Mice with an RGS-insensitive Gα_i2 protein show growth hormone axis dysfunction

Highlights

• Alteration in the functional state of the GH-axis in growth-retarded mice bearing a mutation of Gα_i2 that rends this G protein insensitive to RGS.

• Homozygous GS/GS mice revealed a decrease in expression levels for pituitary receptors and hormones.

• Heterozygous (GS/+) mice exhibit lower stimulated pituitary GH release and a decrease in circulating levels of GH, IGF-1 and IGFBP-3.

Abstract

Mice carrying an RGS-insensitive Gαi2 mutation display growth retardation early after birth. Although the growth hormone (GH)-axis is a key endocrine modulator of postnatal growth, its functional state in these mice has not been characterized. The present study was undertaken to address this issue. Results revealed that pituitary mRNA levels for GH, prolactin (PRL), somatostatin (SST), GH-releasing-hormone receptor (GHRH-R) and GH secretagogue receptor (GHS-R) were decreased in mutants compared to controls. These changes were reflected by a significant decrease in plasma levels of GH, IGF-1 and IGF-binding protein-3 (IGFBP-3). Mutants were also less responsive to GHRH and ghrelin (GhL) on GH stimulation of release from pituitary primary cell cultures. In contrast, they were more sensitive to the inhibitory effect of SST. These data provide the first evidence for an alteration of the functional state of the GH-axis in Gαi2G184S mice that likely contributes to their growth retardation.

Introduction

Signal transduction via G protein coupled receptors (GPCR) is essential for controlling growth hormone (GH)-axis activity. Many neuropeptides and neurotransmitters involved in neuroendocrine regulation of the GH-axis target these receptors, which principally signal through four types of G proteins defined as Gi/o, Gs, Gq/11 and G12/13, according to the G alpha subunits (Wettschureck and Offermanns, 2005). Classically, GPCR-mediated signaling is initiated by receptor-ligand interaction that promotes the exchange of GDP to GTP on the α subunit of a G protein heterotrimer, resulting in dissociation of the Gα subunit from the βγ dimer. This is followed by activation or inhibition of downstream signaling effectors (enzymes, kinases and ion channels) and subsequent cellular responses (Sprang, 1997; Johnston and Siderovski, 2007). Signaling is then terminated when Gα-bound GTP is hydrolyzed to GDP by an intrinsic GTPase activity, allowing the Gα subunit to reassociate with the βγ dimer (Ross, 2008).

To appropriately terminate signaling, the Gα_i and Gα_q proteins require regulators of G protein signaling (RGS) which enhance the intrinsic GTPase activity by interacting with the Gα subunit, leading to more rapid G protein deactivation and termination of G protein signaling. We have previously reported enhanced Gα_i-mediated signaling in embryonic fibroblasts from mice carrying a genetic modification introducing a point mutation Gα_i2(G184S) that rends Gα_i2 protein insensitive to RGS effects (Huang et al., 2006). This results in a gain-of-function phenotype G_i2 signaling. These mice exhibit a complex phenotype affecting multiple organ systems, including heart, myeloid, skeletal, and central nervous system. They also display resistance to obesity. But the most apparent physiological phenotype of these mutant mice is their short stature. Specifically, they show growth retardation characterized by decreased body weight and length (Huang et al., 2006). To date, the mechanism underlying these observations is still unknown. More importantly, how prolonged Gα_i2 activation leads to growth retardation is not clear.

It is well known that GH plays a relevant role in postnatal growth. Humans and mice with GH deficiency show impaired longitudinal bone growth (Rosenfeld et al., 1994; Zhou et al., 1997; Ohlsson et al., 1998; Sjogren et al., 2000), and children of pathologically short stature are commonly treated with human GH (Labarta et al., 2009). The main source of this hormone is the somatotroph pituitary cells, where it is synthesized and then secreted in a pulsatile manner under the stimulatory effect of GH releasing hormone (GHRH) and the inhibitory action of somatostatin (SST), both delivered from hypothalamus. Ghrelin (GhL) expressed from the stomach and hypothalamus is also involved in hormonal regulation of GH release from the pituitary (Kineman and Luque 2007). Beside these three majors hypothalamic regulators, other hypothalamic peptides and neurotransmitters are involved in the modulation of GH secretion either at the pituitary level or via the hypothalamus, interacting with the GHRH or SST neurons (Muller et al., 1999a, Muller et al., 1999b).

Once released into the systemic circulation, GH regulates growth, either directly via its receptor or indirectly by inducing hepatic or local production of IGF-1 (Wood et al., 2005). The importance of indirect regulation in the postnatal growth is well demonstrated by genetic manipulation in mice. Indeed, most IGF-1 knockout mice die, and survivors display severe postnatal growth retardation (Liu et al., 1993; Baker et al., 1993). Interestingly, mice with a liver-specific knocking-out of IGF-1 showed intrauterine growth retardation, while their postnatal growth is normal due to GH-induced IGF-1 local production (Le Roith et al., 2001). Collectively, these observations establish the fundamental importance of the GH-axis in postnatal growth and prompted us to characterize the functional state of this axis in mice with the RGS-insensitive Gα_i2 protein to test the hypothesis that their growth retardation is associated with an aberration in the GH-axis function.