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 pituitary cells and their roles in signaling and hormone secretion, focusing on the transcriptome profiles of secretory and non-secretory cell types using single cell RNA sequencing (scRNAseq) of freshly dispersed pituitary cells from adult female rats. We identified six hormone-producing cell types melanotrophs, corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. We also identified four nonhormonal cell types folliculostellate cells (FSCs), pituicytes, and vascular endothelial cells and pericytes. All hormone-producing cells expressed common genes related to secretory functions, such as genes coding for regulated endocrine-specific protein 18, Resp18, Chga, Chgb, Scg2, Snap25, and Uchl1, as well as the cell type-specific hormone and hormone receptor genes. No evidence was found for the existence of multihormonal - multireceptor types of cells. Genes expressed specifically among all vascular and nonhormonal cell types, but not hormone-producing cells, could also be identified, such as follistatin-related protein 1, Fstl1. FSCs and pituicytes both expressed S100b, historically viewed as a definitive marker for FSCs in the rat anterior pituitary. Both cell types also expressed Aldoc, a recently reported marker for FSCs in both mouse and rat. Notably, S100b and Aldoc are also markers of astrocytes. The corticotroph transcriptome profile was most comparable to melanotrophs and generally agrees with previous basic and clinical work with these cells. However, data on pituitary scRNAseq from several species indicate homogeneity of postnatal corticotrophs, forming a distinct cluster of cells, compared to melanotrophs, other hormone-producing cells, and non-hormonal pituitary cells. Genes specific for corticotrophs include Clrn1, Chrna1, Adh1, Angptl8, Hspb3, Lmx1a, Scube2, and Trdn. The roles of these genes in corticotroph functions have not been characterized. Certainly, the most critical genes for corticotroph functions are Crhr1 and Avpr1, encoding G protein-coupled receptors CRHR1 and AVPR1b, activated by hypothalamic CRH and AVP, respectively. Avpr1b is specifically expressed in corticotrophs, whereas Crhr1 has been detected in some melanotrophs as well. Other genes are specific for melanotrophs, including Oacyl, Pax7, Esm1, and Pcsk2. Moreover, scRNAseq data provide a wealth of new information regarding the expression of a number of common genes encoding other G protein-coupled receptors and enzyme-linked plasma membrane receptors, and their signal transduction pathways, which have not previously been reported to be expressed in the pituitary gland. The expression pattern of these receptors and their ligands highlight the importance of autocrine/paracrine regulation of pituitary cell function and the modulating role of peripheral glands through nuclear receptors. Contrary to the hypothesis of pituitary cell plasticity, gonadotrophs also appear as a single cluster of homogeneous cells, uniquely expressing Fshb, Lhb, and Gnrhr. These cells also specifically express other genes, including Chrna4, Cnga1, Dmp1, Dusp15, Icam5, Lama1, Nhlh2, Nr5a1, Pitx3, Spp1, Tgfbr3l, and Vash2. Some of these genes are clearly expressed in a sex-specific manner, like Dmp1 and its sister gene Spp1. In general, the specific roles of these genes in gonadotroph functions have not yet been elucidated. Our studies point to the gonadotroph-specific patterns of gene expression and hormone secretion. The LH secretory profiles appear to reflect depletion of prestored LH in secretory vesicles by regulated exocytosis. In contrast, FSH is predominantly released by constitutive exocytosis, and secretory activity reflects the kinetics of Fshb gene expression controlled by GnRH, activin and inhibin. Consistent with the role of activin and inhibin on Fshb expression, pituitary cells express three inhibin subunit genes: Inha is expressed in all hormone-producing cell types and FSCs in the anterior pituitary and pituicytes in the posterior pituitary; Inhba is expressed only in FSCs and pituicytes, and Inhbb is expressed in gonadotrophs, corticotrophs, FSCs, pituicytes, and pituitary endothelial cells. In contrast to corticotrophs and gonadotrophs, the Pou1f1-derived cell types contained fewer dominant and specific genes. The 13 lactotrophs-specific genes we identified included not only Prl, Drd2, and Agtr1b, the well-known marker genes for these cells, but also Krt25, a type-I keratin gene; Asic4, an acid sensing ion channel subunit; Irx6, Iroquois homeobox 6; and Klk1b3, kallikrein 1-related peptidase B3. Lactotrophs and other hormone-producing cells also express phosphatidylinositol (PI) kinase genes Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c. In further studies, we analyzed the contribution of phosphatidylinositol kinases to calcium-driven prolactin (PRL) release in pituitary lactotrophs: PI4Ks - which control PI4P production, PIP5Ks - which synthesize PI(4, 5)P2 by phosphorylating the D-5 position of the inositol ring of PI4P, and PI3KCs which phosphorylate PI(4, 5)P2 to generate PI(3, 4, 5)P3. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Wortmannin, a PI3K and PI4K inhibitor, but not LY294002, a PI3K inhibitor, blocked spontaneous action potential driven PRL release with a half-time of 20 min when applied in 10 M concentration, leading to accumulation of intracellular PRL content. Wortmannin also inhibited increase in PRL release by high potassium, the calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanying calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium-driven PRL secretion without affecting calcium signaling and Prl expression. In contrast, PIK93, a specific inhibitor of PI4KB, and ISA2011B and UNC3230, specific inhibitors of PIP5K1A and PIP5K1C, respectively, did not affect PRL release. These experiments revealed a key role of PI4KA in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4, 5)P2-dependent calcium signaling. Recently, we presented scRNAseq and immunohistofluorescence analyses of pituitary cells of adult female rats with a focus on the 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; FS1 contained more cells but lower genetic diversity, while FS2 contained proliferative cells, shared genes with HPCs, and expressed genes consistent with stem cell niche formation, regulation of cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. FS1 cells were randomly distributed in the anterior and intermediate lobes, while FS2 cells were localized exclusively in cells of the marginal zone between the anterior and intermediate lobes. These data indicate the identity of the FSCs as specialized anterior pituitary-specific astroglia, with FS1 cells representing differentiated cells with transcriptomes consistent with classical FSC roles and FS2 cells exhibiting additional stem cell-like features.