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K. Song, M.J. Kelley, T.S. Acott; Effects of TNF– and IL–1 on Responsive Elements in Human MMP–3 Promoter in Trabecular Meshwork . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1356.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Stromelysin–1 or matrix metalloproteinase–3 (MMP–3) expression is stimulated by TNF–α and IL–1α in trabecular meshwork (TM) cells. Previous work revealed different induction patterns with these two cytokines. Further studies were conducted to characterize responsive elements specific to TNF–α or IL–1α in the MMP–3 promoter. Methods: A construct with a secreted alkaline phosphatase (SEAP) reporter driven by a 2.3 kb MMP–3 promoter, MMP3p–SEAP, was made. Mutants were constructed that removed a SPRE or Ets–1 site. A series of several 5’–deletion mutants were generated including a 434 bp fragment containing both Ets–1 and AP–1 sites and a 174 bp fragment containing only an AP–1 site. Cells were transfected with the full length MMP3p–SEAP or the various mutant or deletion constructs. After TNF–α or IL–1α treatment, the conditioned media was assayed for SEAP activity with a chemiluminescent assay. Results: The stimulative effect of either TNFα or IL–1α was significantly reduced by the deletion of Ets–1, but not by the deletion of SPRE. In TNF–α treated cells, 5’–deletion beyond the Ets–1 and AP–1 sites did not diminish promoter activity in comparison with the 2.3 kb full length promoter. The various 5' deletions produced a complex pattern of reductions in the IL–1α response. Only modest activity was detected with TNF–α or IL–1α using the 174 bp promoter fragment containing the AP–1 site. Conclusions: The Ets–1 site is critical to TNF–α or IL–1α activation of the MMP–3 promoter but the SPRE site is not. However, elements including Ets–1 and AP–1 sites present in the 434 bp fragment provided full activation for TNF–α. In contrast to TNF–α, the 5’–deletions of the MMP–3 promoter from regions distal to the Ets–1 and AP–1 sites caused significant changes in IL–1α induction. This study suggests that different elements of the MMP–3 promoter are involved in TNF–α and in IL–1α regulation of MMP–3 expression.
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