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Phosphate-induced ORAI1 expression and store-operated Ca2+ entry in aortic smooth muscle cells

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Abstract

Compromised renal phosphate elimination in chronic kidney disease (CKD) leads to hyperphosphatemia, which in turn triggers osteo-/chondrogenic signaling in vascular smooth muscle cells (VSMCs) and vascular calcification. Osteo-/chondrogenic transdifferentiation of VSMCs leads to upregulation of the transcription factors MSX2, CBFA1, and SOX9 as well as tissue-nonspecific alkaline phosphatase (ALPL) which fosters calcification by degrading the calcification inhibitor pyrophosphate. Osteo-/chondrogenic signaling in VSMCs involves the serum- and glucocorticoid-inducible kinase SGK1. As shown in other cell types, SGK1 is a powerful stimulator of ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by the Ca2+ sensor STIM1. The present study explored whether phosphate regulates ORAI1 and/or STIM1 expression and, thus, SOCE in VSMCs. To this end, primary human aortic smooth muscle cells (HAoSMCs) were exposed to the phosphate donor β-glycerophosphate. Transcript levels were estimated by qRT-PCR, protein abundance by western blotting, ALPL activity by colorimetry, calcification by alizarin red S staining, 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. As a result, β-glycerophosphate treatment increased ORAI1 and STIM1 transcript levels and protein abundance as well as SOCE in HAoSMCs. Additional treatment with ORAI1 inhibitor MRS1845 or SGK1 inhibitor GSK650394 virtually disrupted the effects of β-glycerophosphate on SOCE. Moreover, the β-glycerophosphate-induced MSX2, CBFA1, SOX9, and ALPL mRNA expression and activity in HAoSMCs were suppressed in the presence of the ORAI1 inhibitor and upon ORAI1 silencing. In conclusion, enhanced phosphate upregulates ORAI1 and STIM1 expression and store-operated Ca2+-entry, which participate in the orchestration of osteo-/chondrogenic signaling of VSMCs.

Key messages

• In aortic SMC, phosphate donor ß-glycerophosphate upregulates Ca2+ channel ORAI1.

• In aortic SMC, ß-glycerophosphate upregulates ORAI1-activator STIM1.

• In aortic SMC, ß-glycerophosphate upregulates store-operated Ca2+-entry (SOCE).

• The effect of ß-glycerophosphate on SOCE is disrupted by ORAI1 inhibitor MRS1845.

• Stimulation of osteogenic signaling is disrupted by MRS1845 and ORAI1 silencing.

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Acknowledgments

The authors gratefully acknowledge the meticulous preparation of the manuscript by Lejla Subasic.

Funding

This work has been supported in part by the European Union Seventh Framework Programme (FP7/2007-2013), Systems Biology to Identify Molecular Targets for Vascular Disease Treatment (SysVasc, HEALTH-2013 603288). Ping Liu and Hang Cao are supported by the Chinese Scholarship Council, Tamer al-Maghout by the Deutsche Akademische Austausch-Dienst (DAAD).

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Authors and Affiliations

  1. Department of Pharmacology & Experimental Therapy, University of Tübingen, 72076, Tübingen, Germany

    Ke Ma, Ping Liu, Tamer Al-Maghout, Basma Sukkar & Hang Cao

  2. Institute for Physiology and Pathophysiology, Johannes Kepler University Linz, 4040, Linz, Austria

    Jakob Voelkl & Ioana Alesutan

  3. Department of Internal Medicine and Cardiology, Charité University Medicine, Campus Virchow-Klinikum, Berlin, Germany

    Jakob Voelkl, Ioana Alesutan & Burkert Pieske

  4. DZHK (German Centre for Cardiovascular Research), Partner Site, Berlin, Germany

    Jakob Voelkl, Ioana Alesutan & Burkert Pieske

  5. Department of Nephrology and Medical Intensive Care, Charité University Medicine, Berlin, Germany

    Jakob Voelkl

  6. Berlin Institute of Health (BIH), Berlin, Germany

    Ioana Alesutan & Burkert Pieske

  7. Department of Internal Medicine and Cardiology, German Heart Center Berlin (DHZB), Berlin, Germany

    Burkert Pieske

  8. Department of Vegetative and Clinical Physiology, University of Tübingen, Wilhelmstr. 56, 72074, Tübingen, Germany

    Florian Lang

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  1. Ke Ma
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Contributions

KM, PL, TaM, BS and HC performed experiments and analyzed data; FL JV, BP, and IA designed research, FL drafted and wrote the manuscript. All authors corrected and approved the manuscript.

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Correspondence to Florian Lang.

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Ma, K., Liu, P., Al-Maghout, T. et al. Phosphate-induced ORAI1 expression and store-operated Ca2+ entry in aortic smooth muscle cells. J Mol Med 97, 1465–1475 (2019). https://doi.org/10.1007/s00109-019-01824-7

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