O-13: Na+/K+-ATPase Alpha1 Isoform Mediates Ouabain-Induced Expression of Cyclin D1 and Proliferation of Rat Sertoli Cells

Background: Novel roles for the interaction of cardiotonic steroids to Na+/K+-ATPase have been established in recent years. The aim of the present study was to investigate the intracellular signaling events downstream the action of ouabain on Na+/K+-ATPase in Sertoli cell obtained from immature rats. Treatment of Sertoli cells with ouabain (1 μM) induced a rapid and transient increase in the extracellular signal-regulated kinase (ERK1/2 or MAPK3/1) and phosphatidylinositol 3-kinase (PI3K)/serine-threonine protein kinase (AKT) phosphorylation. Also, ouabain upregulated the expression of cyclin D1 and incorporation of [methyl- 3H]thymidine, both of which were dependent on MAPK3/1 but not AKT intracellular cascade, as shown by pretreatment with MEK (MAP2K1/2) inhibitor U0126 and PI3K inhibitor wortmannin, respectively. Moreover, the effect of ouabain on these proliferation parameters was completely prevented by phospho-CREB (cyclic AMP response element- binding protein)/CREB binding protein (CPB) complex inhibitor KG501 and only partially by NFκB nuclear translocation inhibitor SN50. Pretreatment with estrogen receptors antagonist ICI 182,780 showed that MAPK3/1 activation by ouabain does not involve this receptor. The Na+/K+-ATPase α1 isoform, but not α4 was detected in Sertoli cells, suggesting that ouabain effects in Sertoli cells are mediated via α1. Taken together, these results show a rapid ouabain action in the Sertoli cells, which in turn can modulate nuclear transcriptional events essential for Sertoli cell proliferation in a critical period of testicular development. Our findings are important to understand the role of ouabain in the testis and its possible implications in male infertility. Materials and Methods: Primary cultures of Sertoli cell were obtained from 15-day old male Wistar rats and incubated in the absence (control) or presence of ouabain for detection of cyclin D1, phospho- and total extracellular signal-regulated kinase (ERK1/2 or MAPK3/1) and phosphatidylinositol 3-kinase (PI3K)/serine-threonine protein kinase (AKT) by Western blot. In some experiments the cells were pretreated for 30 minutes with the estrogen receptor antagonist ICI 182,780, MAP2K1/2 inhibitor U0126, PI3K inhibitor wortmannin, disruptor of the CREB:CBP complex KG501 or translocation inhibitor of the NFκB active complex into the nucleus SN50. For proliferation assays, incorporation of [methyl-3H]thymidine into cell DNA was estimated in Sertoli cells. Results: Treatment of Sertoli cells with ouabain (1 microM) induced a rapid and transient increase in MAPK3/1 and PI3K/AKT phosphorylation. Also, ouabain upregulated the expression of cyclin D1 and incorporation of [methyl-3H] thymidine, both of which were dependent on MAPK3/1 but not AKT intracellular cascade, as shown by pretreatment with U0126 and wortmannin, respectively. Moreover, the effect of ouabain on these proliferation parameters was completely prevented by phospho-CREB (cyclic AMP response element-binding protein)/CREB binding protein (CPB) complex inhibitor KG501 and only partially byNFκB nuclear translocation inhibitor SN50. Pretreatment with estrogen receptors antagonist ICI 182,780 showed that MAPK3/1 activation by ouabain does not involve this receptor. The Na+/K+-ATPase alpha1 isoform, but not alpha4 was detected in Sertoli cells, suggesting that ouabain effects in Sertoli cells are mediated via alpha1. Conclusion: Taken together, these results show a rapid ouabain action in the Sertoli cells, which in turn can modulate nuclear transcriptional events essential for Sertoli cell proliferation in a critical period of testicular development. Our findings are important to understand the role of ouabain in the testis and its possible implications in male infertility.