Molecular Mechanism Of The Paradoxical Induction Of Cytosolic Phospholipase A_2α By Glucocorticoids In Human Amnion Fibroblasts

Context and Aims:Cytosolic phospholipase A2α(cPLA2α) is the enzyme catalyzing the formation of the rate-limiting substrate, arachidonic acid, for prostaglandin synthesis. The increasing expression of cPLA2αtowards term gestation in human amnion fibroblasts is believed to be the crucial event in parturition. In contrast to the well-described down-regulation of cPLA2α, up-regulation of cPLA2αby glucocorticoids has been reported in human amnion fibroblasts, which may play a key role in parturition. The mechanisms underlying this paradoxical induction of cPLA2αby glucocorticoids remain largely unknown. Elucidation of this mechanism may help us to understand better the molecular mechanisms underlying human parturition.Materials and methods:By using real time PCR, Western blotting, luciferase reporter assays, siRNA interference and overexpression assays, co-immunoprecipitation assays, chromatin immunoprecipitation assay and enzyme immunoassays, we investigated the molecular mechanisms underlying this paradoxical induction of cPLA2αby glucocorticoids in primary human amnin fibroblasts prepared from term amnion, and made comparison study with human fetal lung fibroblast cell line (HFL-1), which has classical response to glucorticoids.Results:Firstly, we have demonstrated that cortisol (0.01-1μM) has paradoxical dose-dependent stimulating effects on cPLA2αlevels and PGE2 level in primary human amnion fibroblasts, in contrast to the inhibiting effects on cPLA2αlevels and PGE2 level in HFL-1 cells. This paradox of cortisol-induced cPLA2αlevel could be blocked by GR antagonist RU486 (1μM), the messenger RNA transcription inhibitor DRB (75μM) and the protein synthesis inhibitor CHX (10μM), suggesting that the increase of cPLA2αlevel by cortisol is dependent on ongoing transcription mediated by GR and synthesis of at least one other protein in human amnion fibroblasts. Concurrently cortisol (1μM) decreased the proinflammatory cytokine mRNA levels including interleukin 1-β(IL1-β), interleukin 6 (IL6) and tumor necrosis factor a (TNFa) in a dose dependent manner, which could be blocked by RU486 (1μM) in human amnion fibroblasts. In addition, cortisol could also increase the level of IκBαprotein, the inhibitoryκB factor, in human amnion fibroblasts, suggesting the stimulation of cPLA2a expression by cortisol in human amnion fibroblasts is not due to the distorted classical anti-inflammatory pathways in the cells.Secondly, we demonstrated that cultured human amnion fibroblasts mainly expressed the glucocorticoid receptor a subunit, the progesterone receptor A form (PRA) at term and the cells was capable of de novo synthesis of progesterone at a level of 18±1.9 pg/106 cells/ml over 24 h incubation. Knockdown of PRA expression or inhibition of endogenous progesterone production with trilostane significantly enhanced the induction of cPLA2αby cortisol (1μM), whereas overexpression of PRA attenuated the induction of cPLA2αby cortisol (1μM). Coimmunoprecipitation assay revealed PRA in the GR protein complex. Although exogenous progesterone did not alter cPLA2αexpression under basal conditions, it attenuated cortisol-induced cPLA2αexpression at concentrations about 10 fold higher, which might be achieved by competition with cortisol for GR. These results suggest that the high progesterone and inverted PRB/PRA ratio profiles at late human gestation comprised the the paradoxical induction of cPLA2a by cortisol rather than accounting for the paradoxical induction of cPLA2αby cortisol in human amnion fibroblasts. Thirdly, we demonstrated that cortisol stimulated cPLA2αpromoter activity in human amnion fibroblasts, whereas inhibited cPLA2αpromoter activity in HFL-1 cells. The induction of cPLA2αpromoter activity in human amnion fibroblasts by cortisol was abolished by mutagenesis of a glucocorticoid response element (GRE) at-490bp to-485bp in the promoter. The same GRE was found mediating the classical inhibition of cPLA2αexpression by cortisol in HFL-1 cells. Cortisol(1μM) increased Gαs expression in amnion fibroblasts but not in HFL-1 cells. Inhibition of Gαs with NF449 (20μM) attenuated the phosphorylation of CREB-1 and the induction of cPLA2αby cortisol in amnion fibroblasts. Both GR and CREB-1 were found bound to the GRE upon cortisol stimulation of human amnion fibroblasts with chromatin immunoprecipitation assay and co-immunoprecipitation study. Furthermore, the induction of cPLA2αby cortisol was blocked by a protein kinase A inhibitor H89 or dominant negative CREB-1.Conclusions:Cortisol paradoxically stimulated cPLA2αexpression independent of the distorted of the classical anti-inflammatory pathway of cortisol and the unique progesterone profiles in human amnion fibroblasts. Instead, cortisol activates cAMP/PKA/CREB-1 pathway via Gαs induction, and the subsequently phosphorylated CREB-1 interacts with GR at the GRE in the cPLA2αpromoter to promote its expression in human amnion fibroblasts.