Ultraviolet irradiation represses TGF-β type II receptor transcription through a 38-bp sequence in the proximal promoter in human skin fibroblasts.

Abstract

Transforming growth factor-β (TGF-β) is a major regulator of collagen gene expression in human skin fibroblasts. Cellular responses to TGF-β are mediated primarily through its cell surface type I (TβRI) and type II (TβRII) receptors. Ultraviolet (UV) irradiation impairs TGF-β signalling largely due to reduced TβRII gene expression, thereby decreasing type I procollagen synthesis, in human skin fibroblasts. UV irradiation does not alter either TβRII mRNA or protein stability, indicating that UV reduction in TβRII expression likely results from transcriptional or translational repression. To understand how UV irradiation regulates TβRII transcription, we used a series of TβRII promoter-luciferase 5'-deletion constructs (covering 2kb of the TβRII proximal promoter) to determine transcriptional rate in response to UV irradiation. We identified a 137-bp region upstream of the transcriptional start site that exhibited high promoter activity and was repressed 60% by UV irradiation, whereas all other TβRII promoter reporter constructs exhibited either low promoter activities or no regulation by UV irradiation. Mutation of potential transcription factor binding sites within the promoter region revealed that an inverted CCAAT box (-81bp from transcription start site) is required for promoter activity. Mutation of the CCAAT box completely abolished UV irradiation regulation of the TβRII promoter. Protein-binding assay, as determined by electrophoretic mobility-shift assays (EMSAs) using the inverted CCAAT box as probe (-100/-62), demonstrated significantly enhanced protein binding in response to UV irradiation. Super shift experiments indicated that nuclear factor Y (NFY) is able to binding to this sequence, but NFY binding was not altered in response to UV irradiation, indicating additional protein(s) are capable of binding this sequence in response to UV irradiation. Taken together, these data indicate that UV irradiation reduces TβRII expression, at least partially, through transcriptional repression. This repression is mediated by a 38-bp sequence in TβRII promoter, in human skin fibroblasts.