcoated slides (Oucor. 20. Anchorage-independent gr-owth assays were performed essen- tially as described' by Guadagno and Assoian (1991).11TUnoprec::ipitation and pepsin digestion were adjusted so that equivalent amounts of total incorporated C5S]meth. These tissue samples were immediately placed in 4. Also.5-kh open reading frame fragment at the 3' end (AT'CC No. The slides were dipped in photographic emulsion (Ilford K-5. 1993) and found that dermal fibroblasts in sc1er:oderma lesions overexpress CTGF (Igarashi et ai. but CTGF does not substitute for TGF-{3 in this assay.986). The cd. The human fibronectin probe was a O. 200 or 800 ng ofPDGF 10.-type 1 human collagen probe was derived from a J. Assoian ct al.

The purity of the rCTGF is demonstrated in lane 2 of the fiji pond where 5 /Lgof rCTGF are compared to Bio-Rad protein standards (5 /Lg/ea). Both of these peptides are A '70 -e)( B 100 '7 0 C -+ Heparin( 10 ug/ml) '7 75 E >< ~ 30 -a. Q.. . . Because CTGF exhibited a high affinity for heparin. Comparison ofrCTGF with CTGF secreted by human skin fibroblasts (HSF) after activation with TGF-/3 and human umbilicalvein endothelial cells (HUVE) and rPDGF BB (right panel]. WilHams 5. The indicated amounts of CTGF were added to the media alone (0) or in the presence of EGF (0.) or EGF (0. A large synergistic stimulation occurred with low concentrations ofEGF. we examined the effects of heparin 011 CTGF activity. o 20 "4 10 01'" 0 -2 --- CTGF 20 ng/ml - -0- No Additions 4 10 20 30 40 50 60 00 25 50 [CTGF] (ng/mll [CTGFJ (ng/mll [TGF-BJ (ng/ml) Figure 2. The doublet pattern observed in the recombinant material is similar to that of the natural product isolated from human skin fibroblast or endothelial cell-conditioned medium (Fig 1).). 1984).i:. LL en o fo. 110 other growth factor has been found to possess this unique activity.406 FRAZIER ET ilL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY so ~ u. Gel electrophoretic analysis of CTGF. 0 l- 2o I- o W U.. mean:': SD. . As shown in Fig 1.Lg heparin per ml to the medium resulted in a significant increase in the amount of mitogenic activity exhibited by CTGF (3-fold over non=beparin-treated) with no significant change in the concentrations of CTGF required to give half-maximal or maximal stimulation of DNA synthesis (Fig 2B). (B) Effectof heparin on the mitogenic activity of CTGF on NRK cells. Mitogenic activity of CTGF. 1984).. as has been. -0- No Additions 75 100 125 i >. 0). based on amino acid cornposmon and peptide sequence analysis. The indicated amounts of CTGF were added alone (0) or in the presence of heparin (10 peg per 1111. of the baculoviral polyhedron gene promoter. Inability of CTGF to Stimulate Anchorage-Independent Growth or Act as a Growth Inhibitor for Epithelial Cells TGF-f3 was originally identified due to its ability to stimulate the anchorage-independent growth of normal fibroblasts (Delarco and Todaro. (A) Mitogenic activity of CTGF alone and in the presence of EGF for NRK fibroblasts.2 ng per ml.) Synergism ofTGF-B1 and EGF but not CTGF to stimulate DNA synthesisof NRK cells in monolayer cultures. The CTGF is greater than 95'Yc. Peptides were detected after transfer to nitrocellulose by an anti-human PDGF IgG and an alkaline phosphatase-conjugated second antibody as described in Materials alld Methods. 1978).. with a maximal stimulation of 6-fold over non-treated cultures at concentrations of 20-50 ng rCTGF per 1111(Fig 2A). Cells Were assayed as in A.<: I- >< E 0.. All samples represent approximately 10 ng of CTGF or PDGF. two peptides are detected that co-migrate with bands detected in a parallel immunoblot performed on the same material.<: l- E o Q. The presence ofCTGF stimulated only a slight increase in the ability of TGF-f3 to induce DNA synthesis in these cultures (the arroul in the figure indicates baseline of CTGF activity with no TGF-/3 present). TGF-/3 alone exhibited little activity in onr DNA synthesis assay.. CTGF stimulated a concentration-dependent increase in DNA synthesis in the monolayer cultures. . Segarurn P.2 ng/ml w . . . Addition of 10 /. ill the presence ofCTGF (20 ng per ml.. A'TOIII points to baseline of CTGF stimulation. CTGF.2 ng per ml. LL u.. >. This activity was enhanced by the presence of small amounts of EGF (Fig 2A). The purity of the rCTGF was determined by SDS-PAGE and staining with Coornassie Blue K-250. we compared the activity of TGF-/3 alone or in combination with either EGF or CTGF in the NIUZ monolayer mitogenic assay (Fig 2C). Lastly. The indicated amounts of pure TGF-/3were added alone (0). although less than that observed when EGF was added with TGF-f3 (Fig 2C).. 1978. but that there is no synergistic activity between TGF-f3 and CTGF in this 'Issay. rCTGF produced in High Five cells by a bacculovirus expression system was purified by affinity chromatography on heparin Sepharose. CTGF was purified by a one-step isolation procedure using hcparin-Sepharose affinity chromatography as described in Materials and Methods. The basis for the multiple bands appears to be due to differences in glycosylation as detected by differences in reactivity with concanavalin A (Grotendorst GR. previously reported (Delarco and Todaro. While other growth factors are required for cell division under these conditions (Assoian e/ al. o u. i >. unpublished observations).. TGF-/3 has also been shown to act as a specific inhibitor of epithelial cells 9745- 302114Coornassie R-250 Imrnunoblot anti-PDGF Figure 1. > o .. rJ) ::::> C U .. . Cells were assayed as described in Materials and Methods. and because previous studies have demonstrated that heparin-binding growth factors such as acidic FGF can exhibit much higher mitogenic activity in tbe presence of heparin.EGF 0. Error bars..<: IM o -. I I oo l- C) I- rn m a. Assoian et at. pure based on quantitative scans of the Coornrnassic-stained gels. These data suggest that at least some part of the synergistic action ofTGF-f3 and EGF is shared by CTGF and EGF. . Mitogenic Activity of CTGF We initially tested the biologic activity of the CTGF with NRK fibroblasts using a mitogenic assay performed on monolayer cultures.

5 5.//Ods.pare the activity of CTGF with that of TGF-/3.2 12-fold levels of induction (400 to 1200%). lOng TGF-{3 per rnl .0 2.'scribed . The results of these studies demonstrate that both type I collagen and fibronectin synthesis were stimulated by TGF-/3 and CTGF compared to control cultures. Norchenn blot analysis for extracellular matrix protein in cultured fibroblasts. Northern blot analysis was then performed on totall(_NA a. Confluent monolayer cultures of N1U{ fibroblasts were stimulated for 24 h with either TGF-{3 (10 ng per ml) or CTGF (20 ng per 111. These data. NO.1) pulse labeled for 2 It with [. The rate of synthesis of collagen and fibronectin was then examined in both TGF-{3.pare the ability ofCTGF with TGF-/3 to elevate mRNA coding for cd type I collagenvfibronectin. Stimulation of fibroncctin and type I collagen synthesis by TGF-f3 and CTGF. TGF-/3 stimulated the growth of NRK fibroblasts in a concentration-dependent manner (Fig 3A). and the CTGF transcript was induced 20-fold (2000%) after TGF-{3 treatment compared to control cultures. or 10 ng rCTGF per ml for 24 h.5 10. Control experiments examining ribosomal RNA by ethidium bromide staining indicated that equivalent amounts of total RNA were present in each of the samples. En'or bars. fibroblasts and inhibition of growth of MvlLu lung epithelial cells.3 0. As expected.VOL. fibronectin. Quantitative densitometric analysis of the films indicate that.s:: a. Two separate experiments were performed and arc indicated as experiments 1 and 2.and CTGF-treated monolayers ofNRK fibroblasts.1 0. Fibronectin and collagen synthesis was determined as described in Materiats and Methods. whereas CTGF had 110 effect on the growth of the lung epithelial cell line (el. TGF-{3 also stimulated a large increase in the level of CTGF transcripts consistent with our previous observations. Figure 5. We wanted to com. Takehara et al. mean ± SD. TGF-{3 was a potent and effective inhibitor of proliferation of these lung epithelial cells in monolayer culture. (B) Growth inhibition assays were performed as described by Ogawa and Seyedin (J 991). followed by SDS-polyacrylamide gel electrophoresis and autoradiography (Fig 5). Type I collagen and fibronectin were followed by either immunoprecipitation (fibronectin) or pepsin digestion (collagen). addition of CTGF with TGF-/3 did not augrnent the activity found with TGF-{3 alone. Figure 3. based on the TYPE I COLLAGEN FIBRONECTIN 0< 5 INTEGRIN CTCF ACTIN Figure lllRNA NO ADD TGF-B CTGF Experiment # Fibronectin 121212 TYPE I collagen 4. (A) Anchorage-independent growth assays were performed essentially as described by Guadagna anti Assoian (1991). ]. Quantitative scans of the blots demonstrate a variation of ± 15% in the actin signal. fibronectin. Treatment of cells with TGF-{3 and CTGF induced a marked upregulation of cd -type I collagen. CTGF had 110 activity in this assay. Cells were starved in serum-free DMEM containing ITS for 18 h. both in an ancl~orage-il1dependent growth assay with NRK fibroblasts and a growth inhibition assay with Mv l Lu epithelial cells.07. (Tucker et al. The transcript for as integrin was induced 3-fold by TGF-/3 and nearly IS-fold by CTGF. whereas CTGF had no effect either as a growth inhibitor or growth stitnulator. and integrin genes in fibroblasts. TGF-{3 caused a concentration-dependent inhibition of the growth of the Mv l Lu cells (0). which served as a second control for RNA loading. 1984). No detectable changes in CTGF message were observed in the CTGFtreated cultures. Muller et al.4 0. Mntcrials and 1I11'i.I: C. Elevation of Matrix Protein Transcripts and Matrix Protein Synthesis by rCTGF TGF-/3 stimulates the expression of collagen. 0 "0 -"O-TGF-~ 'u « [Growth Factor] (ng/rnl) 7. TGF-{3 induced anchorage-independent growth in a conccntration- '" . A similar outcome occurred in the growth-inhibitory assay using MvlLu cells as targets (Fig 3B). 1987). demonstrate that CTGF cannot substitute for TGF-/3 in either of these assays and indicate that TGF-/3 exhibits biologic activities that are distinctfrom those of CTGF. Confluent cultures of N1U{ fibroblasts were placed in serum-free media for 24 h prior to the addition of either buffer (control lane 1). .15S]l11crhiolline (50 !LCi per ml) in and methionine-free media. 3 SEPl'EMBER '1996 CTGF STIMULATION OF HBROPLASLA 407 A B "' « 0 0. and vascular endothelial cells (Heimark et al. 1986. cells. Effect of TGF-f3 and CTGF on anchorage-independent growth of NRK.0 -e--CTGF '" -S 11) Ql .0 [Growth Factor] (ng/l11l) dependent manner (0) whereas CTGF did not (e). The matrix protein-encoding genes (0'1 type I collagen and fibronectin) exhibit 4. We wanted to com. Arrows indicate fibroucctin (lap 1'([1Ic1) or collagen a-chains (botton: pancl}. and as integrin transcripts as detected by northern blot analysis (Fig 4). or as integrin in the monolayer cultures of NRK.

(D) 800 ng TGF-~. Epidermis (E). . 200 /-LU!. Scale bar. (A) saline. (B) 800 ng rPDGF BB. (E) 400 ng rCTGF daily for 3 d. dermis (D). hair follicles (F). and granulation tissue (G) are indicated.408 FRAZIER ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Figure 6. Mice were injected one time daily for 3 d and the tissue was harvested and processed as described in Materials alld Methods. Skin and subcutis from NIH Swiss neonatal mice after injection of saline control or growth factors. (e) 800 ng EGF.

III sit" hybridization for'CTGF tnRNA in TGF~fl-jnjected neonncal muninc skin and subcutis.andCTGF-injected mice and continued to enlarge throughout the remainder of the experimental protocol. We compared the actions of TGF-{3.fibroplasia in the specimens from the TGF-. and CTGF in this illl. Prolonging the interval from the last injection to sacrifice and histologic examination to 7 d in both theTGF-f3. CTGF. (A) TGF-f3 il\jectedmouse skin. hair follicles. 107. or PDGF. sense-orientedCTGF negative control (no signal IS detected).signal IS detected).il!o model of fibrosis and wound rep au' (Fig 6). The area of granulation tissue was measured in slides from the mice injected with TGF-/3. Injection of PDGF BB at 800 ng/site pro- .on for CTGF message on sections of the subcutis obtained fr0111 saline. The average area of. III situ hybridizationusingCTGF riboprobes in both antisense (A) and (B) and sense (C) orientationswas performedunder high stringencyconditionsfollowedby'an 8-d exposure prior to development of the emulsion (see Materials. TGI~-. Both TGF-{3 and CTGF induced similar alterations of the normal tissue.S-fold increase in collagen synthesis. No CTGF transcripts were present in either noninjected skill (not shown) or saline control-injected skin sites (Fig 7B). (C) TGF-f3-ulJectedmouse 5k111. Hybridization controls of TGF-.n parallel.. and the connective tissue in these tissue samples had an appearance similar to tbat of saline-injected controls. Fibroplasia Induced by Subcuraneous Injection of Growth Factors The injection of TGF-{3 into the dermal/subcuticular area of skin in neonatal NIH Swiss mice induces a rapid and dramatic increase in the formation of granulation tissue primarily composed of connective tissue cells and large amounts of extracellular matrix (Roberts et al. The tissue fr0111 the injection site was harvested and prepared 8 h after the second injection. The EGF-injected specimens. alld Methods).B injection Ul mice. gross nodules were apparent in both the TGf-{3. Vivo.and TGF-{3-illjected mice (Fig 7). EGF.B-injected mice was 28. situ hybridizati.VOL. was 3. The amount of granulation tissue formed at sites injected with 800 ng of CTGF was consistently less than in those with 400. O. however.roll~Spoint to somestr.B-injected dermis when hybridization was performed using an antisense CTGF riboprobe (Fig 7A). Lower concentrations of CTGF induced significant granulation tissue formation with some effect detectable at amounts as low as 50 Ilg per injection. granulati. we performed ill. Animals were injected with 800 ng ofTGF-Bl followed by a second injection 24 h later. After two injections. CTGF stimulated a S-fold increase in both fibronectin and. A strong signal was detected in the TGF-.B-injected tissue sections with sense strand CTGF riboprobes were negative.. which is consistent with the previously reported dose response for TGF-. NO.Bstimulated a 3-fold increase in the synthesis of fibronecrin and a 2. TGF-f3 Induction of CTGF mRNA in Skin Fibroblasts III.and C'I'Gl--injecred mice resulted in resorption of granulation tissue. with respect to the induction of m~ltrix production. F. G. (B) An~senseCTGF probe. 111 saline-injected mouse skin (no. 1986). similar to that produced by CTGF levels of 50 rig/site. TGF~Bl (800 ng) was subcutaneously injected into the nape of the neck of neonatal NJH Swissmice daily (sec Materials IIlId Methods).duced only a mild granulation tissue response. Scale bar. ng/site. Thus. Control experiments using saline only were performed i. with no significant background hybridization (Fig 7C). exhibited hyperkeratosis of the epidermis and degenerative changes in the hair follicular epithelium. To determine whether the CTGF transcript was induced in the fibroblasts participating in the formation of the granulation tissue induced by TGF-{3 injection.B and distinct from other growth factors such as PDGl~ or FGF. and in . including large increases in the number of connective tissue cells and extracellular matrix material. Injection of EGF did not result in detectable fibroplasia. CTGF has actions that are similar to the PDGF-injected specimens it. The only cells expressing the CTGF transcripts were connective tissue cells .4 1111112.ontissue.8 n11112. Dosage studies indicated that the optimal concentration of CTGF for induction of fibroplasia was 400 ng/site compared with 800 ng/site for TGF-l3. the C'TGf=injected mice it was 22.3 SEPTEMBEIJ.ongly positivefibroblasts granulatiou tissue.4 mm ". collagen. 50 Mm. demonstrating that the EGF was biologically active and produced effects on the epidermal cells at the sites of injection. PDGF BB. average of the two experiments. 1996 CTGF STIMULATION OF FIBROPLASIA 409 Figure 7.

CilM 1'0'"1115)'1111' 57:115-129. KF.410 FRAZlER et AL THE JOURNAL OF INVESTIGATIVE DER. fibrotic liver disease. Kingston I'-E.1984 Ausubel PM. RQssR: Transforming grQwf. 1986. 1990). 1993) helps to functionally link the inflammatory phase (where TGF-{3 is expressed) and the granulation tissue formation phase (where CTGF is expressed) of this complex process.h factor beta induces bimodal proliferation of connective tis. such as PAI-1. 1989). These observations support a cascade model for connective tissue formation. nor does it act to inhibit the growth of epithelial cells whose growth is inhibited by TGF-{3. and growth factors must act alone or in concert with factors that are constitutively present in interstitial fluids. Peltonen et ai. Consequently. Por example. it is difficult to determine how much active TGF-/3 is present in a tissue. Because the methods employed to effectively extract growth factorsfrom tissue also convert latent TGF-{3 to the active form. 1995). Seidman JG. This may be a part of the mechanism for TGF-{3 stimulation of connective tissue cell growth. such as scleroderma. the pleotropic actions ofTGF-{3 make it well suited to act as . Furthermore. It is also consistent with the observations that the expression of the TGF-_B and CTGF genes are coordinately linked during normal wound repair. other genes. unpublished observations). unpublished observations). agents that may play important roles during wound healing would not necessarily stimulate a large amount of connective tissue formation in the injection model. Most of the TGF-{3 in tissue and produced by cells in culture is in this latent form (Flaumenhaft et al . 1990 Balmdori L. Thus. The mitogenic activity of CTGF is augmented by EGP and heparin but not by TGF-/3. Previous studies have indicated that TGF-{3 can stimulate the growth of various fibroblastic cell types by the induction ofPDGF genes (Lcof et ai. Current Protocols ill Moicrniar i3io/"SY. Cei! 63:5"15-524. Furthermore. ~r REFEREN CES Arnenro EP. Ishikawa et al. 1990 Bradham OM. the presence of transcripts does not always correlate well with the amount of active TGF-/3 present (Flanders et al. In this regard. the type of tissue induced by PDGF injection has much less extracellular matrix than the tissue induced by TGF-/3 or CTGF injection. In: I-I Piwuicn-Worms. Smith and LeRoy. Grotcndorst GR: Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial ccl. The restricted actions of CTGF suggest this peptide functions as an autocrine and paracrine signaling molecule to maintain and perhaps amplify and synchronize the response of fibroblastic cells in the tissue. This research 'JltlS sllpported by Notiollal Institutes of Healtl.1. '1991 1 Assoian R. Thus. DISCUSSION The data from our studies indicate that CTGF acts to stimulate cell division and extracellular matrix production by NlU{ fibroblasts in culture. is slipported Ill' Nntionnl lnstitntes <1Hea/l" Traillillg Grallt IUW7057-0. Inc. only a minimal amount of trauma occurs at the site. One significant problem with linkil.g the presence of TGF-/3 transcripts in a tissue withT'GF-{3 activity is that TGF-{3 is made in a latent form that must be processed to obtain the active protein (Lawrence et al. New York. Pircher et al. Thus. i995 Batteguy Ej. we find high levels of expression ~!f the CTGF gene ( is related to the SRC-induccd immediate early t. 2..) Purification and transfcctirm of bnculoviral DNA tor generating rcccrubi11:U1t viruses. suggesting a causative association between TGF-{3 and the increased deposition of collagen. So-uh! K (eds. which results in increased synthesis of these proteins. During the course of examination of the expression of growth factor genes in fibrotic disorders we have found CTGF expression to be closely linked to the expression of TGF-{3. Surprisingly. 1994). In summary. Brent R. Cralll CM] 7223. lgurshi A.Cl1C product CEP-'llJ. CTGF appears to mimic some of the actions of TGF-/3 with regard to the stimulation of connective tissue cell growth and the synthesis of extracellular matrix but is not a substitute for TGF-_B in other assays. 'Moore DD.1 146: 1140-1149. Wiley Inrcrscicncc. Seifert itA. 1985). Milder J. The restricted activity of CT'GF on fibroblasts is consistent with tills model. whose transcription is stimulated by TGF-{3 are equally induced by other growth factors such as PDGF (Grotendorst. This may explain the PDGF and EGF results reported here. In tills model. We have not found factors other than TGF-{3 that induce a significaut level of expression of the CTGF gene. alld a gralltfrolll FibroCell.K. Beck LS: TGF-{3 and wound healing./ A 1 alit! (III illstitlltional fellowship FOIil tlie Ullil/ersill' Mialll.'/ Cell Biol l'I4:12851294. 1990. Miller DV. These results indicate that the principal source of CTGF ill 1Ji-IJO are connective tissue cells activated with TGF-{3. 1986. 1993). although the CTGF gene is constitutively expressed by vascular (human umbilical vein) endothelial cells in culture. Odekon et al.". initiators acting during the acute response to injury induce factors whose function is to maintain the repair process and allow for the maturation of the forming connective tissue. Nonetheless. The coordinate expression of TGF-/3 and CTGF during the wound repair cascade (Igarashi et nl. these data indicate that CTGF acts to stimulate fibroblastic cell proliferation and extracellular matrix synthesis. With respect to matrix protein synthesis.10. (to C. 11. however. shared by normal and pathologic processes. These constraints make it all the more remarkable that CTGF induces the formation of connective tissue in a manner that closely resembles that induced by TGF-{3. Smith JA.R. Compared to other known growth factors. we find that CTGF stimulates an increase in the expression of o t-type 1 collagen and fibronectin mRNAs.. CTGF does not substitute for TGF-{3 in anchorageindependent growth assays. it is attractive to speculate that CTGF may function as an autocrine mediator for some of the biologic actions of TGF-{3 on fibroblasts. Injection of CTGF into the skin of neonatal mice stimulates the formation of a matrix-rich granulanion tissue. Raines lEW. 1990.B: Cellular transformation by coordinated action of three peptide growth factors from hurnnn platelets Nnfllre 309:804 -806. C. We feel there is a significant physiologic difference between the injection model [initially described by Roberts et a! (1986) and employed in the studies described here] and wound models. Battegay et al.MATOLOGY (fibroblasts). Because CTGP is selectively induced in connective tissue cells by TGF-{3. growth factors added to wound models in animals with normal healing act in an environment containing a milieu of factors whose primary functions are to stimulate tissue regeneration and repair. Cells in the deeper regions of the skin such as adipocytes were also negative for CTGF transcripts in both control and TGF-/3-injected skin sites. Sporn M. CTGF's biologic actions are most similar to those of TGF-{3. Gold L. and human atherosclerotic plaques.-8. Bowen-Pope DP./ p"tll. a devtlopmenta! tllllardFolil the Svlvcste: Callcer Cell/. 1990. cells via complex sue control of an autocrinc platelet-derived growth factor loop. Overexpression of TGF-{3 also has been reported in scleroderma lesions (Kulozik et al... whereas ill IJiIJO studies indicate that PDGF alone can accelerate granulation tissue formation in normal animals (Grotendorst et al. Grorendorst it does not appear that PDGF plays a direct role in the stimulation of extracellular matrix protein production induced by TGF-{3 (Penttinen et al. Our previous studies on fibroblastic cells in culture and the data reported in this manuscript demonstrate that the expression of CTGF is dependent 011 the presence of active TGF-{3. 1990). Berney M: Active macrophage associated TGF-~ co-localizes with type 1 procollagcu gene expression in atherosclerotic human pulmonary arteries. 1988). we did not detect CTGF transcripts in clearly identifiable endothelial cells in either large vessels or capillaries. 1991 .). 1988). In the injection model. Potter RL. CTGP is overexpressed in fibroblasts in the dermis of patients with scleroderma (Igarashi et ai. No cells in the epidermis were positive for CTGF transcripts in either control or TGF-{3-injected skin. Vol. a systemic fibrotic disorder characterized by the overproduction of collagen (LeRoy et ai.U1 initiator of the repair process.. pp )6. In contrast. 1985. These studies suggest that the presence ofCTGF transcripts may be useful as a marker for the presence of active TGF-{3 in a tissue. Such observations support the possibility that CTGF may playa role in TGF-{3-mediated formation of granulation tissue. in a wide variety of fibrotic conditions in which TGF-{3 is found to be expressed.

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