Nevertheless, only LY294002 suppressed LH induced CYP17A1 mRNA expression, whereas wortman nin did not affect this response. While the reason selleck Ruxolitinib for this apparent discrepancy is not clear, it is worth noting that wortmannin has been reported to be unstable in aqueous solution and less specific than LY294002. Higher concentration of wortmannin induced theca cell detachment and apoptosis in our serum free culture system. Numerous reports have described that an activation of the intracellular signaling is a rapid reaction in most cells. However, in this study, it took 12 h for LH induced increase in phos pho Akt content in theca cells. It is of interest whether PKA pathway, which is considered to be a major mediator of the LH generated signaling, andor the MAPK pathway influence LH induced Akt phosphorylation or not.
Exper iment 4 was performed Inhibitors,Modulators,Libraries to verify this point. androstenedione productionCYP17A1 mRNA expression and Effects of MEK inhibitor on CYP17A1 mRNA expres sion and androstenedione production in bovine theca cells. Subconfluent cultures were pretreated with MEK inhibitor for 30 min. Then they were stimu lated with LH for 12 24 h. Control cells were cultured in the absence of added treatments. RT PCR was conducted Inhibitors,Modulators,Libraries using CYP17A1 and 36B4 primers using total RNA isolated from the cells. The mRNA level of CYP17A1 were expressed as ratio to 36B4 values. Culture media Inhibitors,Modulators,Libraries were also assayed for androstenedione by EIA. Data are the mean SEM. Each experiment was reproduced at least three times. Different let ters represent statistically significant differences of means.
theca cells. Reportedly, LH induced Akt phosphorylation in whole rat ovary, and the PI3K inhibitor, LY294002, suppressed androstenedione Inhibitors,Modulators,Libraries production by theca cells in rat and cattle. It is possible that LH As described earlier, H89, a potent and selective inhibitor of PKA, did not affect LH mediated changes in phospho Akt, indicating that a pathway distinct from that of PKA is involved in LH induced Akt phosphorylation in theca cells. Until recently, the effects of cAMP were generally thought to be mediated by activation of cAMP Inhibitors,Modulators,Libraries dependent PKA, a major cAMP target, followed by phosphorylation of many intracellular targets, such as cAMP responsive ele ment binding protein, resulting in changes in ovarian gene expression such as CYP17A1. Nevertheless, some effects of cAMP appear to be inexplicable by activa tion of PKA.
For instance, TSH and cAMP regulate prolif eration of thyroid cells by mechanisms independent of PKA. Actually, cAMP binds specific guanine nucle otide exchange factors cAMP GEFs. Gonzalez Robayna et al. reported that cAMP GEFs are expressed in rat granulosa cells and that the cAMP GEFs play a role figure 1 in FSH induced activation of the PI3KAkt pathway in gran ulosa cells by PKA independent manner.