Objective To examine whether the abundance, localization, and/or activity of cell cycle regulators CDK2, Cyclin E, p27, and survival proteins AKT and Ras in PCOS-associated endometria (with and without hyperplasia) differ from non-PCOS endometria. Methods The expression of CDK2, Cyclin E, p27, AKT and Ras was measured by immunohistochemistry and/or Western blot in 9 normal endometria (NE), 12 endometria from PCOS patients without endometrial hyperplasia (PCOSE), 7 endometria from PCOS women with endometrial hyperplasia (HPCOSE), and 9 endometria from patients with endometrial hyperplasia (HE). The activity of CDK2 was assessed by an in vitro kinase assay. Results CDK2, Cyclin E and p27 proteins were expressed mainly in the endometrial epithelial cells of the studied groups. No change in the activity of CDK2 was observed in total extracts obtained from the tissue samples. However, the nuclear expression of CDK2 in epithelial cells was slightly elevated in PCOSE and significantly increased in HPCOSE when compared to NE. Higher expression of p27 was detected in the cytoplasm of epithelial cells of PCOSE and HPCOSE when compared to NE. Also, we found an increment in Ser473-AKT phosphorylation and an over-expression of the Ras oncogene in endometria of patients with PCOS. Conclusion The PCOS condition is associated with increased Ser473-AKT phosphorylation, elevated expression of Ras, increased cytoplasmic abundance of p27, and increased nuclear abundance of CDK2 in the endometrial epithelial cells. These biological events could potentially provide a chance for endometrial cells from PCOS patients to exit the controlled cell cycle and become hyperplastic at a later stage.
Nuclear factor kappa B (NF B) is an important intracellular conveyor of extracellular signals and modulates a number of gene responses. Due to the potential significance of NF B in regulating ovarian gene expression, we examined in the rat: (i) whether NF B is activated and developmentally regulated in the corpus luteum (CL) throughout pregnancy; (ii) the proteins forming the NF B complex in luteal cells; and (iii) the role of this transcription factor in luteal function. Western analysis and immunohistochemistry revealed that p65 and p50 were highly expressed throughout pregnancy and were located in both the nucleus and cytoplasm of luteal cells. In addition, because NF B is maintained in the cytoplasm bound to I B, whose phosphorylation allows NF B translocation to the nucleus, we studied the developmental expression of phosphorylated and nonphosphorylated forms of I B . Western analysis revealed that I B was present and phosphorylated throughout pregnancy in the CL whereas by protein/DNA array and electromobility shift assays we found that luteal nuclear extracts bind to an NF B consensus sequence, and that the binding activity decreased along pregnancy. The specific binding was supershifted only by an anti-p65 antibody and not by antibodies against p50, p52, cRel, or RelB. Using day 4 postpartum ovaries, we found higher NF B binding activity in the newly formed CL than in old CL of pregnancy. Furthermore, NF B DNA binding activity was enhanced by prolactin in luteinized granulosa cells. In our first functional study, blockade of NF B/p65 binding to DNA with the sesquiterpene lactone helenalin in luteinized granulosa cells correlated with induction of cell death in a dose-dependent manner. In a second functional study, overexpression of NF B/p65 in luteal cells resulted in inhibition of 20 -hydroxysteroid dehydrogenase (20 HSD) promoter activity as well as endogenous 20 HSD mRNA expression. In summary, we have shown that: (i) NF B is expressed within the CL, primary luteinized granulosa cells, and a rat luteal cell line; (ii) NF B activation within the CL is developmentally regulated in pregnancy, depends on the age of the gland, and can be upregulated by prolactin; (iii) inhibition of NF B/p65 binding to an NF B DNA consensus sequence correlates with induction of cell death in ovarian luteinized granulosa cells; and (iv) overexpression of NF B in luteal cells inhibits 20 HSD gene expression. The results further support a role for NF B as a survival factor in the CL.
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