Mechanisms Of The CAMP/PKA/CREB Pathway Activation In The Induction Of Cyclooxygenase-2 In Human Amnion Fibroblasts

AimsProstaglandins comprise the "final common pathway" in human parturition,so it is essential to clarify the mechanism of the regulation of prostaglandin synthesis for understanding the initiation of parturition.Human amnion fibroblasts are recognized as one of the major sources for prostaglandins.Previous studies have revealed that activation of the cAMP/PKA/CREB signaling pathway by prostaglandin E2(PGE2)is required for glucocorticoids-induced cyclooxygenase-2(COX-2)expression,the rate-limiting enzyme in PGE2 synthesis in human amnion fibroblasts.However,the receptor subtype of PGE2 involved in the activation of the cAMP/PKA signaling pathway and how PKA is anchored in the nucleus to phosphorylate CREB in amnion fibroblasts remain to be determined.Here we addressed these issues in primary human amnion fibroblasts as well as in human amnion tissues obtained from pregnant women with and without labor with an aim to find specific approaches for prevention and treatment of preterm birth.MethodsThe roles of PGE2 receptors(EP2,EP4)and PKA anchoring proteins(AKAP79,AKAP95)in the activation of the cAMP/PKA/CREB signaling pathway and the induction of COX-2 were explored in cultured primary human amnion fibroblasts by using RNA sequencing,overexpression plasmid and siRNA-meidated knock-down,agonists and antagonists of receptors,real time PCR,Western blotting,immunofluorescent staining and enzyme immunoassays.The regulation of EP2 and EP4 receptors,AKAP79 and AKAP95 expression were also studied in human amnion fibroblasts with real time PCR and Western blotting.The amnions obtained from pregnant women with and without labor were used to measure the changes of EP2 and EP4 receptors,AKAP79 and AKAP95 abundance in labor.Results Part 1.Differential roles of prostaglandin EP2 and EP4 receptors in the activation of the cAMP/PKA signaling pathway and the induction of COX-2 expression1.Prostaglandin E receptors include four subtypes(EP1,EP2,EP3 and EP4),RNA sequencing data from amnion fibroblasts and immunohistochemical staining of fetal membranes revealed that EP2 and EP4 receptors were mainly expressed in human amnion fibroblasts,and the expression of EP1 and EP3 receptors were hardly detectable.2.EP2 agonist had no effect on the activity of PI3 K,but caused long-lasting increases in the expression of COX-2 and the phosphorylation of CREB and STAT3,the two key transcription factors downstream to the cAMP/PKA pathway involved in the induction of COX-2 expression in amnion fibroblasts.By constrast,EP4 agonist increased the activity of PI3 K,and induced only transient increases in COX-2 expression and CREB phosphorylation,but had no effect on STAT3 phosphorylation.3.Inhibition of PI3 K not only enhanced the induction of PKA activity and the phosphorylation of CREB and STAT3,but also enhanced the induction of COX-2 by PGE2.4.Inhibition of EP2 receptor blocked the phosphorylation of CREB and STAT3 as well as the induction of COX-2 by PGE2.Overexpression of EP4 receptor attenuated the induction of COX-2 by PGE2,and inhibition of EP4 receptor enhanced the phosphorylation of CREB and STAT3,thus leading to the enhancement of COX-2 induction by PGE2;5.Glucocorticoids and PGE2,the recognized stimulants of COX-2 expression in amnion fibroblasts,increased EP2 but decreased EP4 receptor expression.6.Examination of the amnion tissues obtained from pregnant women with and without labor revealed that the EP2 receptor protein abundance was significantly increased while the EP4 receptor protein abundance was significantly decreased with parturition.Part 2.The role of AKAP79 and AKAP95 in the activation of the cAMP/PKA/CREB pathway and the induction of COX-2 expression1.Western blotting analysis of fractionated protein extracts and immunofluorescence staining of cultured amnion fibroblasts revealed that AKAP95 was detected exclusively in the nucleus whereas AKAP79 was detected mainly in the cell membrane.CREB,a key transcription factor in COX-2 induction was exclusively located in the nucleus and STAT3,another key transcription factor in COX-2 induction was in the cytoplasm.2.Knock-down of AKAP95 significantly decreased the abundance of not only the RIIα subunit but also the Cα subunit of PKA in the nucleus in amnion fibroblasts.In addition,knock-down of AKAP95 reduced the phosphorylated CREB in amnion fibroblasts,whereas the phosphorylation of STAT3 was not affected.3.Knock-down of AKAP95 but not AKAP79 significantly attenuated the induction of COX-2.4.Glucocorticoid,which was essential in the induction of COX-2 in human amnion fibroblasts,increased the abundance of AKAP95;5.The abundances of AKAP95,phosphorylated CREB,and COX-2 were markedly increased in human amnion tissues after labor compared to those in amnion tissues from cesarean sections without labor.ConclusionsEP2 is the dominating PGE2 receptor mediating the activation of cAMP/PKA/CREB signaling pathway by PGE2 in amnion fibroblasts,which can be attenuated by simultaneous activation of PI3 K coupled with the EP4 receptor.Glucocorticoids and PGE2,which were important in up-regulating COX-2 expression in amnion fibroblasts,induce the expression of EP2 receptor whereas inhibit the expression of EP4 receptor,thus leading to an enhancement of activation of cAMP/PKA signaling pathway and induction of COX-2.Nuclear AKAP95-anchored PKA is crucial for the phosphorylation of CREB and subsequent transcriptional induction of COX-2 in human amnion fibroblasts.Glucocorticoids increase the abundance of PKA anchored in the nucleus by inducing AKAP95 expression,thereby enhancing the phosphorylation of CREB in the nucleus and subsequent transcription of COX-2 leading to increased production of PGE2 in human amnion fibroblasts.