Signal transduction associated with cell growth and differentiation in endometrial cell

Ras is an essential component in the transduction of extracellular signals. Ras proteins function as critical relay switches that control signalling pathways. However, Ras functions in endometrial cells that are regulated extracellular signals such as steroid hormones, growth factors and cytokines remain to be established. In the present study, we addressed the role of Ras-mediated signal transduction systems in endometrial cell differentiation, proliferation and apoptosis. First of all, we investigated the implication of Ras protein in endometrial cell differentiation using the RENT4 cells that are precursors of endometrial cells. The RENT4 cells contain mixed characteristics of epithelial and mesenchymal cells. The level of E-cadherin expression that is indicative of the differentiation of epithelial cell lineage was promoted by cAMP. Activated K-Ras protein contributed to the enhanced Ecadherin expression by stimulating c-AMP dependent protein Wnase (PKA) activity. The result suggests that activated K-Ras has a potential to promote the differentiation to epithelial cell lineage by stimulating the cAMP-PKA signal transduction. Steroid hormones such as E2 and P are critical to regulate endometrial cell proliferation. In turn, activation of both Ras-MAPK and Ras-PIK3-Akt signal transduction systems are required for the proliferation. Tims, we investigated to define the implication of estrogen (ER) and progesterone receptors (PR) in the signaling to promote the cell growth using the reconstituted NIH3T3 cells. Expression of [¹²V] K-Ras mutant or both wild type K-Ras and ER resulted in the appearance of tumorigenic K12V and Kwt ER cells, respectively, whereas NIH3T3 cells expressing wt K-Ras or ER individually were nontumorigenic. The transcriptional activity by ER was markedly enhanced in tumorigenic K12V and KwtER cells compared to the nontumorigenic cells in the presence of 10%FCS. Expression of DNER reverted the cell nontumorigenic and markedly induced the senescent cell phenotype. We also investigated the implication of PR in K-Ras mediated signal transduction. The expression levels of PR were significantly suppressed only in tumorigenic K12V and KwtER. In contrast, nontumorigenic cells expressed PR abundantly. Thus, we established the cells (K12VPR) coexpression [¹²Val] K-Ras and PRB. ER activity in the presence of 10%FCS was markedly suppressed in K12VPR cells compared to that in the parent K12V cells. In addition, PR-B expression in tumorigenic K12V cells dominantly suppressed colony formation in soft agar and prolonged the latency period to form palpable tumors in nude mice. These results suggest that transcriptional activation by ER is critical for the K-Ras mediated cell growth promotion and the resultant cell transformation. ER activation by Ras is mediated by the direct activation of ER-AF1 through Ras-MAPK and suppression of PR expression, that competes with ER activation. We focussed on the role of K-and H-Ras proteins in regulation of endometrial cell apoptosis. We used RENT4 cell, human endometrial cancer cell lines, Ishikawa that carried both wt K-and H-Ras and HHUA that carried mutant K-Ras and wt H-Ras, respectively. We transfected activated [¹²Val] K-Ras or [⁶¹Leu] HRas mutant into these cells. An activated K-Ras mutant stimulated apoptosis induction. In contrast, An activated H-Ras sharply suppressed apoptosis of the cells in the presence of apoptotic signals. Expression of dominant negative H-Ras resulted in the marked induction of apoptosis in both Ishikawa and HHUA cells. In addition, a P13 Wnase inhibitor induced apoptosis whereas a MEK inhibitor had no effect. The data are compatible with the idea that the H-Ras-P13-Akt signal transduction has a potential escape from apoptosis. From the present results, we define Ras proteins as a critical regulator of endometrial cell differentiation, proliferation and apoptosis.