Phosphate-induced ORAI1 Expression and Store Operated Ca²⁺ Entry in Megakaryocytes

Scientific Research Question: Chronic kidney disease (CKD) leads to impairment of renal phosphate elimination and thus fosters the development of hyperphosphatemia, which in turn triggers vascular osteogenic signaling eventually resulting in vascular calcification. Osteogenic signaling involves up-regulation of the transcription factor NFAT5, which consequently stimulates the expression of the serum & glucocorticoid-inducible kinase (SGK1). SGK1 up-regulates Orai1, a Ca²⁺-channel accomplishing store operated Ca²⁺-entry (SOCE). Triggering of Orai1 dependent SOCE is a powerful mechanism activating blood platelets (PLTs) which increases risk of thrombo-occlusive formation. In this study we investigated a powerful SGK1 dependent stimulation of Orai1 expression by NFAT5 overexpression in megakaryocytes.

Methodology: Human megakaryocytes were exposed for 24 hours to 2 mM of the phosphate mimic β-glycerophosphate or human PLTs isolated from CKD patients or healthy volunteers. Transcript levels were assessed using q-RT-PCR, SOCE [Ca²⁺] intracellular) by Fura-2-fluorescence and protein levels by Western Blot.

Findings: In this work, increased NFAT5, Orai1, Orai2, Orai3, STIM1 and STIM2 transcript levels, after incubation with β-glycerophosphate as well as SOCE with Fura-2, were shown. Furthermore, we observed enhanced expression of Orai1 induced by NFAT5 overexpression in megakaryocytes. Signaling involved in the upregulation of Orai1 by NFAT5 includes SGK1. Interestingly, the disruption of SGK1 by a specific inhibitor (GSK650394; 10 µM), caused a decrease of the effect on SOCE. In addition, we observed that NFAT5, SGK1, Orai1/2/3, and STIM1/2 transcript levels were significantly higher in PLTs isolated from CKD patients than in PLTs from healthy volunteers. These results indicate that enhanced phosphate triggers a signaling cascade of NFAT5/SGK1/Orai1/STIM1, 2, which leads to up-regulation of store operated Ca²⁺-entry and thus sensitizes PLTs for activation.

Conclusions: The present study discloses a novel effect of the phosphate mimic β-glycerophosphate, i.e., the up-regulation of Orai1, Orai2, Orai3, STIM1, and STIM2 expression in megakaryocytes which is disrupted by pharmacological inhibition of SGK1 and is thus presumably due to upregulation of SGK1 by NFAT5. In view of the upregulation of NFAT5, SGK1, Orai and STIM isoform transcript levels are enhanced in platelets of CKD patients and could thus well contribute to the enhanced cardiovascular risk of those patients.