Role Of STIM1/Orai1-mediated Store-operated Calcium Entry In Airway Smooth Muscle Cell Proliferation

Background:Hyperplasia of airway smooth muscle cells (ASMCs) is a characteristic change of chronic asthma patients. However, the underlying mechanisms that trigger this process are still not completely understood. Ca2+ entry through store-operated Ca2+ (SOC) channels (SOCE) occurs in response to intracellular sarcoplasma reticulum (SR)/endoplasmic reticulum (ER) Ca2+ store depletion. SOCE plays an important role in regulating Ca2+s ignaling and cellular responses of ASMCs. Stromal interaction molecules 1 (STIM1), as an ER/SR Ca2+ sensor, has been proposed to transmit the SR/ER Ca2+ store depletion signal to the SOC channels, interact with Orail and activate SOC channels. STIM1 and Orai1 are important contributors to SOC entry in airway smooth muscle cells and found to be involved in cell proliferation of vascular smooth muscle cells. In this study, we tested whether STIM1/Orai1-regulated SOCE involved in rat airway smooth muscle cell proliferation. Methods:Cell were loaded with fluo-3 and then the intracellular Ca2+ concentration were measured by confocal Ca2+ fluorescence imaging; Real-time PCR were used to half-quantify the mRNA expression of target genes in ASMCs and Western blot analysis were used to determine the protein expression levels of STIM1 and Orai1; the ASMC proliferation was evaluated by an AlamarBlue reduction rate assay and cell counting after trypan blue exclusion; Vector-based short hairpin(sh) RNA were used for RNA interference. Results:(1) We found that SOCE was up-regulated during serum-induced ASMCs proliferation accompanying with a mild increase of STIM1 and a significant increase of Orai1 mRNA expression. (2) The proliferation of ASMCs was partially inhibited by SOC channel blockers SKF-96365 (10μM), Ni2+(100μM)和BTP-2 (100 nM). (3) STIM1 and Orai1 mRNA and protein expression can be inhibited by specific shRNA. (4) Suppressing the mRNA expression of STIM1 or Orai1 with specific shRNA resulted in attenuation of SOCE and serum or PDGF-BB induced ASMCs proliferation. (5) After knockdown of STIM1 or Orail, SOC channel blocker SKF-96365 had no inhibitory effect on the serum or PDGF-BB induced proliferation of ASMCs anymore. Conclusions:These results suggested that STIM1 and Orai1 play an essential role in the regulating SOCE of airway smooth muscle cells. SOCE is important in keeping the calcium homeostasis of airway smooth muscle cells and is involved in airway smooth muscle proliferation.