Intracellular Responses to Gonadotropin-Releasing Hormone in a Clonal Cell Line of the Gonadotrope Lineage

Abstract

We recently derived a GnRH-responsive pituitary cell line of the gonadotrope lineage (alpha T3-1) by targeted oncogenesis in transgenic mice. Here, we report studies characterizing the GnRH receptors present in these cells and the intracellular responses to GnRH treatment. The receptors in alpha T3-1 cells show specificity for different GnRH analogs, with dissociation constants very similar to those found in normal rat and mouse pituitary. The concentration of receptors is within the range found in normal pituitary. The addition of GnRH or GnRH agonists increases phosphoinositide turnover and protein kinase-C translocation to membranes, and enhances activation of voltage-sensitive calcium channels. However, GnRH does not affect cAMP levels. Analysis of alpha-subunit mRNA levels demonstrated induction by GnRH and phorbol esters. Our results indicate that GnRH initiates a cascade of intracellular events that generate a set of second messengers, one or more of which is involved in the regulation of gene expression. The responses of alpha T3-1 cells to GnRH appear to have characteristics equivalent to those of primary pituitary gonadotropes, indicating the utility of this cell line as a model system for the study of GnRH responses.

GHF-1-promoter-targeted immortalization of a somatotropic progenitor cell results in dwarfism in transgenic mice.

“…Mellon, in prep.). Remarkably, these cells are apparently frozen at the step in development at which the specific regulatory region is first activated and maintain the characteristics representing sequential developmental steps in the gonadotropic lineage (Sealfon et al 1990;Windle et al 1990;Horn et al 1991Horn et al , 1992Mellon et al 1991;Schoderbek et al 1992~ Tsutsumi et al 1992. Given that immortalization targeted by the regulatory regions of genes for sequentially expressed known cellular products results in cells representing different steps in a developmental lineage, we reasoned that it might be possible to target successively earlier progenitor cells using the regulatory regions of genes encoding the transcriptional regulators that activate the expression of such cell typespecific markers.…”

Absence of pituitary adenylate cyclase-activating polypeptide-stimulated transcription of the human glycoprotein alpha-subunit gene in LbetaT2 gonadotrophs reveals disrupted cAMP-mediated gene transcription

“…Furthermore, the increase in GSU mRNA transcripts seen in response to PACAP is not altered following PKC depletion (Tsujii et al 1995). Many of these studies have used the murine gonadotroph progenitor cell line, T3-1, which is representative of gonadotroph precursor cells of embryonic day e13·5, expressing SF-1 and the GnRH receptor (Windle et al 1990, Horn et al 1991. Recently, investigations of gonadotrophin gene expression have employed the more differentiated L T2 cell line (Weck et al 1998, Liu et al 2002, representative of murine gonadotrophs at e17·5-18·5.…”

Tissue-specific gene expression in the pituitary: the glycoprotein hormone alpha-subunit gene is regulated by a gonadotrope-specific protein.

“…Most importantly for the studies presented here, the tumors derived from these mice were cultured to derive clonal cell lines (e.g., aT3-1 cells) which continue to produce the endogenous mouse ac subunit and express the human a-Tag transgene. These cells also display functional gonadotropin-releasing hormone receptors (21) (though they do not respond to thyrotropin-releasing hormone), indicating their origin as the gonadotrope lineage (55). We have proposed that these cells represent a precursor to the gonadotrope lineage which is immortalized by the early expression of Tag directed by the al-subunit gene upstream region during development perhaps as early as day ell.5.…”