GnRH Pulse Frequency-Dependent Regulation of FSHβ Expression Is Mediated By Both Gas and Gaq/11 Proteins
Presentation Number: SUN 491
Date of Presentation: April 2nd, 2017
George A. Stamatiades*1, Iain R. Thompson1, Rona S. Carroll2 and Ursula B Kaiser1
1Brigham and Women's Hospital/Harvard Medical School, Boston, MA, 2Brigham and Women's Hospital/Harvard Med School, Boston, MA
The pulsatile release of the hypothalamic decapeptide, GnRH, activates signal transduction cascades in the pituitary gonadotrope to control the synthesis and secretion of FSH and LH, hormones critical for normal reproductive function and fertility. FSH contains a distinct FSHβ subunit, preferentially stimulated at low (every 2 h) rather than high (every 30 min) GnRH pulse frequencies. GnRH binds to its G protein-coupled receptor, GnRHR, which interacts with heterotrimeric G proteins to initiate downstream signaling. It has been demonstrated previously that GnRH stimulates both GnRHR-associated Gas and Gaq/11-mediated signaling pathways in the murine LbT2 gonadotrope-derived cell line. To identify the role of Gas and Gaq/11 proteins in the GnRH pulse frequency-dependent regulation of Fshb expression, knockdown (KD) of Gas and Gaq/11 by lentiviral shRNA transduction was performed in LbT2 cells. Effective KD efficiency was demonstrated by western blot analysis. Gas KD markedly reduced the induction of Fshb mRNA by treatment with continuous GnRH for 8 h, whereas Gaq/11 KD did not significantly alter GnRH-stimulated Fshb mRNA levels, compared to controls. Next, LbT2 cells transduced with scrambled, Gas, or Gaq/11 shRNA were perifused and treated with pulsatile GnRH at varying pulse frequencies (every 30 min or 2 h) for 20 h. As expected, LβT2 cells transduced with a scrambled control shRNA showed a GnRH pulse frequency-dependent pattern of induction of Fshb expression, with significantly greater induction at low than at high GnRH pulse frequency, compared to cells perifused with media only. In contrast, in Gas KD cells, the induction of Fshb mRNA levels was reduced at low GnRH pulse frequency to an extent such that the pulse frequency-dependent pattern of induction was lost. On the other hand, in Gaq/11 KD cells, the induction of Fshb mRNA levels was significantly reduced at high GnRH pulse frequency, with no effect on the response to the low GnRH pulse frequency. In conclusion, these findings suggest that both Gas and Gaq/11 modulate GnRH pulse frequency-dependent Fshb expression in LbT2 cells. Gas-stimulated pathways mediate Fshb expression in response to continuous GnRH as well as to pulsatile GnRH at low GnRH pulse frequencies, whereas induction of Fshb at high frequencies of pulsatile GnRH occurs via Gaq/11-stimulated pathways. These findings offer further insight into the mechanisms by which the gonadotrope decodes the pulsatile GnRH signal to differentially regulate FSH.
Nothing to Disclose: GAS, IRT, RSC, UBK