AKAP95-mediated nuclear anchoring of PKA mediates cortisol-induced PTGS2 expression in human amnion fibroblasts.

Phosphorylation of the transcription factors cyclic adenosine monophosphate response element-binding protein (CREB) and signal transducer and activator of transcription 3 (STAT3) by protein kinase A (PKA) is required for the cortisol-induced production of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in human amnion fibroblasts, which critically mediates human parturition (labor). We found that PKA was confined in the nucleus by A-kinase-anchoring protein 95 (AKAP95) in amnion fibroblasts and that this localization was key to the cortisol-induced expression of PTGS2, the gene encoding COX-2. Cortisol increased the abundance of nuclear PKA by stimulating the expression of the gene encoding AKAP95. Knockdown of AKAP95 not only reduced the amounts of nuclear PKA and phosphorylated CREB but also attenuated the induction of PTGS2 expression in primary human amnion fibroblasts treated with cortisol, whereas the phosphorylation of STAT3 in response to cortisol was not affected. The abundances of AKAP95, phosphorylated CREB, and COX-2 were markedly increased in human amnion tissue after labor compared to those in amnion tissues from cesarean sections without labor. These results highlight an essential role for PKA that is anchored in the nucleus by AKAP95 in the phosphorylation of CREB and the consequent induction of COX-2 expression by cortisol in amnion fibroblasts, which may be important in human parturition.

Autophagic Degradation of Collagen 1A1 by Cortisol in Human Amnion Fibroblasts.

Rupture of fetal membranes can initiate parturition at both term and preterm. Collagen is the crucial factor determining the tensile strength of the membranes. Toward the end of gestation, a feed-forward regeneration of cortisol via 11ß-hydroxysteroid dehydrogenase 1 exists in fetal membranes. It remains undetermined whether cortisol contributes to collagen reduction in fetal membranes. In this study, we have examined whether cortisol accumulation is a causative factor for collagen reduction in human amnion fibroblasts, the major source of collagens in the membranes. Cortisol had no effect on collagen 1A1 (COL1A1) and 1A2 (COL1A2) messenger RNA (mRNA) abundance but decreased their protein abundance. The latter effect was affected by neither mRNA transcription inhibitor nor protein translation inhibitor. Mechanistic studies revealed that the reduction in COL1A1 but not COL1A2 protein by cortisol was blocked by lysosome inhibitor chloroquine or small interfering RNA (siRNA)-mediated knockdown of autophagy-related protein 7, an essential protein for autophagy, whereas the proteasome inhibitors MG132 and bortezomib were ineffective. Further analysis showed that cortisol dose dependently increased the ratio of LC3II/LC3I, a marker of lysosome activation, an effect blocked by the glucocorticoid receptor (GR) antagonist RU486 and siRNA-mediated knockdown of GR. Consistently, cortisol decreased COL1A1 and COL1A2 protein abundance in amnion tissue explants, and decreased COL1A1 and COL1A2 protein abundance was observed at parturition in the amnion tissue. Conclusively, cortisol regeneration in fetal membranes may contribute to rupture of fetal membranes at parturition by reducing collagen protein abundance. Lysosome-mediated autophagy accounts for the reduction in COL1A1 by cortisol, but the mechanism underlying the reduction in COL1A2 awaits further investigation.

Inhibition of Lysyl Oxidase by Cortisol Regeneration in Human Amnion: Implications for Rupture of Fetal Membranes.

The mechanisms underlying human parturition are still not understood, yet we need this knowledge to combat preterm birth. Fetal membranes express abundant 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), which converts inert cortisone to active cortisol. We examined whether cortisol regeneration in the amnion might play a role in human parturition through regulation of lysyl oxidase (LOX), a collagen cross-linking enzyme, thereby contributing to the rupture of fetal membranes. By using cultured human primary amnion fibroblasts, we demonstrated that, in addition to the induction of the key enzymes involved in prostaglandin E2 (PGE2) synthesis, cortisol stimulated 11ß-HSD1 and inhibited LOX reciprocally. These results were reproduced in human amnion tissue explants after cortisol treatment. Cortisone also inhibited LOX expression, which was abolished by the inhibition of 11ß-HSD1. Despite the inhibition of LOX by PGE2, inhibition of the PGE2 pathway failed to block the inhibition of LOX by cortisol. However, inhibition of glucocorticoid receptor and mutation of a negative glucocorticoid response element in LOX promoter abolished the inhibition of LOX by cortisol. Chromatin immunoprecipitation assay revealed that cortisol increased GR binding to the LOX promoter. Moreover, increased cortisol and 11ß-HSD1 abundance and decreased LOX abundance were observed in human amnion tissue after the labor-initiated spontaneous rupture of membranes. These data highlight a crucial role for local cortisol regeneration by 11ß-HSD1 in the down-regulation of LOX expression via glucocorticoid receptor binding to a negative glucocorticoid response element to its promoter in the amnion, which may contribute to rupture of fetal membranes at parturition.