This renewal application proposes extensive studies on the regulation of collagen gene expression, the ultimate goal being delineation of the molecular mechanisms leading to accumulation of collagen in dermal fibrotic diseases. Two hypotheses developed on the basis of past progress in this project will be tested. The first postulates that the critical event leading to fibrotic skin disease is altered growth factor modulation of collagen gene expression at the transcriptional level, with particular emphasis on the antagonistic effects of TGF-beta and TNF-alpha on type I collagen. The second hypothesis suggests that the expression of type VI and possibly type VII collagen genes serves as an early marker of the onset of the fibrotic reaction. To test these hypotheses, the applicant proposes two complementary areas of research: 1) Detailed elucidation of regulatory mechanisms of collagen gene expression in human skin fibroblasts. In these aims the applicant proposes identification of cis-elements responsible for constitutive expression of type I, VI and VII collagen genes, identification of trans-acting factors by gel mobility shift and footprinting analyses, and isolation of such factors by magnetic separation of DNA/protein complexes as well as cloning using the yeast one-hybrid system. Furthermore, growth factor-responsive elements will be identified in the corresponding genes, with particular emphasis on the role of AP1/NF-kB in the regulation of type I collagen gene expression. 2) Development of novel treatment modalities for fibrotic skin diseases. Such approaches include evaluation of novel cytokines that are potentially useful for inhibition of collagen accumulation. The applicant also proposes development of novel molecular approaches towards future gene therapy applications, including utilization of antisense oligonucleotides, triple helix forming oligomers, and expression of mammalian vectors containing antisense mRNA sequences. Collectively, it is expected that these experimental approaches will allow the applicant to define the pathways that are operative in the physiological regulation of collagen gene expression, and to identify the mechanisms of collagen accumulation in fibrotic skin diseases. Such information will permit development of novel approaches to counteract these debilitating diseases by targeting the precise molecular levels of aberration.
Showing the most recent 10 out of 30 publications
