Transforming growth factor-beta (TGF-beta) stimulates the transcription of the alpha2(I) procollagen gene (COL1A2). The intracellular mediators involved in this response remain poorly understood. In this study, we demonstrate that primary human skin fibroblasts express Smads, a novel family of signaling molecules, in vitro in the absence of TGF-beta. The levels of Smad 7 mRNA was rapidly and transiently increased by TGF-beta. Transient overexpression of Smad 3 and Smad 4, but not Smad 1 or Smad 2, caused trans-activation of a CAT reporter gene driven by a 772 bp segment of the human COL1A2 promoter containing putative TGF-beta response elements. Smad stimulation of promoter activity was ligand independent, but was further enhanced by TGF-beta. Overexpression of a phosphorylation-deficient Smad 3 mutant or wild-type Smad 7, which lacks the carboxy-terminal phosphorylation motif, specifically inhibited TGF-beta-induced activation of COL1A2 promoter. A CAGACA sequence shown to be a functional Smad-binding element in the plasminogen activator inhibitor-1 gene promoter was found within the TGF-beta-response region of the proximal COL1A2 promoter. Gel mobility shift assays showed protein phosphorylation-dependent binding activity in fibroblast nuclear extracts specific for this sequence; TGF-beta treatment strongly stimulated the formation of this DNA-protein complex. Smad was identified as a component of the CAGACA-binding transcription complex in TGF-beta-treated fibroblasts by antibody supershifting. These results demonstrate that (i) Smad 3 transmits TGF-beta signals from the receptor to the COL1A2 promoter in human fibroblasts, and is likely to play an important role in stimulation of COL1A2 promoter activity elicited by TGF-beta; (ii) in fibroblasts, Smads appear to function as inducible DNA-binding transcription factors; and (iii) Smad 7 may be involved in autocrine negative feedback in the regulation of COL1A2 promoter activity by TGF-beta.

Among the extracellular signals that modulate the synthesis of collagen, transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma) are preeminent. These two cytokines exert antagonistic effects on fibroblasts, and play important roles in the physiologic regulation of extracellular matrix turnover. We have shown previously that in normal skin fibroblasts, TGF-beta positively regulates alpha2(I) procollagen gene (COL1A2) promoter activity through the cellular Smad signal transduction pathway. In contrast, IFN-gamma activates Stat1alpha, down-regulates COL1A2 transcription, and abrogates its stimulation induced by TGF-beta. The level of integration of the two pathways mediating antagonistic collagen regulation is unknown. We now report that IFN-gamma abrogates TGF-beta-stimulated COL1A2 transcription in fibroblasts by inhibiting Smad activities. IFN-gamma appears to induce competition between activated Stat1alpha and Smad3 for interaction with limiting amounts of cellular p300/CBP. Overexpression of p300 restored COL1A2 stimulation by TGF-beta in the presence of IFN-gamma, and potentiated IFN-gamma-dependent positive transcriptional responses. In contrast to fibroblasts, in U4A cells lacking Jak1 and consequently unable to activate Stat1alpha-mediated responses, IFN-gamma failed to repress TGF-beta-induced transcription. These results indicate that as essential coactivators for both Smad3 and Stat1alpha, nuclear p300/CBP integrate signals that positively or negatively regulate COL1A2 transcription. The findings implicate a novel mechanism to account for antagonistic interaction of Smad and Jak-Stat pathways in regulation of target genes. In fibroblasts responding to cytokines with opposing effects on collagen transcription, the relative levels of cellular coactivators, and their interaction with regulated transcription factors, may govern the net effect.

Transforming growth factor-b (TGF-b) stimulation of Type I collagen gene (COL1A2) transcription involves the Smad signal transduction pathway, but the mechanisms of Smad-mediated transcriptional activation are not fully understood. We now demonstrate that the ubiquitous transcriptional coactivators p300 and CREB-binding protein (CBP) enhanced basal as well as TGF-b-or Smad3-induced COL1A2 promoter activity, and stimulated the expression of endogenous Type I collagen. The adenoviral E1A oncoprotein abrogated stimulation of COL1A2 activity in transfected ®broblasts, and reduced the basal level of collagen gene expression. This e ect was due to speci®c interaction of E1A with cellular p300/CBP because (a) a mutant form of E1A defective in p300 binding failed to abrogate stimulation, and (b) forced expression of p300/CBP restored the ability of TGF-b to stimulate COL1A2 promoter activity in the presence of E1A. The e ect of p300 on COL1A2 transcription appeared to be due, in part, to its intrinsic acetyltransferase activity, as stimulation induced by a histone acetyltransferasede®cient mutant p300 was substantially reduced. Transactivation of COL1A2 by p300 involved the Smad signaling pathway, as Smad4-de®cient cells failed to respond to p300, and stimulation was rescued by overexpression of Smad4. Furthermore, minimal constructs containing only the Smad-binding CAGACA element of COL1A2 were transactivated by p300 in the presence of TGF-b. These results indicate, for the ®rst time, that the multifunctional p300/CBP coactivators play a major role in Smad-dependent TGF-b stimulation of collagen gene expression in ®broblasts.

Objective. In fibroblasts, transforming growth factor ␤ (TGF␤) stimulates collagen synthesis and myofibroblast transdifferentiation through the Smad intracellular signal transduction pathway. TGF␤-mediated fibroblast activation is the hallmark of scleroderma and related fibrotic conditions, and disrupting the intracellular TGF␤/Smad signaling may provide a novel approach to controlling fibrosis. Because of its potential role in modulating inflammatory and fibrotic responses, we examined the expression of the nuclear hormone receptor peroxisome proliferator-activated receptor ␥ (PPAR␥) in normal skin fibroblasts and its effect on TGF␤-induced cellular responses.Methods. The expression and activity of PPAR␥ in normal dermal fibroblasts were examined by Northern and Western blot analyses, immunocytochemistry, flow cytometry, and transient transfections with reporter constructs. The same approaches were used to evaluate the effects of PPAR␥ activation by naturally occurring and synthetic ligands on collagen synthesis and ␣-smooth muscle actin (␣-SMA) expression. Modulation of Smad-mediated transcriptional responses was examined by transient transfection assays using wild-type and dominant-negative PPAR␥ expression constructs. Abnormal synthesis and tissue accumulation of collagen are hallmarks of scleroderma and are responsible for the damage and failure of affected organs. Lesional scleroderma fibroblasts display an activated phenotype characterized by accelerated transcription of genes coding for collagen and other extracellular matrix proteins, increased expression of cell surface receptors for transforming growth factor ␤ (TGF␤), and sustained production of TGF␤, connective tissue growth factor, Supported by grants from the NIH (AR-46390 and AR-42309) and the Scleroderma Foundation. Results

Transforming growth factor-␤ (TGF-␤

Objective. Scleroderma is characterized by excessive synthesis and accumulation of matrix proteins in lesional tissues. Transforming growth factor ␤ (TGF␤) plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of fibroblasts; however, the underlying mechanisms are poorly understood. We undertook this study to examine the expression and function of SMADs, recently characterized intracellular effectors of TGF␤ signaling, in scleroderma fibroblasts.Methods. Primary dermal fibroblasts obtained from 14 patients with scleroderma and from 4 healthy adult volunteers were studied. Northern analysis was used to determine the expression of endogenous SMAD messenger RNA (mRNA), and Western analysis was used to determine SMAD protein expression. Intracellular compartmentalization of cellular SMAD proteins in the presence and absence of TGF␤ was studied by antibody-mediated immunofluorescence confocal microscopy. The effect of TGF␤ blockade on SMAD subcellular distribution was determined using anti-TGF␤ antibodies as well as a dominant-negative TGF␤ receptor type II (TGF␤RII) vector to disrupt TGF␤ responses. SMAD-regulated luciferase reporter expression was examined to investigate the potential functional significance of activation and nuclear accumulation of endogenous SMADs in scleroderma fibroblasts.Results. Protein and mRNA levels of SMAD3, but not of SMAD4 or SMAD7, were variably elevated in scleroderma fibroblasts compared with those from healthy controls. In sharp contrast to control fibroblasts, which displayed predominantly cytoplasmic localization of SMAD3/4 in the absence of exogenous TGF␤, in scleroderma fibroblasts SMAD3 and SMAD4 consistently showed elevated nuclear localization. Furthermore, phosphorylated SMAD2/3 levels were elevated and nuclear localization of phosphorylated SMAD2/3 was increased, suggesting activation of the SMAD pathway in scleroderma fibroblasts. Blockade of autocrine TGF␤ signaling with antibodies or by expression of dominant-negative TGF␤RII failed to normalize SMAD subcellular distribution, suggesting that elevated nuclear SMAD import was due to alterations downstream of the TGF␤ receptors. The activity of a SMADresponsive minimal promoter-reporter construct was enhanced in transiently transfected scleroderma fibroblasts.Conclusion. This study is the first to demonstrate apparently ligand-independent constitutive activation of the intracellular TGF␤/SMAD signaling axis in scleroderma fibroblasts. SMAD signaling may be a mechanism contributing to the characteristic phenotype of scleroderma fibroblasts and playing a role in the pathogenesis of fibrosis.Scleroderma is associated with fibrosis of affected tissues due to excessive synthesis and progressive accumulation of connective tissue macromolecules (1). Scleroderma fibroblasts display features of sustained activation in vitro and in vivo. In culture, these cells show elevated synthesis of collagens, fibronectin, proteoglycans, and tissue inhibitors of metalloproteinases, constitutive secretion of i...

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Transforming growth factor-␤ (TGF-␤) plays a key role in scleroderma pathogenesis. The transcription factor early growth response-1 (Egr-1) mediates the stimulation of collagen transcription elicited by TGF-␤ and is necessary for the development of pulmonary fibrosis in mice. Here, we report that TGF-␤ causes a time-and dose-dependent increase in Egr-1 protein and mRNA levels and enhanced transcription of the Egr-1 gene via serum response elements in normal fibroblasts. The ability of TGF-␤ to stimulate Egr-1 was preserved in Smad3-null mice and in explanted Smad3-null fibroblasts. The response was blocked by a specific mitogen-activated protein kinase kinase 1 (MEK1) inhibitor but not by an ALK5 kinase inhibitor. Furthermore, MEK1 was phosphorylated by TGF-␤, which was sufficient to drive Egr-1 transactivation. Stimulation by TGF-␤ enhanced the transcriptional activity of Elk-1 via the MEK-extracellular signal-regulated kinase 1/2 pathway. Bleomycininduced scleroderma in the mouse was accompanied by increased Egr-1 accumulation in lesional fibroblasts. Furthermore, biopsies of lesional skin and lung from patients with scleroderma showed increased Egr-1 levels, which were highest in early diffuse disease. Moreover, both Egr-1 mRNA and protein were elevated in explanted scleroderma skin fibroblasts in vitro. Together, these findings define a Smadindependent TGF-␤ signal transduction mechanism that underlies the stimulation of Egr-1, demonstrate for the first time sustained Egr-1 up-regulation in fibrotic lesions and suggests that Egr-1 has a role in the induction and progression of fibrosis.