This renewal application proposed continuation of studies towards the overall goal of delineating the molecular mechanisms which lead to accumulation of collagen in dermal fibroses. This sharply focused proposal is based on significant progress made in this project during the previous years of support, implicating pretranslational control of collagen gene expression in these diseases. The studies will utilize skin fibroblast cultures established from patients with fibrotic skin diseases, and will specifically focus on two clinical conditions, keloids and progressive systemic sclerosis, which serve as prototypes of dermal fibrotic conditions. Previous studies have concentrated on type I and type III procollagens which are thought to be the predominant collagens in human dermis. Recent compelling evidence suggests that type VI collagen is a more abundant gene product of human skin fibroblasts than has been previously recognized. Since type VI collagen has been suggested to play a distinct role in collagen fibrillogenesis, the studies proposed here will explore the role of type VI collagen in fibrotic skin diseases in detail. This novel approach has been made feasible by recent cloning of human cDNAs encoding the three constituent polypeptide chains of type VI collagen. Finally, the role of cytokines in fibrotic processes will be examined by evaluating the transcriptional modulation of collagen gene expression by TGF-beta, TNF-alpha, and IL-1, factors that have been previously shown to affect collagen gene expression, yet the exact mechanisms and the level of interference are currently unknown.
The specific aims of the proposal include: 1) Elucidation of the transcriptional control elements of collagen gene expression by utilizing transfection assays with collagen promoter/CAT constructs; 2) Examination of cellular trans-acting regulatory elements by detailed gel retardation and footprinting analyses; 3) Evaluation of cis-acting control elements in collagen genes by deletion analysis of the constructs containing putative regulatory DNA sequences linked to a reporter (CAT) gene; 4) Spatial localization of the enhanced cellular collagen gene expression by in situ hybridizations; 5) Modulation of collagen gene expression in fibroblast cultures by cytokines and novel pharmacologic means. We expect that these experimental approaches will allow us to pinpoint the molecular mechanism leading to accumulation of collagen in fibrotic skin diseases. This information may allow us to develop pharmacologic means which could counteract these debilitating diseases by targeting the precise levels of aberration.
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