Decrease of Store-Operated Ca2+ Entry and Increase of Na+/Ca2+ Exchange by Pharmacological JAK2 Inhibition

Abstract

Background/Aims: Cell proliferation and migration are regulated by cytosolic Ca²⁺ activity ([Ca²⁺]_i). Mechanisms modifying [Ca²⁺]_i include store-operated Ca²⁺-entry (SOCE) accomplished by the pore-forming ion channel unit Orai1 and its regulator STIM1, as well as Ca²⁺ extrusion by Na⁺/Ca²⁺ exchanger NCX1. Kinases participating in the orchestration of cell proliferation include the Janus activated kinase JAK2. The present study explored the impact of pharmacological JAK2 inhibition on SOCE and Na⁺/Ca²⁺ exchange. Methods: MCF-7 breast carcinoma cells were studied in the absence and presence of the JAK2 inhibitors TG101348 (250 nM - 1 µM) or of AG490 (20 - 40 µM). Transcript levels were quantified with RT-PCR, protein abundance with immunoblotting, [Ca²⁺]_i with Fura-2-fluorescence, SOCE from increase of [Ca²⁺]_i following Ca²⁺ re-addition after Ca²⁺-store depletion with sarcoendoplasmatic Ca²⁺-ATPase (SERCA) inhibitor thapsigargin (1 µM), and Na⁺/Ca²⁺ exchanger activity from increase of [Ca²⁺]_i as well as Ca²⁺ current in whole cell patch clamp following extracellular Na⁺ removal. Migratory activity was determined by a wound healing assay. Results: JAK2 inhibitor TG101348 (1 µM) decreased Orai1 and STIM1 protein abundance, increased NCX1 transcript levels, decreased Ca²⁺ release from intracellular stores, decreased SOCE, increased Ca²⁺ entry as well as Ca²⁺-current following extracellular Na⁺-removal, and decreased migration. Similar effects on Ca²⁺ release, SOCE, and Ca²⁺-entry following extracellular Na⁺-removal were observed following treatment with AG490. Conclusion: The present observations disclose a novel powerful mechanism regulating intracellular Ca²⁺ release, cellular Ca²⁺ entry, cellular Ca²⁺ extrusion and cell migration.