Signaling and Secretion in Neuroendocrine Cells
- Stanko S. Stojilkovic, PhD, Head, Section on Cellular Signaling
- Stephanie Constantin, PhD, Staff Scientist
- Naseratun Nessa, PhD, Visiting Fellow
- Sonja Sivcev, PhD, Visiting Fellow
- Kosara Smiljanic, PhD, Visiting Fellow
The Section investigates cell-signaling cascades, gene expression, and secretion in hypothalamic and pituitary cells, with special emphasis on interactions between electrical events at the plasma membrane and receptor-controlled pathways. Specifically, we address how these neuroendocrine cells use ion channels and G protein–coupled receptors (GPCRs) as signaling platforms for efficient information processing. For this purpose, we characterize both natural and recombinant receptors and channels cloned from neuroendocrine cells. In the past, our work has focused on voltage- and ligand-gated ion channels, the cell type–specific patterns of electrical activity and channels involved, the physiological relevance of such activity, and the crosstalk between GPCRs and ion channels. Our current work focuses on age-, sex-, and tissue structure–specific gene expression, signaling and secretion, heterogeneity of secretory cells that reflect their postnatal genesis, and cell type–specific exocytic pathways. Ongoing and proposed projects include the use of transgenic and conditional knockout mouse models, and the research depends in part on the use of equipment at NICHD's Microscopy and Imaging Facility and Molecular Genomics Core.
Single-cell RNA sequencing (scRNA-seq) of pituitary cells
We continued investigations on genes expressed in mammalian pituitary cells and their role in cell signaling and function. We contributed to the work of our collaborator Prashant Chittiboina on scRNA-seq studies of human hormone-producing pituitary adenomas causing Cushing’s disease. The analysis included over 25,000 cells and identified a Cushing’s disease adenoma transcriptomic signature, as compared with adjacent normal cells, with validation by bulk RNA-seq, DNA methylation, qRT-PCR, and immunohistochemistry. Cushing’s disease adenoma cells include a subpopulation of proliferating, terminally differentiated corticotrophs. In Cushing’s disease adenomas, we found recurrent promoter hypomethylation and transcriptional upregulation of PMAIP1 (encoding proapoptotic BH3-only bcl-2 protein noxa), but paradoxical noxa downregulation. Using primary Cushing’s disease adenoma cell cultures and a corticotroph-enriched mouse cell line, we found that selective proteasomal inhibition with bortezomib stabilizes noxa and induces apoptosis, indicating its utility as an anti-tumor agent [Reference 1].
Our recent experiments on transcriptome profiles of secretory and non-secretory cell types using scRNA-seq 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, vascular pericytes, and endothelial cells. Initially, we characterized cell type– and sex-dependent transcriptome profiles of rat anterior pituitary cells. More recently, we summarized scRNA-seq and immunohistofluorescence analyses of adult female rat pituitary with a focus on transcriptomic profiles of non-hormonal cell types. Samples obtained from whole pituitaries and separated anterior and posterior lobe cells contained all expected resident pituitary cell types and a lobe-specific subpopulation of vascular cells. FSCs and pituicytes expressed S100B, ALDOC, EAAT1, ALDH1A1, and VIM genes and proteins, as well as several other astroglial marker genes, some common and some cell type–specific. We also found that the SOX2 gene and protein are 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 are randomly distributed in the anterior and intermediate lobes, while FSC2 are localized exclusively in the marginal zone between the anterior and intermediate lobes. These data indicate that FSCs are 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 [Reference 2].
Roles of protein tyrosine phosphatase receptor N and N2 genes in mice reproduction
All pituitary hormone–producing cells express common genes associated with secretory functions, such as the sister genes encoding 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 genes. 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, we observed delayed puberty and a lack of ovulation, complemented by changes in ovarian gene expression and steroidogenesis. In contrast, testicular gene expression, steroidogenesis, and the development of reproductive organs were not significantly affected in DKO males. However, hypothalamic Gnrh1 and Kiss1 gene expression was reduced in DKO females and males. In parallel, we detected a significant reduction in the density of immunoreactive GnRH and kisspeptin fibers in the hypothalamic arcuate nucleus of DKO females and males. Female-specific immunoreactivity of the neuromodulator kisspeptin in the rostral periventricular region of the third ventricle was also reduced in DKO females but not in DKO males (Figure 1). Furthermore, in both sexes, pituitary luteinizing hormone (LH) beta gene expression and LH level, as well as follicle-stimulating hormone beta gene and gonadotropin-releasing hormone (GnRH) gene were reduced, while the calcium-mobilizing and LH secretory actions of GnRH were preserved (Figure 2). These data indicate a critical role of Ptprn and Ptprn2 in kisspeptin–GnRH neuronal function and sexual dimorphism and in the threshold levels of GnRH required to preserve reproductive functions [Reference 3]. Ongoing experiments on this topic focus on the physiological status of anterior pituitary corticotrophs and intermediate lobe–located melanotrophs of DKO mice.
Figure 1. Common and female-specific effects of DKO on the hypothalamic kisspeptin-secreting neurons
Click image to view.
(A–D) Kisspeptin immunoreactivity in the rostral periventricular region of the third ventricle. Pattern of kisspeptin immunoreactivity of WT (A) and DKO males (B), and WT (C) and DKO females (D). (E–H) Kisspeptin immunoreactivity in the arcuate nucleus. Patterns of kisspeptin immunoreactivity of WT (E) and DKO males (F) and WT (G) and DKO females (H). Horizontal bars of 200 μm apply to all panels. Arrows indicate representative fiber density in the rostral periventricular region of the third ventricle (RP3V) (A–D) and the arcuate nucleus (E–H). (I) Quantification of kisspeptinergic cell bodies (left panel) and fiber densities (right panel) in the RP3V region of WT and DKO females whose representative matched sections are shown in C–D. (J) Quantification of kisspeptinergic fiber densities in the arcuate nucleus of WT and DKO males (left panel) and females (right panel), whose representative matched sections are shown in E–F and G–H, respectively.
Figure 2. Reduced LH levels in the pituitary gland of DKO animals
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(A) Western blot analysis of LHbeta (LHB) of male (top panel) and female (bottom panel) pituitaries in WT and DKO mice. The data shown are representative from five experiments.
(B and C) Pituitary LH content in pituitary tissue (B) and cultured pituitary cells (C) from WT and DKO mice assessed by ELISA.
(D) Basal LH release by cultured pituitary cells during a four-hour incubation.
(E) Time course of GnRH–stimulated LH release in cultured pituitary cells.
(F) Pituitary LH content (left panel) and serum LH concentration (right panel) 90 minutes after the intraperitoneal injection of saline (S) or 0.5 µg buserelin acetate (B), a GnRH receptor agonist, in WT and DKO mice. The bars shown are mean ± SEM values; number of replicates are indicated above the bars; asterisks denote P<0.01 between pairs. The circles indicate mean values from duplicate determination.
PI(4,5)P2–dependent and –independent roles of PI4P in pituitary cell function
In collaboration with Tamás Balla’s group, we are also studying the functions of three phosphoinositides, PI4P, PI(4,5)P2, and PI(3,4,5)P3, in cell signaling and exocytosis, focusing on hormone-producing pituitary cells. PI(4,5)P2, which acts 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 experiments also showed the expression of several PI lipid kinase genes, 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, a hormone that controls lactation. Using a pharmacological approach to specifically inhibit these enzymes, we showed that PI4P, made in the plasma membrane by PI4KA, is critical for exocytosis, without affecting the calcium signals that drive secretion. Our experiments also indicate that inhibition of the PI4KB enzyme, which 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 [Reference 4].
Our recently published study on this topic 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 elevated. Knockout of PI4KA also did not alter embryonic establishment and neonatal development or 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 low synthesis of gonadotropins, but not of other pituitary lineage–specific genes and their hormones. The postnatal gonadotroph population also progressively declined, reaching approximately one third 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 that PI4KA plays a key role in the postnatal development and maintenance of a functional gonadotroph population [Reference 5]. Ongoing experiments on this topic focus on the role of PI4KA in the postnatal development and function of pituitary lactotrophs and hypothalamic GnRH–secreting neurons.
Publications
- Asuzu DT, Alvarez R, Fletcher PA, Mandal D, Johnson K, Wu W, Elkahloun A, Clavijo P, Allen C, Maric D, Ray-Chaudhury A, Rajan S, Abdullaev Z, Nwokoye D, Aldape K, Nieman LK, Stratakis C, Stojilkovic SS, Chittiboina P. Pituitary adenomas evade apoptosis via noxa deregulation in Cushing's disease. Cell Rep 2022 40:111223.
- Fletcher PA, Smiljanic K, Prévide RM, Constantin S, Sherman AS, Coon SL, Stojilkovic SS. The astroglial and stem cell functions of adult rat folliculostellate cells. Glia 2023 7:205–228.
- Sokanovic SJ, Constantin S, Lamarca Dams A, Mochimaru Y, Smiljanic K, Bjelobaba I, Previde RM, Stojilkovic SS. Common and female-specific roles of protein tyrosine phosphatase receptors N and N2 in mice reproduction. Sci Rep 2013 13(1):355.
- Stojilkovic SS, Balla T. PI(4,5)P2-dependent and -independent roles of PI4P in the control of hormone secretion by pituitary cells. Endocrinol (Lausanne) 2023 14:1118744.
- Constantin S, Sokanovic SJ, Mochimaru Y, Smiljanic K, Sivcev S, Previde RM, Wray S, Balla T, Stojilkovic SS. Postnatal development and maintenance of functional pituitary gonadotrophs is dependent on PI4-kinase A. Endocrinology 2023 164(12):doi:10.1210/endocr/bqad168.
Collaborators
- Tamás Balla, MD, PhD, Section on Molecular Signal Transduction, NICHD, Bethesda, MD
- Prashant Chittiboina, MD, PhD, Neurosurgery Unit for Pituitary and Inheritable Diseases, NINDS, Bethesda, MD
- Patrick A. Fletcher, PhD, Laboratory of Biological Modeling, NIDDK, Bethesda, MD
- Arthur Sherman, PhD, Laboratory of Biological Modeling, NIDDK, Bethesda, MD
Contact
For more information, email stojilks@mail.nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/stojilkovic.
