Gene expression profiling of progestin in stromal endometrial cells: mechanism of PR-induced cell proliferation.

GRISELDA VALLEJO, INTI TARIFA, ANA CECILIA MESTRE CITRINOVITZ, MIGUEL BEATO, PATRICIA SARAGÜETA.

Woods Hole – Masssachusetts - USA

Simposio; Frontiers in Reproduction XIII Annual Symposium.; 2010

MBL

We have previously reported that progestin R5020 (10-10M) induces proliferation of UIII rat endometrial cells through Erk1-2 and Akt activation via crosstalk of PR with ER. The aim of this project was to identify the immediate early genes regulated by activated Erk1-2 and Akt mediating progestin-dependent proliferation. We characterized the expression of the cell cycle regulator Ccnd1 and found that R5020 10-9M induce transiently and maximally Ccnd1 transcript levels at 45 minutes through classic PR. We hybridized oligo microarrays with cDNA from cells cultured with R5020 10-10M or vehicle during 45 minutes and identified 97 up and 341 downregulated genes. In silico analysis revealed that the over-represented molecular function was transcription factor activity. In Cellular Proliferation and Cell Cycle ontology terms, 39% were transcription factors, 65% were downregulated genes, and half of latter were associated to inhibitory functions. We validated the expression of seven genes by real time PCR. JunD and Usf1 were chosen as transcription factors, Crebbp and Gfi1 were chosen as cell cycle regulators and transcription factors, and Cyr61, Pten and Cdkn1b were chosen as cell cycle regulatorsonly. Pretreatment with specific inhibitors showed that a) activated Erk1-2 and Akt were involved in Cyr61, JunD, Usf1 and Crebbp up-, and Gfi1 and Cdkn1b down-regulation, b) activated Erk1-2 mediated Pten repression and c) activated Akt was required for Ccnd1 progestin-dependent induction. We observed that cyclin E, a Crebbp targetgene, is induced at 6hs by R5020 10-10M and that Cdc2, a Usf1 target gene, is up-regulated at early (45 min) and later time (12-24 hs) of treatment with progestin. Our findings suggest that the progestin-dependent proliferation mediated by PR in stromal endometrial cells could be due to a) the action of immediate early regulated transcription factors targeting cell cycle regulatory molecules and b) the inhibition of repressors of transcription and cell cycle.