In normal adult fibroblasts, transforming growth factor-␤ (TGF␤) induces the expression of connective tissue growth factor (CTGF). CTGF independently promotes fibroblast proliferation and matrix deposition, and in acute models of fibrosis promotes cell proliferation and collagen deposition acting synergistically with TGF␤. In contrast to normal fibroblasts, fibroblasts cultured from fibrotic tissues express high basal levels of CTGF, even in the absence of added TGF␤. Induction of transcription by TGF␤ requires the action of SMAD proteins. In this report we have investigated the role of SMADs in the TGF␤-induction of CTGF in normal fibroblasts and in the elevated levels of CTGF expression found in dermal fibroblasts cultured from lesional areas of patients with scleroderma, a progressive fibrotic disorder that can affect all organs of the body. We have identified a functional SMAD binding site in the CTGF promoter. TGF␤-induction of CTGF is dependent on SMAD3 and SMAD4 but not SMAD2 and is p300-independent. However, mutation of the SMAD binding site does not reduce the high level of CTGF promoter activity observed in dermal fibroblasts cultured from lesional areas of scleroderma patients. Conversely, the previously termed TGF␤RE in the CTGF promoter is required for basal CTGF promoter activity in normal fibroblasts and for the elevated level of CTGF promoter activity in scleroderma fibroblasts. Thus, the maintenance of the fibrotic phenotype in scleroderma fibroblasts, as visualized by excess CTGF expression, appears to be independent of SMAD-dependent TGF␤ signaling. Furthermore, given CTGF's activities, the high level of CTGF expression observed in scleroderma lesions may contribute to the excessive scarring observed in this disorder.

The endothelins are a family of endothelium-derived peptides that possess a variety of functions, including vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and promotes myofibroblast contraction and migration, hence contributing to matrix remodeling during tissue repair. Here, we show that addition of ET-1 to normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including ␣-smooth muscle actin (␣-SMA), ezrin, moesin, and paxillin. We confirm that ET-1 enhances the ability of lung fibroblasts to contract extracellular matrix, a function essential for tissue repair, through induction of de novo protein synthesis. Blockade of the Akt/ phosphoinositide 3-kinase (PI3-kinase) pathway with LY294002 and wortmannin prevents the ability of ET-1 to induce ␣-SMA, ezrin, paxillin, and moesin and to promote matrix contraction. Dominant negative rac and Akt blocked the ability of ET-1 to promote formation of ␣-SMA stress fibers. Using specific ET-1 receptor inhibitors, we show that ET-1 induces collagen matrix contraction through the ETA, but not the ETB, receptor. Relative to normal pulmonary fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease systemic sclerosis (scleroderma) show enhanced ET-1 expression and binding. Systemic sclerosis lung fibroblasts show increased ability to contract a collagen matrix and elevated expression of the procontractile proteins ␣-SMA, ezrin, paxillin, and moesin, which are greatly reduced by antagonizing endogenous ET-1 signaling. Thus, blocking ET-1 or the PI3-kinase/Akt cascades might be beneficial in reducing scar formation in pulmonary fibrosis. INTRODUCTIONA complex histological and architectural structure is a prerequisite for effective lung function. In the lung, specialized structures, the alveoli, increase the surface area of the lung, allowing for efficient gas exchange. The maintenance of these specialized structures is in turn dependent on the underlying connective tissue, comprised principally of fibroblasts and extracellular matrix (ECM; for review, see Gadek et al., 1984), which is essential for the mechanical and structural integrity of the lung. As a response to environmental insults, or as a consequence of local inflammatory processes, structural damage to the lung can occur, resulting in a wound healing response. This response consists of an integrated series of biochemical, immunological, and structural changes that result in the de novo synthesis of a new epithelium, blood vessels, and connective tissue (Razzaque and Taguchi, 2003). The proper repair of connective tissue requires synthesis of new ECM components, such as collagen and fibronectin (Badylak, 2002). In addition, repair of connective tissue requires the proper reconstitution of its support function; that is, an appropriate tensile strength must be recreated. This tensile strength results from the remodeling of the newly formed ECM through a combination of cell locomotion and translocation of the flexible collage...

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Connective tissue growth factor (CTGF) is overexpressed in a variety of fibrotic disorders, presumably secondary to the activation and production of transforming growth factor-␤ (TGF-␤), a key inducer of fibroblast proliferation and matrix synthesis. The CTGF gene promoter has a TGF-␤ response element that regulates its expression in fibroblasts but not epithelial cells or lymphocytes. Recent studies have shown that the macrophage-produced cytokine tumor necrosis factor ␣ (TNF␣) is necessary to promote inflammation and to induce genes, such as matrix metalloproteinases, involved with the early stages of wound healing. In this study, we examined the ability of TNF␣ to modulate CTGF gene expression. TNF␣ was found to suppress the TGF-␤-induced expression of CTGF protein in cultured normal fibroblasts. The activity of TNF␣ was blocked by NF-B inhibitors. We showed that sequences between ؊244 and ؊166 of the CTGF promoter were necessary for both TGF-␤ and TNF␣ to modulate CTGF expression. There was a constitutive expression of CTGF by scleroderma fibroblasts that was increased by TGF-␤ treatment. Although TNF␣ was able to repress TGF-␤-induced CTGF and collagen synthesis both in normal and scleroderma skin fibroblasts, fibroblasts cultured from scleroderma patients were more resistant to TNF␣ as TNF␣ was unable to suppress the basal level of CTGF expression in scleroderma fibroblasts. Thus, we suspect that the high level of constitutive CTGF expression in scleroderma fibroblasts and its inability to respond to negative regulatory cytokines may contribute to the excessive scarring of skin and internal organs in patients with scleroderma.

This study examines endothelin-induced modulation of extracellular matrix synthesis and remodeling by fibroblasts, and its potential role in the pathogenesis of systemic sclerosis (scleroderma). Endothelin-1 promoted fibroblast synthesis of collagen types I and III, but not fibronectin, by a mechanism dependent upon both ETA and ETB receptors. Conversely, endothelin-1 inhibited both protein expression of matrix metalloproteinase 1 and zymographic activity exclusively via ETA receptors. A dual regulatory role for endothelin-1 in transcriptional regulation was suggested by the ability of endothelin-1 to enhance steady-state levels of collagen mRNA and activate the proalpha2(I) collagen (Col1a2) promoter, but in contrast to reduce matrix metalloproteinase 1 transcript expression and suppress transcription of a human matrix metalloproteinase 1 promoter reporter construct in transient transfection assays. Although endothelin-1 significantly enhanced remodeling of three-dimensional collagen lattices populated by normal fibroblasts, this was not observed for lattices populated by systemic sclerosis fibroblasts. Promotion of matrix remodeling was dependent upon ETA receptor expression and was blocked by specific inhibitors of tyrosine kinases or protein kinase C. Reverse transcriptase polymerase chain reaction, S1 nuclease, and functional cell surface binding studies showed that normal and systemic sclerosis fibroblasts express both ETA and ETB receptors (predominantly ETA), but that ETA receptor mRNA levels and ETA binding sites on fibroblasts cultured from systemic sclerosis skin biopsies are reduced by almost 50%. Endothelin-1 is thus able to induce a fibrogenic phenotype in normal fibroblasts that is similar to that of lesional systemic sclerosis fibroblasts. Moreover, reduced responsiveness to exogenous endothelin-1 in systemic sclerosis suggests that downstream pathways may have already been activated in vivo. These data further implicate dysregulated endothelin-receptor pathways in fibroblasts in the pathogenesis of connective tissue fibrosis.

In vivo, CCN2 (connective tissue growth factor) promotes angiogenesis, osteogenesis, tissue repair, and fibrosis, through largely unknown mechanisms. In vitro, CCN2 promotes cell adhesion in a variety of systems via integrins and heparin sulfate proteoglycans (HSPGs). However, the physiological relevance of CCN2-mediated cell adhesion is unknown. Here, we find that HSPGs and the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) mitogenactivated protein kinase cascade are required for adult human dermal fibroblasts to adhere to CCN2. Endogenous CCN2 directly binds fibronectin and the fibronectin receptors integrins ␣4 ␤1 and ␣5 and syndecan 4. Using Ccn2؊/؊ mouse embryonic fibroblasts, we show that loss of endogenous CCN2 results in impaired spreading on fibronectin, delayed ␣-smooth muscle actin stress fiber formation, and reduced ERK and focal adhesion kinase phosphorylation. These results suggest that a physiological role of CCN2 is to potentiate the ability of fibroblasts to spread on fibronectin, which may be important in modulating fibroblast adhesion to the provisional matrix during tissue development and wound healing. These results are consistent with the notion that a principal function of CCN2 is to modulate receptor/ligand interactions in vivo. INTRODUCTIONHuman skin, the largest organ in the body, acts as a protective barrier between the internal organs and the external environment (Chuong et al., 2002). The barrier function of skin is provided by both the epidermis, which consists of extracellular lipid-like substances and intracellular insoluble keratin (Fuchs and Byrne, 1994), and the dermis, which consists of fibroblasts that produce extracellular matrix (ECM; Kielty and Shuttleworth, 1997).As a response to environmental insults or trauma, or as a consequence of local inflammatory processes, structural damage to skin can occur. The resultant wound-healing response consists of an integrated series of biochemical, immunological, and structural changes culminating in the de novo synthesis of new epithelia, blood vessels, and connective tissue (Werner and Grose, 2003). During this process, fibroblasts synthesize new ECM components, such as collagen and fibronectin (Badylak, 2002). In addition, fibroblasts proliferate and repopulate the wound. These fibroblasts attach to, remodel, and contract the newly synthesized ECM, providing the tensile strength required for the support function of the dermis (Clark, 1985). Thus, understanding the mechanism underlying the ability of fibroblasts to adhere to ECM components such as fibronectin is a necessary prerequisite to understanding how the new dermis and other connective tissues are being synthesized.Connective tissue growth factor (CCN2), a member of the CCN family of matricellular proteins (Bork, 1993;Lau and Lam, 1999;Moussad and Brigstock, 2000;Perbal, 2001;, is a cysteine-rich protein that is secreted via a 37-amino acid signal sequence (Chen et al., 2001b). Secondary sequence structure predictions have suggested that CC...

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Objective. No therapy for fibrotic disease is available. The proadhesive matricellular protein connective tissue growth factor CCN2 is a marker of fibrotic cells; however, the specific role of CCN2 in connective tissue biology in general and in fibrogenesis in particular is unclear. The aim of this study was to assess whether adult mice bearing a smooth muscle cell/fibroblastspecific deletion of CCN2 are resistant to bleomycininduced skin scleroderma.Methods. Cutaneous fibrosis was induced in mice by subcutaneous injection of bleomycin. Untreated control groups were injected with phosphate buffered saline. Mice bearing a fibroblast/smooth muscle cellspecific deletion of CCN2 were investigated for changes in dermal thickness, collagen content, and the number of ␣-smooth muscle actin (␣-SMA)-positive cells. Dermal fibroblasts were isolated to assess whether the induction of collagen and ␣-SMA messenger RNA in response to transforming growth factor ␤ (TGF␤) was impaired.Results. The loss of CCN2 resulted in resistance to bleomycin-induced skin fibrosis. In response to bleomycin, wild-type mice possessed, but CCN2-deficient mice lacked, abundant ␣-SMA-expressing myofibroblasts within fibrotic lesions. Fibroblast responses to TGF␤, a potent inducer of myofibroblast differentiation, were not affected. Collectively, these results indicate that CCN2 is essential for bleomycin-induced skin fibrosis, likely due to a defect in myofibroblast recruitment.Conclusion. These data indicate that therapeutic strategies that involve blocking CCN2 in vivo may be of benefit in combating fibrotic skin disease.