The uncontrolled tissue accumulation of Type I collagen characteristic of scleroderma is attributed to increased transcription of the collagen genes in scleroderma fibroblasts. TGF-Beta, a potent inducer of collagen synthesis, is strongly implicated in the development of pathological fibrosis in scleroderma. Other cytokines such as interferon-gamma antagonize the effects of TGF-Beta, and are likely to be important for prevention of scarring. Little is known about the intracellular signaling pathways involved in the physiologic regulation of collagen synthesis by cytokines, and the cis-acting elements of the collagen genes that are targets for these pathways. This information is of crucial importance for gaining a better understanding of the pathogenesis of fibrosis. We have shown that TGF-Beta stimulates transcription of the alpha1 (I) collagen gene (COL1A1) in fibroblasts. During the previous period of funding, we have identified cis-acting elements and their cognate transcription factors that play roles in regulating basal COL1A1 transcription. Our long-term goal is to understand the cellular mechanisms for modulation of collagen transcription in response to stimulatory and inhibitory extracellular signals, and to delineate alterations in the intracellular signaling pathways that result in constitutive up-regulation of the expression of collagen genes in scleroderma. In Specific Aim 1, we will ask which regions of the human COL1A1 promoter (and first intron) are responsive to TGF-Beta in fibroblasts, and what trans-acting proteins bind to these elements? We will confirm the functional role of TGF-Beta response elements in vivo by gene transfer in mice. In Specific Aim 2, we will examine the role of a novel family of intracellular signaling proteins in activation of collagen transcription by TGF-Beta in vitro. By gain- of-function and loss-of-function experiments, we will ask which Smad proteins are involved, and whether the Smads function as DNA-binding transcription factors in fibroblasts. We will ask if the Smads are molecular targets for antagonistic regulation by cytokines with opposing effects on collagen transcription. In Specific Aim 3, we will ask whether aberrant or deregulated Smad signaling underlies the constitutive up-regulation of collagen transcription in scleroderma fibroblasts. These studies should better define the signaling mechanisms that are important in regulating collagen transcription in normal and fibrotic fibroblasts. The results will facilitate the design of interventions to selectively inhibit this process.