Intracellular Signaling In Endocrine Cells

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

We continue investigations on receptors and channels expressed in neuroendocrine and endocrine cells and their roles in signaling, gene transcription, and hormone secretion. To gain better understanding of the cell type-specific expression and role of these and other proteins in anterior pituitary cell functions and related disorders, we extended our single cell RNA sequencing on freshly dispersed pituitary cells from adult female rats to characterize the transcriptome profiles of secretory and non-secretory cell types, focusing on their heterogeneity, comparison between folliculostellate cells (FSCs) and pituicytes, and stem cell identification. Hormone-producing cells were identified by common expression of dense core secretory vesicle protein genes, and the cell type-specific expression of genes encoding hormones and G protein-coupled receptors; no evidence was found for the existence of multihormonal/multireceptor types of cells. A common feature of non-hormonal FSCs, pituicytes, pericytes, and endothelial cells is the expression of S100a genes and genes encoding endogenous ligands and extracellular matrix proteins. FSCs are heterogeneous, with at least two subclusters: the more differentiated FSC1 cell, and the less differentiated FSC2 cells. Sox2-positive cells were also heterogeneous populations, expressed in most FSC1, FSC2, and pituicytes. These cells also express Sox9, Hes1, Cd9, and Vim, while Prop1 and Prrx1 are expressed in FSCs, and Lhx2 and Tbx3 in pituicytes only. Further comparison of FSCs and pituicytes revealed high astroglial marker gene scores for both cell types, including the expression of S100b. These results indicate cell-type-specific differentiation of hormone-producing cells, genetic and functional similarity between FSCs and pituicytes, including their potential capacity to regenerate and transit to other cell types, and the role of pericytes and endothelial cells in pituitary functions other than blood supply. In mammalian cells, extracellular protons act as orthosteric and allosteric ligands for multiple receptors and channels. The aim of our recent study was to identify proton sensors in the rat pituitary gland. qRT-PCR analysis indicated the expression of G protein-pituitary cells and Asic1 and Asic2 in immortalized GH3 pituitary cells. Asic1a and Asic2b were the dominant splice isoforms. Single anterior pituitary cell RNA sequencing and immunocytochemical analysis showed that nonexcitable FSC express GPR68 gene and protein, whereas excitable secretory cells express ASIC genes and proteins. Asic1 was detected in all secretory cell types, Asic2 in gonadotrophs, thyrotrophs, and somatotrophs, and Asic4 in lactotrophs. Extracellular acidification activated two types of currents in a concentration-dependent manner: a fast-developing, desensitizing current with an estimated EC50-value of pH 6.7 and a slow-developing, non-desensitizing current that required a higher proton concentration for activation. The desensitizing current was abolished by removal of bath sodium and application of amiloride, a blocker of ASIC channels, whereas the non-desensitizing current was amiloride insensitive and voltage dependent. Activation of both currents increased the excitability of secretory pituitary cells, consistent with their potential physiological relevance in control of voltage-gated calcium influx and calcium-dependent cellular functions. In further studies, we studied the role of phosphatidylinositol (PI) kinases to calcium driven prolactin release in pituitary lactotrophs: PI4 kinases - which control PI4P production, PIP5 kinases - which synthesize PI(4,5)P2 by phosphorylating the D- 5 position of the inositol ring of PI4P, and PI3 kinases which phosphorylate PI(4,5)P2 to generate PI(3,4,5)P3. To do this, we used common and PI kinase-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Gene expression was analyzed by single cell RNA sequencing and qRT-PCR analysis; intracellular and released hormones were assessed by radioimmunoassay and ELISA; and single cell calcium signaling was recorded by Fura 2 imaging. Single cell RNA sequencing revealed the expression of Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in at least 10% of lactotrophs. Wortmannin, a PI3 kinase and PI4 kinase inhibitor, but not LY294002, a PI3 kinase inhibitor, blocked the spontaneous action potential driven prolactin release with a half-time of 20 min when applied in 10 M concentration, leading to accumulation of intracellular prolactin content. Wortmannin also inhibited increase in prolactin release by high potassium, calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanied calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium driven prolactin secretion without affecting calcium signaling and prolactin expression. In contrast, PIK93, a specific inhibitor prolactin 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. Thyrotropin (TSH) is well known as the hormone of the anterior pituitary thyrotrophs responsible for acting in the thyroid gland, where it stimulates synthesis and release of thyroid hormones through Gs and Gq/11 protein coupled TSH receptors. In a recent study, we examined whether the functional TSH receptors are also expressed in cultured rat pituitary cells, using double immunocytochemistry, quantitative reverse transcription-polymerase chain reaction analysis, cAMP and hormone measurements, and single-cell calcium imaging. Double immunocytochemistry revealed the expression of TSH receptors in cultured corticotrophs and melanotrophs, in addition to previously identified receptors in FSCs. The functional coupling of these receptors to the Gq/11 signaling pathway was not observed, as demonstrated by the lack of TSH activation of IP3-dependent calcium mobilization in these cells when bathed in calcium-deficient medium. However, TSH increased cAMP production in a time- and concentration-dependent manner and facilitated calcium influx in single corticotrophs and melanotrophs, indicating their coupling to the Gs signaling pathway. Consistent with these findings, TSH stimulated adrenocorticotropin and beta-endorphin release in male and female pituitary cells in a time- and concentration-dependent manner without affecting the expression of proopiomelanocortin gene. These results indicate that TSH is a potential paracrine modulator of anterior pituitary corticotrophs and melanotrophs, controlling the exocytotic but not the transcriptional pathway in a cAMP/calcium influx-dependent manner. Multiple sclerosis is an autoimmune disease that usually occurs during the reproductive years in both sexes. Many male patients with multiple sclerosis show lower blood testosterone levels, which was also observed in male rats during experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. To better understand the causes of decreased testosterone production during autoimmune encephalomyelitis, we investigated the expression status of genes and proteins associated with steroidogenesis in the testes. No changes in the number of interstitial cells were observed in autoimmune encephalomyelitis animals, but the expression of the insulin-like 3 gene was reduced at the peak of the disease, implying that the Leydig cell functional capacity was affected. Consistent with this finding, the expression of most steroidogenic enzyme genes and proteins was reduced during autoimmune encephalomyelitis, including StAR, CYP11A1, CYP17A1 and HSD3B. No signs of testicular inflammation were observed. Recovery of steroidogenesis was observed after injectio