We continue investigations on receptors and channels expressed in neuronal and endocrine cells and their roles in signaling, gene transcription, and hormone secretion. Our collaborative work with Dr. Arthur Sherman was focused on common and diverse elements of ion channels and receptors underling electrical activity in secretory pituitary cells: corticotrophs, melanotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. All these cell types are electrically excitable, and voltage-gated calcium influx is the major trigger for their hormone secretion. Along with hormone intracellular content, G-protein-coupled receptor and ion channel expression can also be considered as defining cell type identity. While many aspects of the developmental and activity dependent regulation of hormone secretion and G-protein-coupled receptor expression have been elucidated, much less is known about the regulation of the ion channels needed for excitation-secretion coupling in these cells. We compare the spontaneous and receptor-controlled patterns of electrical signaling among endocrine pituitary cell types, including insights gained from mathematical modeling. We argue that a common set of ionic currents unites these cells, while differential expression of another subset of ionic currents could underlie cell type-specific patterns. We tested these ideas using a generic mathematical model, showing that it reproduces many observed features of pituitary electrical signaling. Mapping these observations to the developmental lineage suggests possible modes of regulation that may give rise to mature pituitary cell types. We also continued our investigations on purinergic P2X receptor (P2XR) channel focusing on their functions. In collaboration with Dr. Claudio Coddou we utilized electrophysiology and mutagenesis to test the relevance of intracellular ATP and calcium in receptor gating, an issue that has been previously addressed for other plasma membrane channels, but not for P2XRs, that are gated by extracellular ATP. We found that P2X2R gating is dependent on both intracellular ATP and calcium, having these ligands opposite effects in receptor desensitization, with calcium accelerating and ATP impeding desensitization. The action of intracellular calcium is allosteric, whereas intracellular ATP acts covalently, as the phosphate source for receptor phosphorylation. We also identified the relevance of the C-terminal S363 residue for intracellular ATP regulation. These data suggest that P2X2Rs represent an important functional crosslink between extracellular and intracellular ATP and calcium signaling and provide integration of cellular responses to these ligands. We also summarized the current knowledge about interactions of pannexin1 channels with purinergic and N-methyl-D-aspartate receptor channels. Pannexins are a three-member family of vertebrate plasma membrane spanning molecules that have homology to the invertebrate gap junction forming proteins, the innexins. However, pannexins do not form gap junctions but operate as plasma membrane channels. The best-characterized member of these proteins, pannexin1 was suggested to be functionally associated with purinergic P2XR and N-methyl-D-aspartate receptor channels. Activation of these receptor channels by their endogenous ligands leads to cross-activation of pannexin1 channels. This in turn potentiates P2XR and N-methyl-D-aspartate receptor channel signaling. Two potentiation concepts have been suggested: enhancement of the current responses and/or sustained receptor channel activation by ATP released through pannexin1 pore and adenosine generated by ectonucleotidase-dependent dephosphorylation of ATP. In collaboration with Dr. Tamas Balla, we examined effects of Schwann-cell-specific deletion of hosphatidylinositol 4-kinase III alpha (PI4KA) on cellular functions. PI4KA is an essential cofactor of hepatitis C virus (HCV) replication. We initiated this study to determine whether HCV directly engages PI4KA to establish its replication. PI4KA kinase activity was found to be absolutely required for HCV replication using a small interfering RNA transcomplementation assay. Moreover, HCV infection or subgenomic HCV replicons produced a dramatic increase in phosphatidylinositol 4-phosphate (PI4P) accumulation throughout the cytoplasm, which partially colocalized with the endoplasmic reticulum. In contrast, the majority of PI4P accumulated at the Golgi bodies in uninfected cells. The increase in PI4P was not observed after infection with UV-inactivated HCV and did not reflect changes in PI4KA protein or RNA abundance. In an analysis of U2OS cell lines with inducible expression of the HCV polyprotein or individual viral proteins, viral polyprotein expression resulted in enhanced cytoplasmic PI4P production. Increased PI4P accumulation following HCV protein expression was precluded by silencing the expression of PI4KA. Silencing of this enzyme expression also resulted in aberrant agglomeration of viral replicase proteins, including NS5A, NS5B, and NS3. NS5A alone, but not other viral proteins, stimulated PI4P production in vivo and enhanced PI4KA kinase activity in vitro. Lastly, PI4KA coimmunoprecipitated with NS5A from infected Huh-7.5 cells and from dually transfected 293T cells. These results suggest that HCV NS5A modulation of PI4KA-dependent PI4P production influences replication complex formation. To gain better understanding of anterior pituitary cell functions and related disorders, we performed single cell RNA sequencing on freshly dispersed anterior pituitary cells from adult male and female rats. We identified cell type-specific gene expression in endocrine cells and folliculostellate cells, examined cellular heterogeneity in these populations, and compared them with pituitary-nonspecific cell populations: endothelial cells, erythrocytes, and leukocytes. Comparison of male and females, the latter in diestrus phase of estrous cycle, allowed us to identify sex-specific gene expression profiles in pituitary cell types. Our differential expression analysis identified several groups of genes significantly upregulated in folliculostellate cells, endocrine cells or both: development/regeneration, neuronal/neuroendocrine, endogenous ligands, detoxification enzymes, extracellular matrix proteins, and cell adhesion molecules. We examined in detail the expression pattern of these genes in pituitary-specific cell types. This work provides a comprehensive transcriptional overview of the anterior pituitary gland that will facilitate future experimental and clinical investigations in this field.

Project Start
Project End
Budget Start
Budget End
Support Year
25
Fiscal Year
2018
Total Cost
Indirect Cost
Name
U.S. National Inst/Child Hlth/Human Dev
Department
Type
DUNS #
City
State
Country
Zip Code
Alvarez-Prats, Alejandro; Bjelobaba, Ivana; Aldworth, Zane et al. (2018) Schwann-Cell-Specific Deletion of Phosphatidylinositol 4-Kinase Alpha Causes Aberrant Myelination. Cell Rep 23:2881-2890
Zemková, Hana; Stojilkovic, Stanko S (2018) Neurotransmitter receptors as signaling platforms in anterior pituitary cells. Mol Cell Endocrinol 463:49-64
Rokic, Milos B; Castro, Patricio; Leiva-Salcedo, Elias et al. (2018) Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel. Int J Mol Sci 19:
Bjelobaba, Ivana; Stojilkovic, Stanko S; Naor, Zvi (2018) Editorial: Gonadotropin-Releasing Hormone Receptor Signaling and Functions. Front Endocrinol (Lausanne) 9:143
Stojilkovic, Stanko S (2018) Signaling pathways regulating pituitary functions. Mol Cell Endocrinol 463:1-3
Fletcher, Patrick A; Sherman, Arthur; Stojilkovic, Stanko S (2018) Common and diverse elements of ion channels and receptors underlying electrical activity in endocrine pituitary cells. Mol Cell Endocrinol 463:23-36
Li, Shuo; Bjelobaba, Ivana; Stojilkovic, Stanko S (2018) Interactions of Pannexin1 channels with purinergic and NMDA receptor channels. Biochim Biophys Acta Biomembr 1860:166-173
Coddou, Claudio; Sandoval, Rodrigo; Castro, Patricio et al. (2017) Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372. Pain 158:2155-2168
Stojilkovic, Stanko S; Bjelobaba, Ivana; Zemkova, Hana (2017) Ion Channels of Pituitary Gonadotrophs and Their Roles in Signaling and Secretion. Front Endocrinol (Lausanne) 8:126
Janjic, Marija M; Stojilkovic, Stanko S; Bjelobaba, Ivana (2017) Intrinsic and Regulated Gonadotropin-Releasing Hormone Receptor Gene Transcription in Mammalian Pituitary Gonadotrophs. Front Endocrinol (Lausanne) 8:221

Showing the most recent 10 out of 74 publications