| 19538480 |
Borel V, Marceau G, Gallot D, Blanchon L, Sapin V: Retinoids regulate human amniotic tissue-type plasminogen activator gene by a two-step mechanism. J Cell Mol Med. 2009 Jun 16.
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 fetal membranes rupture. Nevertheless, the regulation of t-PA gene expression in extra-embryonic developmental contexts remains unknown. The aim of our study is to propose the retinoic acids as molecular regulators of t-PA expression in fetal membranes. Retinoic acid (RA) induced t-PA mRNA and proteins in a time-dependent manner in amniotic membrane explants and 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 retinoids receptors (RARs and RXRs) with a DR5 response element located at -7kb from the transcription site. Site-directed mutagenesis of this region of the t-PA promoter showed that SP1 factor was also indispensable for this 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: RARalpha / RXRalpha bound DR5 motif before and up to 12 hrs of RA exposure, and RARbeta / RXRalpha bound DR5 response element after 12 hrs of RA treatment. Finally, experiments using shRNA and RARbeta-specific antagonist revealed that reducing RARbeta induction decreased t-PA induction. Altogether, our results established that the RA-mediated regulation of t-PA in human fetal membranes occurred through two steps with a major role played by RARbeta. |
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