GNRH Induces the Unfolded Protein Response in the LβT2 Pituitary Gonadotrope Cell Line

Abstract

The neuropeptide GNRH 1 stimulates the secretion of the reproductive hormone LH in pituitary gonadotropes. Other secretory cell types depend on the unfolded protein response (UPR) pathway to regulate protein synthesis and protect against endoplasmic reticulum (ER) stress in response to differentiation or secretory stimuli. This study investigated the role of the UPR in GNRH action within the LbetaT2 gonadotrope model. Cells were treated with GNRH, and the activation of UPR signaling components and general translational status was examined. The ER-resident stress sensors, Atf6, Eif2ak3, and Ern1, are all present, and GNRH stimulation results in the phosphorylation of eukaryotic translation initiation factor 2A kinase 3 and its downstream effector, eukaryotic translation initiation factor 2A. Additionally, activation of the UPR was confirmed both in LbetaT2 as well as mouse primary pituitary cells through identifying GNRH-induced splicing of Xbp1 mRNA, a transcription factor activated by splicing by the ER stress sensor, ER to nucleus signaling 1. Ribosome profiling revealed that GNRH stimulation caused a transient attenuation in translation, a hallmark of the UPR, remodeling ribosomes from actively translating polysomes to translationally inefficient ribonucleoprotein complexes and monosomes. The transient attenuation of specific mRNAs was also observed. Overall, the results show that GNRH activates components of the UPR pathway, and this pathway may play an important physiological role in adapting the ER of gonadotropes to the burden of their secretory demand.

GnRH Regulates Gonadotropin Gene Expression Through NADPH/Dual Oxidase-Derived Reactive Oxygen Species

“…Mice were sacrificed using CO 2 asphyxiation followed by cervical dislocation, in accordance with UCSD Institutional Animal Care and Use Committee regulations. Whole pituitaries were isolated into ice-cold PBS and then dispersed as previously described (32). After plating in 48-well Costar tissue culture plates (Corning Life Sciences, Tewksbury, MA) precoated with poly-d-lysine (Sigma-Aldrich, St. Louis, MO) to aid adherence, the cells were allowed to recover for at least 48 hours at 37 C and 5% CO 2 in culture medium before studies were conducted.…”

Translational control of gene expression in the gonadotrope

“…EIF2AK3 phosphorylation is induced by GNRH in LβT2 cells, but is unique in that the overall level of activation is moderate in comparison to the typical pharmacological insult used in most studies (Do et al, 2009). Similarly, EIF2A phosphorylation is moderate as well.…”

“…Accordingly, both Atf4 mRNA and its target gene Ddit3 (also called CHOP) are increased in response to GNRH stimulation, and have a peak activation level under tonic rather than pulsatile stimulation, indicating an elevated stress response under tonic stimulation (Lawson et al, 2007). Interestingly, GNRH induction of the UPR results in an overall remodeling of the polyribosome profile in LβT2 cells that is also modest in comparison to pharmacological induction of the UPR by dithiothreitol(Do et al, 2009). Both Cga and Lhb mRNAs are targeted by this inhibition, as would be expected of secretory proteins passing though the ER.…”

Polyribosome and ribonucleoprotein complex redistribution of mRNA induced by GnRH involves both EIF2AK3 and MAPK signaling

“…Another regulatory cascade initiated by GnRH treatment is the unfolded protein response, or UPR (Do et al, 2009). This pathway involves both transcriptional and protein synthesis regulatory arms that serve to minimize endoplasmic reticulum (ER) stress.…”

“…We have shown that GnRH induces the UPR in gonadotropes and in the cultured gonadotrope cell line LβT2 (Do et al, 2009). Induction of the UPR is transient in LβT2 cells, resolving within 60 minutes.…”