Intracellular Signaling In Endocrine Cells

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

We continue investigations on genes expressed in mammalian pituitary cells and their role in pituitary cell signaling and function. Our experiments on transcriptome profiles of secretory and nonsecretory cell types using single cell RNA sequencing (scRNAseq) of freshly dispersed pituitary cells revealed the presence of six hormone-producing cell types melanotrophs, corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. We also identified four non-hormonal cell types folliculostellate cells (FSCs), pituicytes, and vascular endothelial cells and pericytes. We recently summarized scRNAseq and immunohistofluorescence analyses of pituitary cells of adult female rats with a focus on transcriptomic profiles of nonhormonal cell types. Samples obtained from whole pituitaries and separated anterior and posterior lobe cells contained all expected pituitary resident cell types and lobe-specific vascular cell subpopulations. FSCs and pituicytes expressed S100B, ALDOC, EAAT1, ALDH1A1, and VIM genes and proteins, as well as other astroglial marker genes, some common and some cell type specific. We also found that the SOX2 gene and protein were expressed in 15% of pituitary cells, including FSCs, pituicytes, and a fraction of hormone-producing cells, arguing against its stem cell-specificity. FSCs comprised two Sox2-expressing subclusters; FSC1 contained more cells but lower genetic diversity, while FSC2 contained proliferative cells, shared genes with hormone-producing cells, and expressed genes consistent with stem cell niche formation, regulation of cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. FSC1 cells were randomly distributed in the anterior and intermediate lobes, while FSC2 cells were localized exclusively in cells of the marginal zone between the anterior and intermediate lobes. These data indicate the identity of these cells as specialized anterior pituitary-specific astroglia, with FSC1 representing differentiated cells with transcriptomes consistent with classical FSC roles and FSC2 exhibiting additional stem cell-like features. All pituitary hormone-producing cells expressed common genes related to secretory functions, such as sister genes coding for regulated endocrine-specific protein 18, Resp18, and the protein tyrosine phosphatase receptor genes Ptprn and Ptprn2, as well as Chga, Chgb, Scg2, Snap25, and Uchl1. Unlike cell type-specific hormone and hormone receptor genes, the roles of these common genes are not well characterized. Our recent studies confirmed that simultaneous knockout of the neuroendocrine marker genes Ptprn and Ptprn2 causes infertility in female mice while males are fertile. To elucidate the mechanism of the sex-specific roles of Ptprn and Ptprn2 in mouse reproduction, we further analyzed the effects of their double knockout (DKO) on the hypothalamic-pituitary-gonadal axis. In DKO females, delayed puberty and lack of ovulation were observed, complemented by changes in ovarian gene expression and steroidogenesis. In contrast, testicular gene expression, steroidogenesis, and reproductive organs development were not significantly affected in DKO males. However, in both sexes, pituitary luteinizing hormone (LH) beta gene expression and LH levels were reduced, as well as follicle-stimulating hormone beta gene and gonadotropin-releasing hormone (GnRH) gene, while the calcium-mobilizing and LH secretory actions of GnRH were preserved. Hypothalamic Gnrh1 and Kiss1 gene expression was also reduced in DKO females and males. In parallel, a significant decrease in the density of immunoreactive GnRH and kisspeptin fibers was detected in the hypothalamic arcuate nucleus of DKO females and males. The female-specific kisspeptin immunoreactivity in the rostral periventricular region of the third ventricle was also reduced in DKO females, but not in DKO males. These data indicate a critical role of Ptprn and Ptprn2 in kisspeptin-GnRH neuronal function and sexual dimorphism in the threshold levels of GnRH required to preserve reproductive functions. Ongoing experiments on this topic are focuses on the physiological status of anterior pituitary corticotrophs and intermediate lobe-located melanotrophs of DKO mice. In collaboration with the group of Dr. Balla at the NICHD, we are also studying the functions of three phosphoinositides, PI4P, PI(4,5)P2, and PI(3,4,5)P3, in cellular signaling and exocytosis, focusing on hormone-producing pituitary cells. PI(4,5)P2, acting as a substrate for phospholipase C, plays a key role in the control of pituitary cell functions, including hormone synthesis and secretion. PI(4,5)P2 also acts as a substrate for class I PI3-kinases, leading to the generation of two intracellular messengers, PI(3,4,5)P3 and PI(3,4)P2, which act through their intracellular effectors, including Akt. PI(4,5)P2 can also influence the release of pituitary hormones acting as an intact lipid to regulate ion channel gating and concomitant calcium signaling, as well as the exocytic pathway. Recent findings also showed the expression of several PI lipid kinases, including Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in pituitary lactotrophs, which are responsible for the secretion of prolactin (PRL), a hormone controlling lactation. Using a pharmacological approach to specifically inhibit these enzymes we show that PI4P made in the plasma membrane by PI4KA is critical for exocytosis without affecting the calcium signals that trigger secretion. Our experiments also indicate that inhibition of the PI4KB enzyme that generates PI4P in the Golgi is dispensable for the exocytic step. These experiments revealed a key role of PI4KA-derived PI4P in the plasma membrane in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4,5)P2-dependent calcium signaling. The ongoing study on this topic is focused on the role of PI4KA in gonadotroph function by knocking out this enzyme in cells expressing the GnRH receptor. Knockout mice were infertile, reflecting underdeveloped gonads and reproductive tracts, and lack of puberty. The number and distribution of hypothalamic GnRH neurons and Gnrh1 expression in postnatal knockouts were not affected, while Kiss1/kisspeptin expression was increased. Knockout of PI4KA also 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 and Lhb, accompanied by low synthesis of gonadotropins, but not of other pituitary lineage-specific genes and their hormones. The postnatal gonadotroph population also progressively declined, reaching approximately 25% of that observed in controls at 100 days of age. In these residual gonadotrophs, GnRH-dependent calcium signaling, and calcium-dependent membrane potential changes were lost, but intracellular administration of inositol-1,4,5-trisphosphate rescued this signaling. These results indicate a key role for PI4KA in the postnatal development and maintenance of a functional gonadotroph population.