Analysis of Tumor Necrosis Factor Promoter Responses to Ultraviolet Light

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Abstract

Ultraviolet (UV) light induces the biosynthesis of chloramphenicol acetyltransferase (CAT) in the skin of mice bearing the CATTNF reporter transgene.Moreover, nuclear run-on assays indicate that UV light induces transcription of the TNF gene in RAW 264.7 macrophages. These observations suggest that the TNF gene (and/or its mRNA product) responds to signals elicited by UV light. To identify transcriptional UV response elements within the TNF promoter, and to determine whether a posttranscriptional response might also exist, a series of reporter constructs using a CAT coding sequence attached to various portions of the TNF promoter and 3' untranslated region were devised and transfected into several cultured cell lines. All cells tested were found to be UV responsive, and in NIH 3T3 cells, induction was found to depend upon two general regions of the promoter. The more distal region encompassed nucleotides (nt) -1059 through -451 with respect to the cap site, while the more proximal region spanned nt -403 through -261. A negative element, blocking the UV response, was interposed (nt -451 through -403). As with the response to LPS, the response to UV irradiation appears to involve translational activation in macrophages. However, the UV and LPS signaling pathways have little in common with one another, as indicated by three observations. First, no difference in responsiveness was observed on comparison of TNF gene induction in macrophages derived from C3H/HeN as opposed to C3H/HeJ mice. Second, cell fusion studies showed that while the LPS signaling pathway is extinguished by fusion of RAW 264.7 cells with NIH 3T3 cells, the UV signaling pathway remained intact. Finally, induction did not depend upon the NF-kappaB binding sites that are known to be required for LPS response in macrophages, since mutation of these sites did not impair the UV response. (J. Clin. Invest. 1994. 93:56-62.) Key words: lipopolisaccharide. mouse. signal transduction. NF-kappaB. transcription

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