Signaling mechanism for inducing PP2A nitration and morphological change during decidualization of human endometrial stromal cells

Abstract

By providing the landing area for the implantation of embryo, decidualization of endometrial stroma makes profitable pregnancy environment of uterus through morphological change of hESCs. So, cellular morphology has been observed as the main parameter of decidualization. However, the detailed mechanisms of morphological alteration have not been enough understood yet. We previously reported that a metabolite of phospholipase D1 (PLD1), phosphatidic acid (PA) induces decidualization of hESCs, but we did not determine morphology-related functions of decidualization marker genes. In present study, we aim to elucidate the mechanism how to occur PA-induced cytoskeletal rearrangement during decidualization of hESCs by staining with F-actin. First, we confirmed that decidualization marker genes, IGFBP1 and prolactin, does not related with cytoskeletal rearrangement during PA-induced decidualization. Transfection of dominant-negative Src (DN-Src) prevents PA-mediated cytoskeletal rearrangement. Likewise, the morphological changes were suppressed by treatment of PF-573228 (an inhibitor of FAK phosphorylation). Moreover, RhoA-ROCK pathway, as the downstream effectors of FAK, regulated PA-induced cytoskeletal rearrangement during decidualization of hESCs. Taken together, we provide that a novel signaling mechanism for the cytoskeletal rearrangement through PLD1-PA-Src-FAK-RhoA-ROCK during decidualization of hESCs, and we suggest that morphological alteration of hESCs is not directly related with expression of marker genes.; Decidualization of endometrial stroma is an essential differentiation process for embryo implantation and maintaining pregnancy. We previously reported that protein phosphatase 2A (PP2A) acts as a key mediator during cAMP-induced decidualization of human endometrial stromal cells (hESCs). However, its activation mechanism has been remained veiled in this model. In present study, we aimed to reveal the mechanism which induces the nitration of PP2A catalytic subunit (PP2Ac) during cAMP-induced decidualization of hESCs. First, cAMP-induced PP2Ac nitration and nitric oxide (NO) synthesis was significantly repressed by L-NAME, an inhibitor of nitric oxide synthase (NOS). Among several NOS isoforms, only inducible NOS (iNOS) was highly expressed in this model, indicating iNOS directly regulates the nitration of PP2Ac. Second, cAMP-induced iNOS expression and PP2Ac nitration were decreased by treatment of TSA, an inhibitor of histone deacetylase 5 (HDAC5). And cAMP-induced phosphorylation of CaMKⅡ and HDAC5 were suppressed by treatment of U73122 (an inhibitor of phospholipase C) or transfection of PLCε siRNA. Finally, small G protein Rap1 and its guanine nucleotide exchange factor Epac1 were involved in cAMP-induced PP2A activation. Taken together, our results suggest that PP2Ac nitration during cAMP-induced decidualization of hESCs is induced through the Epac1-Rap1-PLCε-CaMKⅡ-HDAC5-iNOS signaling pathway. |By providing the landing area for the implantation of embryo, decidualization of endometrial stroma makes profitable pregnancy environment of uterus through morphological change of hESCs. So, cellular morphology has been observed as the main parameter of decidualization. However, the detailed mechanisms of morphological alteration have not been enough understood yet. We previously reported that a metabolite of phospholipase D1 (PLD1), phosphatidic acid (PA) induces decidualization of hESCs, but we did not determine morphology-related functions of decidualization marker genes. In present study, we aim to elucidate the mechanism how to occur PA-induced cytoskeletal rearrangement during decidualization of hESCs by staining with F-actin. First, we confirmed that decidualization marker genes, IGFBP1 and prolactin, does not related with cytoskeletal rearrangement during PA-induced decidualization. Transfection of dominant-negative Src (DN-Src) prevents PA-mediated cytoskeletal rearrangement. Likewise, the morphological changes were suppressed by treatment of PF-573228 (an inhibitor of FAK phosphorylation). Moreover, RhoA-ROCK pathway, as the downstream effectors of FAK, regulated PA-induced cytoskeletal rearrangement during decidualization of hESCs. Taken together, we provide that a novel signaling mechanism for the cytoskeletal rearrangement through PLD1-PA-Src-FAK-RhoA-ROCK during decidualization of hESCs, and we suggest that morphological alteration of hESCs is not directly related with expression of marker genes.