Kelly E. Mayo

International Union of Pharmacology. XXXV. The Glucagon Receptor Family

Peptide hormones within the secretin-glucagon family are expressed in endocrine cells of the pancreas and gastrointestinal epithelium and in specialized neurons in the brain, and subserve multiple biological functions, including regulation of growth, nutrient intake, and transit within the gut, and digestion, energy absorption, and energy assimilation. Glucagon, glucagon-like peptide-1, glucagon-like peptide-2, glucose-dependent insulinotropic peptide, growth hormone-releasing hormone and secretin are structurally related peptides that exert their actions through unique members of a structurally related G protein-coupled receptor class 2 family. This review discusses advances in our understanding of how these peptides exert their biological activities, with a focus on the biological actions and structural features of the cognate receptors. The receptors have been named after their parent and only physiologically relevant ligand, in line with the recommendations of the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR).

Photic and circadian regulation of c-fos gene expression in the hamster suprachiasmatic nucleus

Photic information entrains a circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the mammalian hypothalamus to environmental light/dark cycles. To determine whether light regulates c-fos gene expression in the SCN, we have measured c-fos mRNA levels in the SCN of the golden hamster. We report that, during the subjective night, light causes a rapid increase in levels of c-fos mRNA in the SCN. Light pulses of 5 min duration are sufficient to induce c-fos mRNA, and the highest mRNA levels occur 30 min following the onset of light. The minimum level of illumination required to induce an increase in c-fos mRNA is indistinguishable from the minimum irradiance that produces a phase shift in the hamsters circadian rhythm of activity. In addition, the induction of c-fos mRNA in the SCN by light is itself under circadian regulation. Light induction of c-fos mRNA occurs only during the subjective night, at circadian times when photic phase shifting of activity occurs. Taken together, these data suggest that c-fos may be a molecular component of the photic pathway for entrainment of mammalian circadian rhythms.

Targeted Disruption of the Estrogen Receptor-α Gene in Female Mice: Characterization of Ovarian Responses and Phenotype in the Adult1

Targeted disruption of the mouse estrogen receptor-α gene (estrogen receptor-α knockout; ERKO) results in a highly novel ovarian phenotype in the adult. The ERKO mouse model was used to characterize ERα-dependent processes in the ovary. Visualization of the ovaries of 10-, 20-, and 50-day-old wild-type (WT) and ERKO mice showed that the ERKO phenotype developed between 20 and 50 days of age. Developmental progression through the primordial, primary, and antral follicle stages appeared normal, but functional maturation of preovulatory follicles was arrested resulting in atresia or in anovulatory follicles, which in many cases formed large, hemorrhagic cysts. Corpora lutea were absent, which also indicates that the normal biochemical and mechanical processes that accomplish ovulation were compromised. Northern and ribonuclease protection analyses indicated that ERKO ovary FSH receptor (FSHR) messenger RNA (mRNA) expression was approximately 4-fold greater than in WT controls. Ovarian LH receptor (LHR) mRNA ...

Light, Immediate-Early Genes, and Circadian Rhythms

Many diverse behaviors exhibit clear circadian rhythms in their expression. In mammals, these rhythms originate from a neural circadian clock located in the suprachiasmatic nuclei (SCN). Recently, signaling pathways activated by light in the SCN have begun to be identified. A specific set of immediate-early genes is induced by light in the SCN, and their expression is correlated with the resetting of circadian behavioral rhythms. These light-regulated immediate-early genes offer multiple inroads into the biology of the SCN: first, they are functional markers for the activation of SCN neurons by light; second, they can direct us to the upstream light-activated (and clock-regulated) signal transduction pathways which mediate their induction; and finally, they encode transcription factor proteins which may play a role in the molecular mechanism of resetting the circadian clock.

Effects of aging on light-induced phase-shifting of circadian behavioral rhythms, Fos expression and creb phosphorylation in the hamster suprachiasmatic nucleus

Aging is associated with a variety of alterations in circadian rhythms, including changes in the response to environmental stimuli. The underlying causes for these age-related changes in the circadian system remain unknown. Recent studies have demonstrated that light induces the expression of Fos and phosphorylation of the cyclic-AMP response element-binding protein in the rodent suprachiasmatic nuclei, the location of a master circadian pacemaker in mammals, suggesting that these transcription factors may mediate the effects of light on the circadian clock. The purpose of this study was to determine the effects of aging upon light-induced phase-shifting of circadian locomotor activity rhythms, Fos protein expression and cyclic-AMP response element-binding protein phosphorylation in the suprachiasmatic nuclei. Young (three to four months) and old (18-22 months) male golden hamsters free-running in constant darkness were exposed to 5-min monochromatic light pulses of different irradiance levels, at circadian time 19, after which either steady-state phase shifts of locomotor activity rhythms were measured, or else immunocytochemistry for Fos or for phospho-cyclic-AMP response element-binding protein was performed. Old hamsters were approximately 20 times less sensitive to the phase-shifting effects of light on the activity rhythm, and the photic irradiance threshold for Fos-like immunoreactivity induction in the suprachiasmatic nuclei was elevated when compared to young animals. Aging was also associated with a deficit in cyclic-AMP response element-binding protein phosphorylation by light. These data indicate that there are dramatic changes in light-activated molecular responses in the suprachiasmatic nuclei of old hamsters, and suggest that these molecular changes may underlie age-related changes in the effects of light on the circadian clock system.

Growth hormone-releasing hormone: synthesis and signaling.

The molecular characterization of GHRH and the GHRH receptor provides a framework for understanding the hypothalamic regulation of pituitary somatotroph function. The signaling events discerned from our investigation of GHRH receptor structure and function form the basis of a model for GHRH action, which is shown in Fig. 20. GHRH interaction with its seven transmembrane domain Gs-coupled receptor on the somatotroph (step 1) leads to the release of growth hormone from secretory granules (step 2), which is likely to involve a G protein-mediated interaction with ion channels, and to a stimulation of intracellular cAMP accumulation (step 3) (Mayo, 1992; Lin et al., 1992; Gaylinn et al., 1993). In several cell types tested, elevated cAMP leads to the phosphorylation and activation of the transcription factor CREB by protein kinase A (Gonzalez and Montminy, 1989; Sheng et al., 1991), and one target gene for CREB action is the pituitary-specific transcription factor Pit-1 or GHF-1 (step 4) (Bodner et al., 1988; Ingraham et al., 1988; McCormick et al., 1990). Pit-1 is a prototypic POU domain protein that is required for the appropriate regulation of the growth hormone gene in somatotroph cells, thus providing a pathway by which a GHRH signal can lead to increased growth hormone synthesis in the pituitary (step 5). In addition, Pit-1 is likely to directly regulate the synthesis of the GHRH receptor (step 6), in that the receptor is not expressed in the pituitary of dw/dw mice that lack functional Pit-1 (Lin et al., 1992), and a cotransfected Pit-1 expression construct can activate the GHRH receptor promoter in transiently transfected CV1 cells (Lin et al., 1993). It remains to be determined whether additional direct regulation of the GHRH receptor gene in response to the cAMP signaling pathway occurs (step 7). The inhibitory peptide somatostatin presumably interacts with this same signaling pathway through G protein-mediated suppression of the cAMP pathway (Tallent and Reisine, 1992; Bell and Reisine, 1993). In agreement with the importance of this signaling system for normal growth, a transgene encoding a nonphosphorylatable mutant CREB protein, which blocks the function of the endogenous CREB protein, is able to cause somatotroph hypoplasia and dwarfism in mice when its expression is targeted to pituitary somatotrophs (Struthers et al., 1991). Several steps in the signaling pathway leading to growth hormone secretion are subject to disruption, resulting in growth hormone deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)

Suppression of Notch Signaling in the Neonatal Mouse Ovary Decreases Primordial Follicle Formation

Notch signaling directs cell fate during embryogenesis by influencing cell proliferation, differentiation, and apoptosis. Notch genes are expressed in the adult mouse ovary, and roles for Notch in regulating folliculogenesis are beginning to emerge from mouse genetic models. We investigated how Notch signaling might influence the formation of primordial follicles. Follicle assembly takes place when germ cell syncytia within the ovary break down and germ cells are encapsulated by pregranulosa cells. In the mouse, this occurs during the first 4-5 d of postnatal life. The expression of Notch family genes in the neonatal mouse ovary was determined through RT-PCR measurements. Jagged1, Notch2, and Hes1 transcripts were the most abundantly expressed ligand, receptor, and target gene, respectively. Jagged1 and Hey2 mRNAs were up-regulated over the period of follicle formation. Localization studies demonstrated that JAGGED1 is expressed in germ cells prior to follicle assembly and in the oocytes of primordial follicles. Pregranulosa cells that surround germ cell nests express HES1. In addition, pregranulosa cells of primordial follicles expressed NOTCH2 and Hey2 mRNA. We used an ex vivo ovary culture system to assess the requirement for Notch signaling during early follicle development. Newborn ovaries cultured in the presence of gamma-secretase inhibitors, compounds that attenuate Notch signaling, had a marked reduction in primordial follicles compared with vehicle-treated ovaries, and there was a corresponding increase in germ cells that remained within nests. These data support a functional role for Notch signaling in regulating primordial follicle formation.

Gonadal Pathologies in Transgenic Mice Expressing the Rat Inhibinα -Subunit

Inhibin and activin are structurally related dimeric peptide hormones and are members of the TGF-beta superfamily of proteins. In the accompanying paper, we describe transgenic mice that overexpress the inhibin alpha-subunit gene from a metallothionein-I promoter (MT-alpha) and examine the effects of the MT-alpha transgene on gonadotropin levels and fertility. To characterize the effects of increased inhibin alpha-subunit on gonadal morphology and function, in this report we investigate gonadal histology, steroid hormone levels, and the basis of ovarian cyst formation in MT-alpha transgenic mice. MT-alpha transgenic female mice develop large fluid-filled ovarian cysts of follicular origin as early as 3 months of age. By 12 months of age, more than 92% of female MT-alpha transgenic mice develop ovarian cysts compared with less than 25% of wild-type littermates. Ovarian cysts form unilaterally or bilaterally, and cystic ovaries often have a greatly expanded bursal sac. Additionally, the ovaries of MT-alpha transgenic mice contain polyovular follicles and have fewer mature antral follicles and corpora lutea. MT-alpha female mice exhibit abnormal steroid hormone production, with increased serum T levels and reductions in serum E with corresponding reductions in uterine mass. In the MT-alpha transgenic males, testis size was decreased by 20-40% compared with control males, and there is a corresponding reduction in seminiferous tubule volume. After a chronic treatment with a GnRH antagonist, MT-alpha female mice continued to develop ovarian cysts and bursal sac expansions, although the cysts were markedly reduced in size. These results indicate that the expression of the rat inhibin alpha-subunit in mice results in significant ovarian pathology, reduced testicular size, and altered ovarian steroidogenesis. The antagonist studies are consistent with a direct ovarian effect of the alpha-subunit transgene product mediated by changes in the inhibin-to-activin ratio in these mice.

The Rat Growth Hormone-Releasing Hormone Receptor Gene: Structure, Regulation, and Generation of Receptor Isoforms with Different Signaling Properties

The interaction of GHRH with membrane-bound receptors on somatotroph cells of the anterior pituitary is an important step in the regulation of GH synthesis and secretion. The identification of a G protein-coupled receptor for GHRH has made it possible to investigate the pathway by which GHRH regulates pituitary somatotroph cell function. To initiate an analysis of the mechanisms regulating expression and function of the GHRH receptor, the structure of the gene and its promoter region were analyzed. The coding sequence of the rat GHRH receptor gene is contained within 14 exons spanning approximately 15 kb of genomic DNA. Four transcription start sites are located within 286 bp upstream of the initiation codon. The 59 flanking region of the GHRH receptor gene acts as a functional promoter in rat pituitary tumor GH3 cells, and basal promoter activity is enhanced in GH3 and COS7 cells by cotransfection of an expression construct encoding the pituitary-specific transcription factor Pit-1. The rat GHRH receptor gene is subject to at least 1 alternative RNA processing event that generates 2 receptor isoforms differing by 41 amino acids within the third intracellular loop (IL) of the protein. The short isoform of the GHRH receptor is predominant in pituitary cells. The MtT/S pituitary tumor cell line was found to express the GHRH receptor, and different populations of these cells produce predominantly the long or short isoforms of the receptor messenger RNA, suggesting that the alternative splicing can be regulated. Functional analysis of the two GHRH receptor isoforms demonstrates that both bind GHRH, but only the short isoform signals through a cAMPmediated pathway. Neither receptor isoform is able to stimulate calcium mobilization from internal stores after GHRH treatment. Our findings indicate that the pituitary-specific transcription factor Pit-1 is involved in the somatotroph-specific expression of the GHRH receptor gene and that functionally distinct receptor proteins are generated by an alternative RNA processing mechanism. (Endocrinology 140: 4152‐ 4165, 1999)

ROLES FOR TRANSFORMING GROWTH FACTOR BETA SUPERFAMILY PROTEINS IN EARLY FOLLICULOGENESIS

Primordial follicle formation and the subsequent transition of follicles to the primary and secondary stages encompass the early events during folliculogenesis in mammals. These processes establish the ovarian follicle pool and prime follicles for entry into subsequent growth phases during the reproductive cycle. Perturbations during follicle formation can affect the size of the primordial follicle pool significantly, and alterations in follicle transition can cause follicles to arrest at immature stages or result in premature depletion of the follicle reserve. Determining the molecular events that regulate primordial follicle formation and early follicle growth may lead to the development of new fertility treatments. Over the last decade, many of the growth factors and signaling proteins that mediate the early stages of folliculogenesis have been identified using mouse genetic models, in vivo injection studies, and ex vivo organ culture approaches. These studies reveal important roles for the transforming growth factor beta (TGF-beta) superfamily of proteins in the ovary. This article reviews these roles for TGF-beta family proteins and focuses in particular on work from our laboratories on the functions of activin in early folliculogenesis.