Retinoids regulate human amniotic tissue‐type plasminogen activator gene by a two‐step mechanism

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Abstract
The collagenolytic effects of the tissue‐type plasminogen activator (t‐PA) leading to extracellular matrix degradation are clearly involved in the physiopathology of human foetal membranes rupture. Nevertheless, the regulation of t‐PA gene expression in extraembryonic developmental contexts remains unknown. The aim of our study is to propose the retinoic acids (RAs) as molecular regulators of t‐PA expression in foetal membranes. RA induced t‐PA mRNA and proteins in a time‐dependent manner in amniotic membrane explants and Wistar Institute Susan Hayflick (WISH) cells. Furthermore, the use of cycloheximide revealed a two‐step regulation of t‐PA gene. Gene reporter assays confirmed that the RA‐induced t‐PA gene expression occurred through interactions of retinoid receptors (RARs and RXRs) with a DR5 response element located at –7 kb from the transcription site. Site‐directed mutagenesis of this region of the t‐PA promoter showed that SP1 factor was also retinoid‐mediated induction, and immunoprecipitation assays revealed that SP1 and RAR/RXR interacted physically. Chromatin immunoprecipitation demonstrated that interactions between RARs, RXRs and t‐PA promoter were time dependent: RAR‐α/RXR‐α bound DR5 motif before and up to 12 hrs of RA exposure, and RAR‐β/RXR‐α bound DR5 response element after 12 hrs of RA treatment. Finally, experiments using shRNA and RAR‐β‐specific antagonist revealed that reducing RAR‐β induction decreased t‐PA induction. Altogether, our results established that the RA‐mediated regulation of t‐PA in human foetal membranes occurred through two steps, with a major role played by RAR‐β.

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