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

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