Stimulation of Fibroblast Cell Growth, Matrix Production, and Granulation Tissue Fortnation by Connective Tissue Growth Factor Ken Frazier, Shawn Williams, Devashish Kothapallj, Helene Klapper, and Gary R . Gwtendorst 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 pulse labeling of cells with [35S]methionine. Subcu­ 38-kDa peptide that is selectively induced by trans­ taneous injection of TGF-J3 and CTGF into neonatal forming growth factor-J3 (TGF-J3) in fibroblastic cell NIH Swiss mice resulted in a large stimulation of types. We compared the biologic activities of CTGF granulation tissue and fibrosis at the site of injection. with TGF-J3 on fibroblasts in culture and in animal In sitll hybridization studies revealed that TGF-J3 models of fibroplasia. CTGF was active as a mitogen injection induced high levels of CTGF mRNA in the in monolayer cultures of normal rat kidney fibro­ dermal fibroblasts at the injection site, demonstrat­ blasts. CTGF did not stimulate anchorage-indepen­ ing that TGF-{3 can induce the expression of CTGF in dent growth ofNRK fibroblasts, however, or inhibit connective tissue cells ill vivo. No CTGF transcripts the growth of mink lung epithelial cells, distinguish­ were detected in the epidermal cells in either control ing CTGF's growth-regulatory activities from those or TGF-J3-injected skin or in fibroblasts in control ofTGF-J3. In NRK fibroblasts, both TGF-J3 and CTGF (saline-injected) skin. These results demonstrate that, significantly increased the transcripts encoding at like TGF-J3, CTGF can induce connective tissue cell type I collagen, as integrin, and fibronectm. Stimu­ proliferation and extracellular nlatrix synthesis. Key lation of type I collagen and fibronectin protein words: TGF-J3/collagell/wolUld 1.epairlfi/wosis. J Invest Del'­ synthesis by TGF-J3 and CTGF was confirmed by IIIatol 107:404-411, 1996 issue regeneration and repair proceed in a cascade fibroblast proli feration (DeLarco and Todaro, 1978; Assoian cf ai, fas hion beginning w ith a coagulati o n and infl amma­ 1984; Leof c/ ai, 1986; Soma and Grotendo rst, ] 989; Ishikawa e/ ,II, tOl'y phase, fo ll owed by granulation tissue formation 1990; Battegay e/ ai, 1990); (ii) elevated synthesis of extracellular and fi nally extraceIJul ar matrix deposition and termj­ matrix components inclu diJlg fibronecrin, typc I collagen, ill.tcgrins, nation of the respo nse. Peptide growth factors playa laminin, and glycosaminoglycalls productio n (Ignotz and Mas­ ceTntral role in this process. ft is li kely that f.1ctOI·S released by sague, '1986; Roberts eI ai, 1986; R aghow ct ai, 1987; Varga ct ai , pl atelets and inflamm atory cell s serve as initiators of the regenera­ 1987; Penttinen ct ai, 1988), and (iii) decrcased degradatio n of tion/repair response. Similarly, wound repair disorders, as wcll as extracellular m atrix due to direct inhibition of protease activity and organ-specifi c fibrosis, may be ca used by dysfunctional cascades. stimulation of the synthesis of protease iJlhibito rs (Laiho et ai, 1986; O ne of the principal regula tory factors that appears to function as an Lund et ai, 1987; Kerr ct ai, 1990). Previous studies have demon­ ini tiator in these processes is transforming growth factor-{3 strated that 'I large po rtion of the TGF-,B induction of matrix (TGF-(3) (Amcnto and Beck, 1991; Raghow, 1991; Wahl, 1992; protein synthesis is not shared by other growth factors such as R.oberts and Spo.", 1993). fibroblast growth factor (FGF) o r platelet deri ved growth factor TG F-{3 has been sho wn to act as a potent stimulatory signal for (PDGF) (Ignotz and Massague, 1986; R o berts el ai, 1986; Penttinen connective tissue formation during wound repair and in fibrotic et ai, 1988). conditions. Elevated TGF-{3 mRNA or protein Icvels have becn Connecti ve tissue growth factor (CTGF) is a cysteine-rich documented in tissue during normal wound repair (I garashi eI (II, mitogenic peptide that was o ri gin ally identificd as a growth factor 1993; Levine e ( ai, 1993), and fib rotic disorders ofch e skjn (Kulozik secreted by vascular endothe li al cell s in culture (Bradham el a/. et ai, 1990; Peltonen ct ai, 1990; Smith and LeRoy, 1990) and 1991 ). CTGF is selectively induced in fibroblasts aftel' acti vation internal organs and tissues (Nagy c ( ai, 1991 ; Kagami e/ ai, 1993; with TGF-{3 (Soma and Grotcndo rst, 1989; Igarashi ef ai, 1993). Bahadori et ai, 1995). T he increased fi brotic tiss ue has been CTGF is a m ember of a tamily of peptides that include serum­ attributed to severa l actions of TGF- {3, including: (i) increased induced gene products ceftO (Simmons et ai, 1989), cyr61 (O'Bri en et ai, 1990), flsp1 2/ {31G M 1 (Brunner el ai, 1991; R yseck et ai , '1991) Manuscript received February 23, 1996; revised April 29, 1996; accepted and a chi cken transforming gene, nov Oo li ot et 1992). CTGF fo r publication May 21.1 996. ai, Reprint requests to: Dr. Gary R . Grotcndorst, Department of Cdl also shares significant sequence homology w ith a Drosophila gene Biology and Anatomy, University of Miami School of Medicin e (R-124), product, twisted gastrulation (twg) (Mason et ai, 1994), that 1600 NW 10th Avenue, Miami, FL 33'136. determines cell fates during do rsa l/ventral pa ttern formation in the Abbreviations: CTGF, connective ti ss ue growth fa ctor; r TGF, recom­ cmbryo. Previo us studies have dcmonstratcd coordinate expression binant connective tisslI e growth flletor. of TGF-,Bl and CTGF in granul;ltion tiss ue bcds during wound 0022-202X/96/S10.50 • Copyri ght lid 1996 by T he Society for In vestigative Dermatology, Ill c. 404 VOL. 107, NO.3 SEPTEMB ER 19% e T C I: STIMULATION OF FIBROPLASIA 405 repair (Igarashi el n/, 1993) and found th at d e rmal fibroblasts in 49F fibroblasts as ta rget cell s as described previously (Soma and Groten­ sc1et-o d erma lesions overexpress CTGF (Ig arashi c( (/ 1, 1995). T h e do rst, 1989). Anchorage-independent growth assays 'were pcrfornlcd essen­ CTGF mRNA is selectively induced in fibroblasts b y TGF- {3, but tia ll y as described by Guadagno and Assoian (1991). not by oth e r g rowth fa c tors, su c h as epiderm a l growth f.,cto r Growth Inhibition Assays T he mink lung epithelial cell line. Mv1Lu (EGF), PDGF, or FGFs (acidic FGF, basic .FGF) (Igarashi el n/, (ATTC No. CCL 64), was used as C<l.rget for growth-inhibitory assays with 1993)- Prote in synthesis is n ot required for TGF-{3 stimulation of TGF-{3 and CTGF. Assays were perfomled as described by Ogawa and CTGF gen e expressio n (Igarashi eI aI, 1993; G rotendorst c( ai, Seyedin (1991). 1996), indicating that the CTGF gen e is directly regulate d by Extracellular Matrix Protein mRNA Induction Assays NRK rat TGF-/3- We h ave now ide n tifi e d a n ovel TGF-/3 resp o n se e le m e nt fibro blasts 'were g ro\vn to conRucn cc in DMEM \~' i th 50/0 feta] b ovin e serun1 (T/3RE) in th e CTGF promoter (Grotendorst c( n/, 1996), w hic h and then serum starved in DMEM with 1% bovine serum albumin for 24 h. begins to define the molecular basis for the selective regulation of Growth f.,cto rs were added to the cell cultures; tota'! cellular RNA was tills gene by TGF-/3_ T h e T/3RE seque n ce present in th e human and extracted after 24 h, and northern blot analysis was perfonn ed as described mou se CTGF genes is not prese nt in the promote rs o f other previously (I garas hi el ai, 1993)_ To ensure that equ.ivalent amo unts of total TGF-f3-regulate d gene that h as b een d escribed to d a te . RNA were added to each lane on a gel, RNA was quantitated by AU ,O / 2S" ratios. and equi valent transfer was ass UJ:ed by comparing ribosom al 28S and Becau se of the pleiotropic action s of TGF-/3 on cells and the 18S RNA bands in each lane after stai ning with ethi dium bromide. As an selective induction ofCTGF by TGF-/3 in fibroblasts, w e wanted to additional con trol, blo ts we re reprobed with an actin cDNA probe. c ompare th e biolog ic activities of CTGF with TGF-/3- Until n ow, Double- stranded cDNA fi'agments used for probes were labeled with the limite d availability of n at m al CTGF made it difficult to p e rform ,n pjdCTP using a ra ndom prime labeling kit (Boehri nger Mannheim. studies o n the biolog ic ac tions of thc m o lecule_ We h ave produced Indianapolis. IN). T he CTGF probe was derived frOI11 a l.l - kb human recombinant CTGF to address this questio n . O ur studies indicate cDNA tragment that encompassed the open reading frame of the CTGF that r ecombina n t CTGF is mitogenic for NRJ< fibroblasts in transcript. T he TGF- f31 probe w as a 1.0-kb Narr fragment derived fro m a monolayer c ulture. TGF- /3 in th e presen ce of EGF and serum is 2.0-kb human TGF-f31 cDNA presen t (G.