Rapid in Vivo Effects of Estradiol-17β in Ovine Pituitary Gonadotropes Are Displayed by Phosphorylation of Extracellularly Regulated Kinase, Serine/Threonine Kinase, and 3′,5′-Cyclic Adenosine 5′-Monophosphate-Responsive Element-Binding Protein

Abstract

We have determined the time course of phosphorylation of MAPK/ERK, cAMP-responsive element-binding protein (CREB), and serine/threonine kinase (Akt) in ovine pituitary gonadotropes after in vivo injection (iv) of either 25 mug estradiol-17beta (E17beta) or vehicle. In ovariectomized ewes, E17beta increased the number of gonadotropes expressing phosphorylated (p)ERK-1/2 and pCREB immunoreactivity (-IR) within 90 min, as assessed by immunohistochemistry. By Western blot, we also showed that pERK-1/2, pCREB, and pAkt (ser 473) proteins were up-regulated by E17beta. In ovariectomized, hypothalamo-pituitary-disconnected animals, gonadotrope function was restored with hourly GnRH pulses (iv), and E17beta injection (iv) reduced LH response within 1 h. Immunohistochemistry showed that E17beta increased pERK-1/2-IR in gonadotropes within 15 min and peak response at 60 min. The number of cells immunoreactive for pCREB was greater in E17beta-treated animals than in vehicle-injected controls at 60 and 90 min. Western blot revealed a pERK-1/2 response within 15 min and pCREB response at 30 min. Up-regulation of pAkt occurred within 60 min of E17beta treatment. Thus, rapid effects of E17beta on gonadotropes involve phosphorylation of second messenger proteins with a time course that may relate to the rapid negative feedback effect to reduce responsiveness to GnRH.

Membrane-initiated actions of estradiol (E2) in the regulation of LH secretion in ovariectomized (OVX) ewes

“…Since an ERE is not involved in mediating the actions of E 2 BSA [24,27,47,48] and a canonical ERE has not been reported in the GnRH receptor gene [47-50], we speculate that a membrane-initiated pathway may activate down-stream events facilitating the transcription of GnRH receptors, as well as inhibiting GnRH-induced secretion of LH. As mentioned above, an increase in phosphorylation level of a number of proteins in the gonadotropes has been reported 15 to 90 minutes after administration of E 2 [42]. The ability of E 2 or E 2 BSA to increase the level of protein phosphorylation for longer periods would be compatible with our speculation on a membrane-initiated pathway as a component in the synthesis of GnRH receptors number, as well as maintaining the inhibition of LH secretion after the initial decrease.…”

“…In that study, the increase in cytoplasmic intracellular free calcium concentration ([Ca ++ ]i) mediating the GnRH-induced release of LH secretion was abolished by 2 min pretreatment with E 2 or E 2 BSA, which agrees with the time frame for the beginning of the rapid decrease of LH secretion induced by estrogens. In hypothalamus-pituitary-disconnected ewes, E 2 increased the number of gonadotropes expressing phosphorylated extracellular signal-regulated-kinases 1 and 2 (pERK-1/2), c-AMP-responsive element-binding protein (pCREB), and serine 473 kinase (pAkt) within 15 to 90 minutes after administration of E 2 [42]. Whether these and/or other upstream signaling molecules contribute to maintain a decreased responsiveness of the pituitary gland to GnRH (for at least 10 h in our previous study [30]) after administration of estrogens is yet to be evaluated.…”

Fetal uninephrectomy in male sheep alters the systemic and renal responses to angiotensin II infusion and AT1R blockade

“…The frozen slice was further subdivided into cortex and medulla (inner and outer combined) to determine tissue levels of renin and ANG II and for Western blot analysis. Protein extraction and Western blot analysis were performed as previously described (19), using rabbit polyclonal anti-AT 1 (40 kDa, 1:500) and anti-AT2 (44 kDa, 1:500) antibodies (Alomone Laboratories). Membranes were stripped and reprobed with anti-␣-smooth muscle actin (␣-SMA, 1:1,000).…”

Does a Nonclassical Signaling Mechanism Underlie an Increase of Estradiol-Mediated Gonadotropin-Releasing Hormone Receptor Binding in Ovine Pituitary Cells?1

“…In the ovine promoter, the CRE is capable of binding CREB and mediating enhanced transcriptional activity in response to cAMP and forskolin [62]. Although the ability of E2 to module transcriptional activity of target genes through cAMP, protein kinase A, and CREB pathways has been well described in neurons [63][64][65][66][67][68][69], recently a 30-min exposure to E2 was shown to increase levels of phosphorylated CREB in gonadotrophs in the ovine pituitary gland [37,47]. Based on these date, we were intrigued with the possibility that E2 may influence transcriptional activity of the GnRHR gene and subsequent protein synthesis through a CREB/ATF-dependent mechanism.…”

“…The ability of membrane forms of ESR1 to integrate signaling through both AP-1 [33,34] and CREB is well established [35,36]. Furthermore, recent work has established that E2 leads to rapid phosphorylation of CREB in ovine gonadotrophs [37]. To determine if this pathway contributes to E2 regulation of GnRHR number, we applied an experimental paradigm based on overexpression of a dominantnegative form of CREB termed A-CREB.…”