FRI286 Stimulation Of Egr1 Expression Is Mediated By A Gα(q/11)-signaling Pathway In A Pattern Distinct From That Of MEK/ERK At High And Low GnRH Pulse Frequencies

Disclosure: G.A. Stamatiades: None. H.K. Kim: None. R.S. Carroll: None. U.B. Kaiser: None. Introduction: The pulsatile release of the hypothalamic decapeptide, GnRH, activates signal transduction cascades in pituitary gonadotropes to control the synthesis and secretion of LH and FSH, hormones critical for normal reproductive function and fertility. LH and FSH release are differentially regulated by varying GnRH pulse patterns, with LH secretion being preferentially stimulated at high (e.g., every 30 min) GnRH pulse frequencies, while FSH secretion is preferentially induced at low (e.g., every 2 hour) GnRH pulses. LH is a heterodimer comprised of a common α subunit and a distinct LHβ subunit, with Lhb gene (encoding LHβ) transcription being preferentially stimulated at the higher GnRH pulse frequencies. It is well established that Egr1 is a key inducer of Lhb expression upon pulsatile GnRH stimulation. In addition, it has been shown that MEKI/II inhibition abrogates the induction of Lhb mRNA levels at both high and low GnRH pulse frequencies. Hypothesis:Egr1 expression is mediated by a Gα(q/11)-signaling pathway that includes MEK/ERK at both high and low GnRH pulse frequencies. Methods: To determine whether a Gα(q/11)-mediated pathway regulates Egr1 expression upon GnRH stimulation, LβT2 cells transduced with lentiviruses encoding scrambled or Gnaq/11 shRNA were perifused and treated with pulsatile GnRH at either high (q30min) or low (q2h) pulse frequencies, or with medium only, for 20 hours. Following the same perifusion paradigm, ERK1/2 phosphorylation was also assessed. Results: LβT2 cells transduced with a scrambled control shRNA showed a GnRH pulse frequency-dependent pattern of induction of Egr1 expression, with significantly greater induction at high than at low GnRH pulse frequency, compared to cells perifused with media only. In addition, Gα(q/11) depletion reduced Egr1 mRNA levels at both high and low GnRH pulse frequencies, but the frequency dependence was maintained. A GnRH pulse frequency-dependent pattern of induction of ERK1/2 phosphorylation was observed, with significantly greater induction at low than at high GnRH pulse frequency in control cells transduced with scrambled control shRNA, consistent with prior studies from our group. Following Gnaq/11 KD, the induction of ERK1/2 phosphorylation was reduced at both GnRH pulse frequencies, with loss of the pulse frequency dependence. Conclusion: Taken together, these findings suggest that a Gα(q/11)-signaling pathway involving Egr1 induces Lhb expression at both pulse frequencies. However, the link between MEK/ERK and Egr1 needs to be further investigated, given the differences in the GnRH pulse frequency dependent patterns of induction. Presentation: Friday, June 16, 2023