Beta-Glycerophosphate-Induced ORAI1 Expression and Store Operated Ca2+ Entry in Megakaryocytes

Lisann Pelzl,Florian Lang,Abdulla Al Mamun Bhuyan,Irene Marini,Tamer Al-Maghout,Flaviana Rigoni,Burkert Pieske,Burkert Pieske,Burkert Pieske,Burkert Pieske,Hang Cao,Ravi Gutti,Basma Sukkar,Ke Ma,Itishri Sahu,Jakob Voelkl,David Heinzmann,Ferruh Artunc,Itishri Sahu,Yamini Sharma,Tamam Bakchoul,Jakob Voelkl,Jakob Voelkl,Meinrad Gawaz

doi:10.1038/s41598-020-58384-x

Abstract

Impairment of renal phosphate elimination in chronic kidney disease (CKD) leads to enhanced plasma and tissue phosphate concentration, which in turn up-regulates transcription factor NFAT5 and serum & glucocorticoid-inducible kinase SGK1. The kinase upregulates ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by Ca2+-sensors STIM1 and/or STIM2. In megakaryocytes and blood platelets SOCE and thus ORAI1 are powerful regulators of activity. The present study explored whether the phosphate-donor ß-glycerophosphate augments NFAT5, ORAI1,2,3 and/or STIM1,2 expressions and thus SOCE in megakaryocytes. Human megakaryocytic Meg01cells were exposed to 2 mM of phosphate-donor ß-glycerophosphate for 24 hours. Platelets were isolated from blood samples of patients with impaired kidney function or control volunteers. Transcript levels were estimated utilizing q-RT-PCR, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). NFAT5 and ORAI1 protein abundance was estimated with Western blots. As a result, ß-glycerophosphate increased NFAT5, ORAI1/2/3, STIM1/2 transcript levels, as well as SOCE. Transcript levels of NFAT5, SGK1, ORAI1/2/3, and STIM1/2 as well as NFAT5 and ORAI1 protein abundance were significantly higher in platelets isolated from patients with impaired kidney function than in platelets from control volunteers. In conclusion, phosphate-donor ß-glycerophosphate triggers a signaling cascade of NFAT5/SGK1/ORAI/STIM, thus up-regulating store-operated Ca2+-entry.

Highlights

Www.nature.com/scientificreports cloned as tonicity responsive enhancer binding protein (TonEBP) stimulated by hyperosmotic cell shrinkage[13,14] and subsequently been shown to be enhanced in several disorders, such as diabetes[15], inflammation[16] and CKD12
Recent observations revealed a powerful serum & glucocorticoid inducible kinase 1 (SGK1) dependent stimulation of ORAI1 expression by nuclear factor of activated T cells 5 (NFAT5) overexpression in megakaryocytes[28]. In view of those observations we hypothesized that the phosphate-donor ß-glycerophosphate mimicking enhanced extracellular phosphate concentration may up-regulate the expression of NFAT5 in megakaryocytes, and that NFAT5 enhances the expression of SGK1, ORAI1 and STIM1 and/or STIM2
The present study explored whether NFAT5, SGK1, ORAI1, ORAI2, ORAI3 STIM1 and/or STIM2 expression in megakaryocytes is sensitive to phosphate-donor ß-glycerophosphate and altered in patients with chronic kidney disease (CKD) incl. dialysis-dependency

Summary

Introduction

Www.nature.com/scientificreports cloned as tonicity responsive enhancer binding protein (TonEBP) stimulated by hyperosmotic cell shrinkage[13,14] and subsequently been shown to be enhanced in several disorders, such as diabetes[15], inflammation[16] and CKD12. Platelets are released into the blood stream by megakaryocytes, which differentiate from hematopoietic progenitor cells in the bone marrow[26,27]. Recent observations revealed a powerful SGK1 dependent stimulation of ORAI1 expression by NFAT5 overexpression in megakaryocytes[28]. In view of those observations we hypothesized that the phosphate-donor ß-glycerophosphate mimicking enhanced extracellular phosphate concentration may up-regulate the expression of NFAT5 in megakaryocytes, and that NFAT5 enhances the expression of SGK1, ORAI1 and STIM1 and/or STIM2. Considering that the abundance of the respective proteins in circulating platelets is a function of protein synthesis in megakaryocytes, enhanced extracellular phosphate may up-regulate the expression of NFAT5, SGK1, ORAI1 and STIM1 and/or STIM2 in circulating blood platelets. The present study explored whether NFAT5, SGK1, ORAI1, ORAI2, ORAI3 STIM1 and/or STIM2 expression in megakaryocytes is sensitive to phosphate-donor ß-glycerophosphate and altered in patients with CKD incl. dialysis-dependency

Methods

Results

Conclusion