Mathematical Modeling of Neurons and Endocrine Cells

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

We are continuing our work with the Stojilkovic lab and the Genomics core of NICHD, providing bioinformatic support for their to single-cell RNAseq analyses of gene expression in the anterior pituitary. This gland is composed of five hormone producing cell types, glia-like folliculostellate cells, and endothelial and blood cells comprising the pituitary sinusoidal capillary network. Some key lines of inquiry in pituitary physiology include three-dimensional organization tissue organization and intercellular communication among cells, development and regeneration of pituitary cells, and the heterogeneity and function of the multiple cell types in the pituitary. The primary function of endocrine pituitary cells is to secrete hormones via regulated exocytosis, for which the triggering role of intracellular calcium is well established. However, this process relies on many calcium independent cellular components. Phosphoinositides, low-abundance membrane lipids, have critical roles in exocytosis due to their interactions with vesicle priming and fusion proteins, but their role in pituitary hormone secretion has received little attention. Seven distinct phosphoinositides can be generated via phosphorylation of phosphatidylinositol by distinct families of kinases. This study found that blockade of phosphoinositide production with Wortmannin, at doses blocking PI3 and PI4 kinases, abolished prolactin secretion from lactotrophs downstream of calcium signaling. The genes coding for all major families of these kinases were detected in all hormone-producing pituitary cell types by scRNAseq and quantitative RT-PCR, so a systematic pharmacological approach was used to identify PI4KA as the essential kinase underlying the prolactin secretion blockade. This establishes PI4KA as a key enzyme regulating prolactin secretion. The work has been published (ref. #1). The adult pituitary gland is made up of hormone-producing cells, vascular endothelial cells and pericytes, posterior lobe pituicytes, anterior lobe folliculostellate cells (FSCs), and Sox2-positive pituitary stem cells (PSCs). Pituicytes are considered astroglial cells of the posterior pituitary, supporting hypothalamic axon terminals. Similarly, FSCs have a range of established functions supporting and coordinating the endocrine functions of the anterior pituitary. The recent identification of PSCs has led to intensive investigation of these cells with modern techniques, including single-cell RNA sequencing (scRNAseq). However, despite their well-established roles in pituitary function, most of these recent studies have not reported on the transcriptomic profiles of FSCs. We carried out scRNAseq and immunohistofluorescence analyses of adult female rat pituitary cells, focusing on FSCs. We found that, much like pituicytes, FSCs expressed many key genes and proteins characteristic of astroglial cells. 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 (HPCs), arguing against its stem cell specificity. FSCs comprised two Sox2-expressing subclusters; one contained more cells but lower genetic diversity, while the other contained proliferative cells, shared genes with HPCs, and expressed genes consistent with stem cell functions. These data indicate the identity of the FSCs as anterior pituitary-specific astroglia, consisting of at least two subpopulations: one representing differentiated cells equipped for classical FSC roles and the other exhibiting additional stem cell-like features. A paper is in press.