NFAT5‐sensitive Orai1 expression and store‐operated Ca ²⁺ entry in megakaryocytes

Abstract

The transcription factor nuclear factor of activated T cells 5 (NFAT5) is up-regulated in several clinical disorders, including dehydration. NFAT5-sensitive genes include serum and glucocorticoid-inducible kinase 1 (SGK1). The kinase is a powerful regulator of Orai1, a Ca channel accomplishing store-operated Ca entry (SOCE). Orai1 is stimulated after intracellular store depletion by the Ca sensors stromal interaction molecule 1 (STIM1), or STIM2, or both. In the present study, we explored whether nuclear factor of activated T cell (NFAT)-5 influences Ca signaling in megakaryocytes. To this end, human megakaryocytic (MEG-01) cells were transfected with NFAT5 or with siNFAT5. Platelets and megakaryocytes were isolated from wild-type mice with either access to water or dehydration by 36 h of water deprivation. Transcript levels were determined with quantitative RT-PCR and protein abundance by Western blot analysis and flow cytometry, cytosolic (intracellular) Ca concentration ([Ca]) by fura-2-fluorescence. SOCE was estimated from the increase of [Ca] following readdition of extracellular Ca after store depletion with thapsigargin (1 µM). Platelet degranulation was estimated from P-selectin abundance and integrin activation from αβ integrin abundance determined by flow cytometry. As a result, NFAT5 transfection or exposure to hypertonicity (+40 mM NaCl) of MEG-01 cells increased Orai1, Orai2, STIM1, and STIM2 transcript levels. Orai1 transcript levels were decreased by NFAT5 silencing. NFAT5 transfection and IκB inhibitor BMS 345541 (5 µM) increased SOCE, whereas NFAT5 silencing and SGK1 inhibitor GSK650394 (10 µM) decreased SOCE. In the mice, dehydration increased NFAT5 and Orai1 protein abundance in megakaryocytes and NFAT5, Orai1, and Orai2 abundance in platelets. Dehydration further augmented the degranulation and integrin activation by thrombin and collagen-related peptide. In summary, NFAT5 is a powerful regulator of Orai1-expression and SOCE in megakaryocytes.-Sahu, I., Pelzl, L., Sukkar, B., Fakhri, H., al-Maghout, T., Cao, H., Hauser, S., Gutti, R., Gawaz, M., Lang, F. NFAT5-sensitive Orai1 expression and store-operated Ca entry in megakaryocytes.

Heterologous calcium-dependent inactivation of Orai1 by neighboring TRPV1 channels modulates cell migration and wound healing

“…Even though all CoIP studies presented here are in agreement with the FRET and super resolution results, we would like to highlight the fact that the molecular weight obtained from Orai1 (≈55 kDa) differs from the predicted molecular weight for this protein (≈33 kDa). Thus, we would like to take the CoIP studies with caution, even though other reports have found similar discrepancies in the molecular weight for Orai1 34,35 . Feasible explanations for the higher molecular weight observed may involve glycosylation, phosphorylation, and other post-translational modifications of Orai1 36–38 .…”

Deficiency of platelet adhesion molecule CD226 causes megakaryocyte development and platelet hyperactivity

“…Many immune regulatory molecules and cytokines are also expressed on platelets (eg, Toll‐like receptors, P‐selectin, CD40L, interleukin‐1β, and transforming growth factor‐β) and have the ability to interact with immune cells, with integrin family member playing key roles, 28,29 such as β2 integrin modulating platelet caspase activation and life span in mice 30 . Autoantibody to α v β 3 was detected in immune thrombocytopenia patients and contributes to thrombocytopenia by blocking the adhesion and migration of MKs 31 .…”

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

“…Consequently, the proteins expressed in megakaryocytes are expected to be transferred into circulating blood platelets 27 . Recent observations revealed a powerful SGK1 dependent stimulation of ORAI1 expression by NFAT5 overexpression in megakaryocytes 28 .…”