Retinoids regulate human amniotic tissue-type plasminogen activator gene by a two-step mechanism.
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-alpha/RXR-alpha bound DR5 motif before and up to 12 hrs of RA exposure, and RAR-beta/RXR-alpha bound DR5 response element after 12 hrs of RA treatment. Finally, experiments using shRNA and RAR-beta-specific antagonist revealed that reducing RAR-beta 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-beta.
Borel
Valerie
V
Génétique Reproduction et Développement (GReD), UMR CNRS 6247, Clermont Université, INSERM U931, Faculté de Médecine, Clermont-Ferrand, France.
Marceau
Geoffroy
G
Gallot
Denis
D
Blanchon
Loïc
L
Sapin
Vincent
V
D000650:Amnion / Q000187:drug effects* / Q000378:metabolism*; D002460:Cell Line; D003984:Dibenzazepines / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D011485:Protein Binding / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D047488:Retinoid X Receptors / Q000378:metabolism; D012176:Retinoids / Q000494:pharmacology*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics
D003984:Dibenzazepines; C104182:LE 135; D034741:RNA, Small Interfering; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D012176:Retinoids; D016329:Sp1 Transcription Factor; C082925:retinoic acid receptor beta; D010959:Tissue Plasminogen Activator