citations

                    Proteins and Autocrine Signaling Differentially Regulate Gonadotropin Subunit Expression in Pituitary Gonadotrope
                

Authors:

Soon-Gang Choi, Jingjing Jia, Robert L. Pfeffer, and Stuart C. Sealfon

In:

Source: J Biol Chem
Publication Date: (2012)
Issue: 287 (25): 21550–21560

Research Area:

Gene Expression

Basic Research

Cells used in publication:

LbetaT2: Species: mouse; Tissue Origin: pituitary gland

Platform:

4D-Nucleofector® 96-well Systems

Experiment

In order to understand the specific Galpha subunit involved in the GnRHR signaling, sveral KO experiences with the different subunit were perfromed using G(alpha)s,q,11,12,G13 siRNAs. 1 million LT2 cells were transfected with 0.5 ug of siRNA using Amaxa 96 wells Shuttle with SG buffer and 96-DS-137 nucleofection program. After siRNA transfection, cells were seeded on a cell culture plate supplemented with 10% FBS DMEM and incubated at 37 ºC in 5% CO2 incubator.

Abstract

Gonadotropin-releasing hormone (GnRH) acts at gonadotropes to direct the synthesis of the gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH). The frequency of GnRH pulses determines the pattern of gonadotropin synthesis. Several hypotheses for how the gonadotrope decodes GnRH frequency to regulate gonadotropin subunit genes differentially have been proposed. However, key regulators and underlying mechanisms remain uncertain. We investigated the role of individual G proteins by perturbations using siRNA or bacterial toxins. In LT2 gonadotrope cells, FSH gene induction depended predominantly on Gq/11, whereas LH expression depended on Gs. Specifically reducing Gs signaling also disinhibited FSH expression, suggesting the presence of aGs-dependent signal that suppressed FSH biosynthesis. The presence of secreted factors influencing FSH expression levels was tested by studying the effects of conditioned media from Gs knockdown and cholera toxin-treated cells onFSH expression. These studies and related Transwell culture experiments implicate Gs-dependent secreted factors in regulating both FSH and LH gene expression. siRNA studies identify inhibin as a Gs-dependent GnRH-induced autocrine regulatory factor that contributes to feedback suppression of FSH expression. These results uncover differential regulation of the gonadotropin genes by Gq/11 and by Gs and implicate autocrine and gonadotrope-gonadotrope paracrine regulatory loops in the differential induction of gonadotropin genes.