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

Abstract

Estradiol-17beta (E2) is the major regulator of GnRH receptor (GnRHR) gene expression and number during the periovulatory period; however, the mechanisms underlying E2 regulation of the GNRHR gene remain undefined. Herein, we find that E2 conjugated to BSA (E2-BSA) mimics the stimulatory effect of E2 on GnRH binding in primary cultures of ovine pituitary cells. The time course for maximal GnRH analog binding was similar for both E2 and E2-BSA. The ability of E2 and E2-BSA to increase GnRH analog binding was blocked by the estrogen receptor (ER) antagonist ICI 182,780. Also, increased GnRH analog binding in response to E2 and the selective ESR1 agonist propylpyrazole triol was blocked by expression of a dominant-negative form of ESR1 (L540Q). Thus, membrane-associated ESR1 is the likely candidate for mediating E2 activation of the GNRHR gene. As cAMP response element binding protein (CREB) is an established target for E2 activation in gonadotrophs, we next explored a potential role for this protein as an intracellular mediator of the E2 signal. Consistent with this possibility, adenoviral-mediated expression of a dominant-negative form of CREB (A-CREB) completely abolished the ability of E2 to increase GnRH analog binding in primary cultures of ovine pituitary cells. Finally, the presence of membrane-associated E2 binding sites on ovine pituitary cells was demonstrated using a fluorescein isothiocyanate conjugate of E2-BSA. We suggest that E2 regulation of GnRHR number during the preovulatory period reflects a membrane site of action and may proceed through a nonclassical signaling mechanism, specifically a CREB-dependent pathway.

Estrogens Induce Expression of Membrane-Associated Estrogen Receptor α Isoforms in Lactotropes

“…Rapid estrogenic actions via mER leading to hormone release and (anti)proliferative responses have been described in both normal and transformed anterior pituitary cells [11], [38]–[41]. Also, E2 activation of the GnRH receptor gene was recently reported to involve mER in ovine pituitary cells [42]. Most studies indirectly infer the involvement of mERα in these estrogenic actions, either because of high expression of this receptor in pituitary cell lines or absence of ERβ in the plasma membrane of normal pituitary cells [11], [38]–[41].…”

Gonadotropes and Gonadotropin-Releasing Hormone Signaling

“…[395][396][397][398][399][400] Moreover, the GnRHR gene promoter is also a target of activin signaling in the gonadotrope. Increased receptor numbers alter the sensitivity and responsiveness of the gonadotrope during physiologically relevant situations such as changes leading to the preovulatory surge of LH and ovulation.…”

Dissecting rapid estrogen signaling with conjugates

“…The conjugates are commercially available, have fluorescently labeled derivatives and have found widespread use as probes of a number of different types of estrogen-induced responses [18-20]. In these studies, the BSA conjugates are used primarily to probe whether the estrogen-induced signal is originating from the plasma membrane, since BSA is assumed to membrane-impermeable.…”

“…Ultimately reverse phase preparative HPLC was required to remove the contaminant [31]. Cursory analysis of work with the BSA-estradiol conjugates, which have already been documented to have some degree of contaminating free ligand [21], suggest that some groups pay great attention to ensuring the purity of the conjugates [37] while many others do not appear to have performed any purification at all. Dialysis can still be an effective method to purify conjugates and is likely sufficient for most studies, but ideally, the purity of the material should be assessed before using.…”

“…There are a number of good examples of purity assessment in the literature with both synthetic conjugates and the BSA conjugates. The most thorough example was reported for testing the purity of the estrogen dendrimer conjugate after a methanol washing process [37]. Prior to purification, the authors spiked the estrogen-dendrimer mixture with a free, radiolabeled version of the ligand prior to dialysis and then monitored the amount of radioactivity that was still left bound to the dendrimer.…”