Darrell A. Austin

Involvement of Both Gq/11 and Gs Proteins in Gonadotropin-releasing Hormone Receptor-mediated Signaling in LβT2 Cells

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) stimulates the synthesis and release of the pituitary gonadotropins. GnRH acts through a plasma membrane receptor that is a member of the G protein-coupled receptor (GPCR) family. These receptors interact with heterotrimeric G proteins to initiate downstream signaling. In this study, we have investigated which G proteins are involved in GnRH receptor-mediated signaling in LβT2 pituitary gonadotrope cells. We have shown previously that GnRH activates ERK and induces the c-fos and LHβ genes in these cells. Signaling via the Gi subfamily of G proteins was excluded, as neither ERK activation nor c-Fos and LHβ induction was impaired by treatment with pertussis toxin or a cell-permeable peptide that sequesters Gβγ-subunits. GnRH signaling was partially mimicked by adenoviral expression of a constitutively active mutant of Gαq(Q209L) and was blocked by a cell-permeable peptide that uncouples Gαq from GPCRs. Furthermore, chronic activation of Gαq signaling induced a state of GnRH resistance. A cell-permeable peptide that uncouples Gαs from receptors was also able to inhibit ERK, c-Fos, and LHβ, indicating that both Gq/11 and Gs proteins are involved in signaling. Consistent with this, GnRH caused GTP loading on Gs and Gq/11 and increased intracellular cAMP. Artificial elevation of cAMP with forskolin activated ERK and caused a partial induction of c-Fos. Finally, treatment of Gαq(Q209L)-infected cells with forskolin enhanced the induction of c-Fos showing that the two pathways are independent and additive. Taken together, these results indicate that the GnRH receptor activates both Gq and Gs signaling to regulate gene expression in LβT2 cells.

Transcriptional Activation of the Ovine Follicle-Stimulating Hormone-β Gene by Gonadotropin-Releasing Hormone Involves Multiple Signal Transduction Pathways

GnRH regulates gonadotrope cells through GnRH receptor activation of the PKC-, MAPK-, and calcium-activated signaling cascades. Due to the paucity of homologous model systems expressing FSHβ, little is known about the specific mechanisms involved in transcriptional regulation of this gene by GnRH. Previous studies from our laboratory demonstrated that the gonadotrope-derived LβT2 cell line expresses FSHβ mRNA. In the present study we characterized the mechanisms involved in GnRH regulation of the FSHβ promoter using this cell model. Using transfection assays, we show that GnRH regulation of the ovine FSHβ promoter involves at least two elements, present between −4152/−2878 and −2550/−1089 bp, in association with one or several elements within the proximal region of the promoter. Surprisingly, the two activating protein-1 sites previously shown to be involved in the FSHβ response to GnRH in heterologous cells do not play a role in GnRH responsiveness in the gonadotrope cell model. Here we demonstrate that ...

PLC-1 Enzyme Activity Is Required for Insulin-Induced DNA Synthesis

Previously, we had shown that inhibition of PLC activity impaired the ability of insulin to activate ERK in 3T3-L1 adipocytes. In this study, we confirmed that the insulin receptor and PLC-1 are physically associated in hIRcB fibroblasts, insulin stimulates PLC-1 enzyme activity, and inhibition of PLC activity impairs activation of ERK. We subsequently investigated whether PLC-1 is required for insulin-stimulated mitogenesis. First, inhibition of PLC activity using U73122 impairs the ability of insulin to stimulate DNA synthesis. Second, disruption of the interaction of the insulin receptor with PLC-1 by microinjection of SH2 domains derived from PLC-1 or Grb2 but not Shc similarly blocks insulin-induced DNA synthesis. Third, microinjection of neutralizing antibodies to PLC-1 blocks DNA synthesis, but nonneutralizing antibodies do not. The blockade in all three cases is rescued by synthetic diacylglycerols but not by inositol-1,4,5trisphosphate, indicating a requirement for PLC enzyme activity. These experimental data point to a requirement for PLC-1 in insulin-stimulated mitogenesis in hIRcB cells. (Endocrinology 143: 655– 664, 2002)

Jun N-terminal protein kinase enhances middle ear mucosal proliferation during bacterial otitis media

ABSTRACT Mucosal hyperplasia is a characteristic component of otitis media. The present study investigated the participation of signaling via the Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase in middle ear mucosal hyperplasia in animal models of bacterial otitis media. Otitis media was induced by the inoculation of nontypeable Haemophilus influenzae into the middle ear cavity. Western blotting revealed that phosphorylation of JNK isoforms in the middle ear mucosa preceded but paralleled mucosal hyperplasia in this in vivo rat model. Nuclear JNK phosphorylation was observed in many cells of both the mucosal epithelium and stroma by immunohistochemistry. In an in vitro model of primary rat middle ear mucosal explants, bacterially induced mucosal growth was blocked by the Rac/Cdc42 inhibitor Clostridium difficile toxin B, the mixed-lineage kinase inhibitor CEP11004, and the JNK inhibitor SP600125. Finally, the JNK inhibitor SP600125 significantly inhibited mucosal hyperplasia during in vivo bacterial otitis media in guinea pigs. Inhibition of JNK in vivo resulted in a diminished proliferative response, as shown by a local decrease in proliferating cell nuclear antigen protein expression by immunohistochemistry. We conclude that activation of JNK is a critical pathway for bacterially induced mucosal hyperplasia during otitis media, influencing tissue proliferation.