Roles of estrogen receptor α and β in modulating urothelial cell proliferation

We reported previously that both subtypes of estrogen receptors, ER alpha and ER beta, are expressed by human urothelial cells and mediate estrogen-induced cell proliferation in these cells. The aim of this study was to determine the extent to which each ER subtype contributes to urothelial cell proliferation and their possible involvement in the regulation of the cell cycle. We compared the expression of ER alpha and ER beta mRNAs and protein quantitatively in primarily cultured human bladder urothelial cells obtained from six individuals with three immortalized urothelial (E6, E7, and UROtsa) and two bladder cancer cell lines (HTB-9 and T24). We found that all these cells express similar levels of ER beta, but immortalized and cancer cells express much higher amounts of ER alpha than primary cells. Higher levels of ERa mRNA were also observed in the biopsies of bladder transitional cell carcinoma compared with sample from the same bladder unaffected by tumor. Using the ER alpha-selective agonist PPT, the ER beta-selective agonist DPN, and specific small interfering RNA against ER alpha or ER beta, we found that ER beta predominantly mediates estrogen-induced G1/S transition and cell proliferation in the primary urothelial cells. By contrast, ERa predominantly mediates estrogen-induced G1/S transition and cell proliferation in bladder cancer cell lines. Furthermore, we found that 17 beta-estradiol (E-2) rapidly induces phosphorylation of extracellular signal-regulated kinases, but U0126, a mitogen-activated protein kinase kinase (MEK) inhibitor, does not affect E-2-induced urothelial cell proliferation. E-2 up-regulated cyclin D1 and cyclin E expression in both the primary and bladder cancer cells, and the cancer cells have higher cyclin D1 and cyclin E expression during G0/G1 phases. Our data suggest that estrogen exerts its effects through different ER subtypes in urothelial cells. Increased expression of ER alpha may contribute to early induction of cyclin D1 and cyclin E during the cell cycle in bladder cancer cells.