Intracellular Signaling In Endocrine Cells

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

We continue to investigate genes expressed in mammalian hypothalamic and pituitary cells and their roles in cellular signaling and function. Our recent work has focused on protein tyrosine phosphatase receptor type N and N2 (PTPRN and PTPRN2) and phosphatidylinositol 4-kinase alpha (PI4KA) genes and their products. PTPRN1 and PTPRN2 are atypical members of the protein tyrosine phosphatase receptors. They are pseudophosphatase due to mutation of two residues in the catalytic domain, but they exhibit other cellular functions, through a still unknown mechanism. These genes are expressed in the hypothalamic and pituitary neuroendocrine cells, but their knockout only affected hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal functions. Both gonadotropin-releasing hormone- (GnRH) and kisspeptin-secreting neurons also express Ptprn+Ptprn, as do pituitary gonadotrophs, but double knockout (DKO) of these genes did not directly affect the function of GnRH neuron and gonadotrophs. However, kisspeptin staining was significantly reduced in both the arcuate nucleus and the RP3V region of DKO female mice. This in turn inhibited the expression of Gnrh1 in GnRH neurons and Lhb, Fshb, and Gnrhr in pituitary gonadotrophs, as well as the accumulation and release of LH. This was accompanied by significant changes in ovarian steroidogenesis and gene expression in DKO females, resulting in the absence of puberty and development of female reproductive organs. In contrast, no changes were observed in testicular steroidogenesis and spermatogenesis, nor in seminal vesicles development in DKO males. In contrast, DKO increased expression of the corticotroph/melanotroph genes Pomc and Tbx19 and the melanotroph-specific gene Pax7, and the effect was observed in both females and males. We also found in vivo and in vitro increased synthesis and release of beta-endorphin, alpha-MSH, and ACTH in DKO mice, which was associated with increased serum corticosterone levels and adrenal mass. DKO also increased expression of other melanotroph-specific genes, but not corticotroph-specific genes. Furthermore, hyperplasia of the intermediate lobe was observed in DKO females and males, accompanied by increased POMC immunoreactivity per cell. These results indicate that PTPRNs contribute to hypothalamic-pituitary-adrenal function by being involved in processes that regulate postnatal melanotroph development and Pomc expression. Therefore, although all hypothalamic and pituitary cells express Ptprn and Ptprn2 gene, their DKO affects only the function of specific cells. This is consistent with the hypothesis that PTPRNs act as transcription factors or upstream elements in the control of gene transcription. The cellular specificity of PTPRNs action is consistent with the specificity of promoter activation and repression for different genes, as suggested by the failure of DKO to increase Pomc expression in the hypothalamus but facilitate its expression in the pituitary. PI4KA is an enzyme that contributes to the production of phosphoinositide PI4P, which serve as a substrate to produce PI(4,5)P2, and PI(3,4,5)P3. We are studying the function of this gene by its specific knockout in hypothalamic and pituitary cells. Knockout of this gene in pituitary gonadotrophs did not alter embryonic establishment and neonatal development and function of the gonadotroph population. However, during the postnatal period, there was a progressive loss of expression of gonadotroph-specific genes, including Fshb, Lhb, and Gnrhr, accompanied by loss of GnRH receptor signaling and reproductive functions in female and male mice. Intracellular administration of inositol-1,4,5-trisphosphate rescued GnRH receptor signaling, indicating that gonadotrophs did not fully dedifferentiate. In further studies on this project, we investigated the role of PI4KA in the postnatal differentiation of gonadotrophs from Sox2-expressing cells. Gonadotrophs and Sox2-expressing cells were visualized by immunostaining, and gonadotrophs were also identified by the specific expression of the fluorescent protein tdTomato during embryonic and postnatal development. Our experiments showed the existence of three populations of Sox2-positive cells in the mouse pituitary: anterior parenchymal Sox2 single and small cluster cells, Sox2 cells in marginal zone located between anterior and intermediate pituitary lobes, and Sox2 expression by pituicytes in the posterior pituitary. Gonadotrophs are localized in the anterior parenchyma separate from Sox2-expressing cells. In the marginal zone, we visualized the functions of Sox2 cells as stem cells for gonadotroph differentiation by the gradual substitution of nuclear Sox2 with tdTomato, and the appearance of LHB in the cytoplasm, as well as the migration of newly formed gonadotrophs into the parenchyma and their transition to secretory cell types. Gonadotroph differentiation was most pronounced during the juvenal-prepubertal period and was virtually silent in the adult pituitary. Juvenile-prepubertal gonadotroph differentiation was decreased by PI4KA knockout in these cells, which caused a significant reduction in the size of the gonadotroph population. This was accompanied by a progressive loss of gonadotroph-specific gene/protein expression and an increase in the number of regressed cells expressing tdTomato. Current studies on this topic are focused on the role of Sox2 -expressing cells in the marginal zone in differentiating into other hormone-producing cells and the relationship between stem cells and folliculostellate cells. We also generated GnRH-neuron-specific Pi4ka knockout (KO) to study the role of PI4KA in these cells. KO animals were healthy and indistinguishable from their control littermates in size. However, they were infertile and lacked external features normally associated with sexual maturity (vaginal opening in females and preputial separation in males). Immunohistochemistry against GnRH revealed the hypothalamus of adult KO mice devoid of immunoreactive GnRH neurons. In contrast, immunohistochemistry against kisspeptin showed a comparable sex-specific pattern of expression in control littermates and KO mice. At 3 days of age, Gnrh1 expression levels were similar in KO mice and control littermates, but expression of this gene progressively declined in KO mice and was undetectable by 45 days of age. Immunofluorescence against GnRH at 10 days of age revealed morphological changes in GnRH neurons that preceded their disappearance. In contrast, Kiss1 expression was similar in KO mice and control littermates until the onset of puberty, when the lack of sex steroid regulation in KO mice affected expression of this gene. The Gnrh1-Cre mouse line used to generate KO mice has strong ectopic expression within the lateral septum, as demonstrated by expression of the tdTomato reporter. However, these cells did not display abnormal morphology in KO mice and persisted into adulthood. Therefore, the PI4KA-dependent signaling pathway is not required for the migration of GnRH neurons into the hypothalamus, but is required for their survival, unlike the ectopic cell type. Current experiments aim to characterize this cell-type-specific signaling pathway.