RETINOIDS AND COLLAGEN TRANSCRIPTION IN LUNG FIBROBLASTS

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Interstitial pulmonary fibrosis (IPF) is characterized by the accumulation of type I collagen in the lung. Retinoids, vitamin A derivatives, are widely used to suppress collagen formation in the skin and have been shown to decrease alpha2(I) collagen gene expression in vitro. My preliminary work indicates that retinoic acid inhibits type I collagen formation in human lung fibroblasts by down regulating alpha1(I) collagen gene transcription. In contrast to described mechanisms of RA-induced gene regulation, my data suggest that RA inhibits alpha1(I) expression through a newly synthesized trans-acting intermediary protein that binds to the RAIE in the proximal portion of the collagen promoter. This grant proposes to examine the mechanism by which RA inhibits type I collagen gene transcri ion. The Specific Aims of this proposal are to define the regulatory element(s) involved in retinoic acid-induced inhibition of the alpha1(I) collagen gene, and to characterize the nuclear protein(s) binding to that element. To localize the RA inhibitory elements(s) (RAIE), the active 900 bp alpha1(I) promoter/reporter gene construct will undergo deletion analysis. Promoter activity will be assessed by transfection studies. To demonstrate that the isolated RAIE confers the RA inhibitory effect, the RAIE will be subcloned into a heterologous SV40-enhancer driven/luciferase reporter construct. In vivo and in vitro competition assays will test the function and binding specificity of the putative RAIE. In vitro, RAIE binding specificity will be determined using DNA mobility shift assays with unlabelled RAIE, unrelated oligonucleotides, and RAIE mutated at potential protein binding sites by methylation interference assay. In vivo, varying concentrations of complementary and mutated RAIE will be co-transfected into cells transiently transfected with the 900 bp alpha1(I)/luciferase construct. DNase I footprinting studies will specify nuclear protein binding regions within the RAIE. To characterize the proteins that bind the RAIE, the sequence will be analyzed for consensus binding motifs for known transcription factors. The effect of site directed mutagenesis on protein binding to any of these putative transcription factor elements will be determined. DNA mobility shift studies will be performed with antibodies and oligonucleotides complementary to the common transcription factors. If no known consensus binding sequence is noted within the RAIE, I will extract and characterize the unique proteins by affinity chromatography using biotinylated RAIE. Taken together, these data will offer insights into collagen regulation in the lung, and possibly new strategies for treatment IPF.