PRL-induced ER alpha gene expression is mediated by janus kinase 2 (Jak2) while signal transducer and activator of transcription 5b (Stat5b) phosphorylation involves Jak2 and a second tyrosine kinase

In the rat corpus luteum of pregnancy, PRL stimulation of ER expression is a prerequisite for E2 to have any luteotropic effect. Previous work from ou r laboratory has established that PRL stimulates ER alpha expression at the level of transcription and that the transcription factor Stat5 (signal tra nsducer and activator of transcription 5) mediates this stimulation. Since it is well established that PRL activates Stat5 through the tyrosine kinase , Janus kinase 2 (Jak2), the role of Jak2 in PRL regulation of ER alpha exp ression was investigated. In primary luteinized granulosa cells, the genera l tyrosine kinase inhibitors, genistein and AG18, and the Jak2 inhibitor, A G490, prevented PRL stimulation of ER alpha mRNA levels, suggesting that PR L signaling to the ER alpha gene requires Jak2 activity. However, using an antibody that recognizes the tyrosine-phosphorylated forms of both Stat5a a nd Stat5b (Y694/Y699), it was found that AG490 could inhibit PRL-induced St at5a phosphorylation only and had little or no effect on Stat5b phosphoryla tion. These effects of AG490 were confirmed in COS cells overexpressing Sta t5b. Also in COS cells, a kinase-negative Jak2 prevented PRL stimulation of ER alpha promoter activity and Stat5b phosphorylation while a constitutive ly active Jak2 could stimulate both in the absence of PRL. Furthermore, kin ase-negative-Jak2, but not AG490, could inhibit Stat5b nuclear translocatio n and DNA binding. Therefore, it seems that in the presence of AG490, Stat5 b remains phosphorylated, is located in the nucleus and capable of binding DNA, but is apparently transcriptionally inactive. These findings suggest t hat PRL may activate a second tyrosine kinase, other than Jak2, that is cap able of phosphorylating Stat5b without inducing transcriptional activity. T o investigate whether another signaling pathway is involved, the src kinase inhibitor PP2 and the phosphoinositol-3 kinase inhibitor (P13K), LY294002, were used. Neither inhibitor alone had any major effect on PRL regulation of ER alpha promoter activity or on PRL-induced Stat5b phosphorylation. How ever, the combination of AG490 and LY294002 largely prevented PRL-induced S tat5b phosphorylation. These findings indicate that PRL stimulation of ER a lpha expression requires Jak2 and also that PRL Gan induce Stat5b phosphory lation through two tyrosine kinases, Jak2 and one downstream of P13K. Furth ermore, these results suggest that the role of Jak2 in activating Stat5b ma y be through a mechanism other than simply inducing Stat5b phosphorylation.