STIM1 regulates platelet-derived growth factor-induced migration and Ca2+ influx in human airway smooth muscle cells.

It is suggested that migration of airway smooth muscle (ASM) cells plays an important role in the pathogenesis of airway remodeling in asthma. Increases in intracellular Ca(2+) concentrations ([Ca(2+)](i)) regulate most ASM cell functions related to asthma, such as contraction and proliferation. Recently, STIM1 was identified as a sarcoplasmic reticulum (SR) Ca(2+) sensor that activates Orai1, the Ca(2+) channel responsible for store-operated Ca(2+) entry (SOCE). We investigated the role of STIM1 in [Ca(2+)](i) and cell migration induced by platelet-derived growth factor (PDGF)-BB in human ASM cells. Cell migration was assessed by a chemotaxis chamber assay. Human ASM cells express STIM1, STIM2, and Orai1 mRNAs. SOCE activated by thapsigargin, an inhibitor of SR Ca(2+)-ATPase, was significantly blocked by STIM1 siRNA and Orai1 siRNA but not by STIM2 siRNA. PDGF-BB induced a transient increase in [Ca(2+)](i) followed by sustained [Ca(2+)](i) elevation. Sustained increases in [Ca(2+)](i) due to PDGF-BB were significantly inhibited by a Ca(2+) chelating agent EGTA or by siRNA for STIM1 or Orai1. The numbers of migrating cells were significantly increased by PDGF-BB treatment for 6 h. Knockdown of STIM1 and Orai1 by siRNA transfection inhibited PDGF-induced cell migration. Similarly, EGTA significantly inhibited PDGF-induced cell migration. In contrast, transfection with siRNA for STIM2 did not inhibit the sustained elevation of [Ca(2+)](i) or cell migration induced by PDGF-BB. These results demonstrate that STIM1 and Orai1 are essential for PDGF-induced cell migration and Ca(2+) influx in human ASM cells. STIM1 could be an important molecule responsible for airway remodeling

Effects of PDGF on Intracellular Ca²⁺ Concentrations.

<p>Effects of PDGF-BB on [Ca²⁺]_i. Representative changes in the F₃₄₀/F₃₈₀ ratio with 10 ng/mL PDGF-BB in the normal solution (<b>A</b>) or in the nominally Ca²⁺-free solution (<b>B</b>). <b>C</b>: The peak F₃₄₀/F₃₈₀ ratios in response to PDGF-BB in the normal solution (control) or the nominally Ca²⁺-free solution. Bars represent the means ± SD (n = 6). *Significantly different from the baseline values without PDGF-BB treatment (P<0.05). <b>D</b>: The sustained F₃₄₀/F₃₈₀ ratios (plateau phases) in response to PDGF-BB in the normal solution (control) or the nominally Ca²⁺-free solution. Effects of EGTA (2 mM) on sustained increase in the F₃₄₀/F₃₈₀ ratio with 10 ng/mL PDGF-BB are also shown. Bars represent the means ± SD (n = 6). *Significantly different from the values with PDGF-BB alone (P<0.05).</p

Role of STIM1 in PDGF-Induced Intracellular Ca²⁺ Elevation.

<p>Roles of STIM1 and Orai1 in [Ca²⁺]_i elevation induced by PDGF-BB. Representative changes in the F₃₄₀/F₃₈₀ ratios with 10 ng/mL PDGF-BB in the cells transfected with siSTIM1 (<b>A</b>) and siOrai1 (<b>B</b>), and siSTIM2 (<b>C</b>) are shown. Transient (peak) (<b>D</b>) and sustained increases (plateau phase) (<b>E</b>) in the F₃₄₀/F₃₈₀ ratio in response to PDGF-BB with or without (control) siRNA treatment are compared. Bars represent the means ± SD (n = 6). *Significantly different from the values of the control cells treated with 10 ng/mL PDGF-BB plus scrambled siRNA (P<0.05).</p

Role of STIM1 and Orai1 in Store-Operated Ca²⁺ Entry.

<p>Roles of STIM1 and Orai1 in SOCE. Representative changes in the F₃₄₀/F₃₈₀ ratio due to 5 μM thapsigargin (TPG) in cells transfected with siRNA targeting STIM1 (<b>A</b>) and Orai1 (<b>B</b>). After the cells were treated with thapsigargin in the nominally Ca²⁺-free solution, 2 mM Ca²⁺ was added to the solution. <b>C</b>: The F₃₄₀/F₃₈₀ ratios in response to 5 μM thapsigargin in the normal solution due to SOCE with or without (control) siRNA treatment. The cells transfected with scrambled siRNA, siSTIM1, siOrail, both siSTIM1 and Orai1, or siSTIM1. Bars represent the means ± SD (n = 6). *Significantly different from the values of the cells transfected with scrambled siRNA (P<0.05).</p

Roles of STIM1 and Orai1 in Cell Migration Induced by PDGF.

<p>Roles of STIM1 and Orai1 in cell migration induced by PDGF-BB (10 ng/mL, 6 h) are shown. Cell migration was assessed by a chemotaxis assay. <b>A</b>: Effects of PDGF-BB on migrated cell numbers with or without (control) 1 mM EGTA treatment (n = 6). Baseline (black column) denotes the time-matched number of cells that migrated without PDGF-BB treatment. Significantly different from the values of the baseline (*) and by PDGF-BB alone (#) (P<0.05). <b>B</b>: Effects of siRNA treatment targeting STIM1, STIM2, Orai1, and both STIM1 and Orai1 on migrating cell numbers induced by PDGF-BB (n = 6). *Significantly different from the values of the time-matched control cells treated with PDGF-BB plus scrambled siRNA (negative control, NC) (P<0.05). Bars represent means ± SD.</p

Expression of STIM1, STIM2, and Orai1.

<p><b>A</b>: Expression of STIM1, STIM2, Orai1, and GAPDH mRNAs detected by RT-PCR in human airway smooth muscle (ASM) cells is shown. Negative indicates a negative control. The product sizes for STIM1, STIM2, Orai1, and GAPDH were 481bp, 498bp, 483bp, and 498bp, respectively. <b>B</b>: Effects of siRNA-targeted knockdown of STIM1, STIM2, and Orai1 mRNAs on the change in mRNA expression over control normalized to the reference gene GAPDH are shown (n = 4). Changes in mRNA expression were assessed by quantitative real-time PCR. Effects of siRNA transfection targeting STIM1 (siSTIM1) (<b>C</b>), STIM2 (siSTIM2) (<b>D</b>), and Orai1 (siOrai1) (<b>E</b>) on changes in protein levels were assessed by Western blot. STIM1, STIM2, and Orai1 protein levels expressed as the target protein/actin ratio in the cells transfected with siSTIM1 (<b>C</b>), siSTIM2 (<b>D</b>), or siOrai1 (<b>E</b>) and scrambled siRNA (negative control) are compared (n = 3). The control value without siRNA transfection is defined as 100%. *Significantly different from the values of the scrambled siRNA condition (P<0.05). Bars represent means ± SD.</p