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A7259 - Differential Estrogen Receptor Signaling Regulates Store Operated Calcium Entry in Human Airway Smooth Muscle
Author Block: R. Kalidhindi1, N. S. Ambhore1, M. Thompson2, C. M. Pabelick2, Y. Prakash2, S. Venkatachalem1; 1Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND, United States, 2Departments of Anesthesiology and Perioperative Medicine, and Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, United States.
Rationale: Store-operated Calcium entry (SOCE) is a ubiquitous calcium selective conductance mechanism promoted by STIM1 and ORAI1 proteins and suppressed via TMEM66 (SARAF). SOCE influences contractility of human airway smooth muscle cells (ASM). Estrogen has been long known to influence intracellular calcium levels in multiple cell types; however, the mechanisms of calcium regulation by estrogens are cell- and context-specific. It is also unclear how differential estrogen receptor signaling (ERα and ERβ) affects intracellular calcium during inflammation and asthma. In the present study, we explored the hypothesis that estrogen receptor signaling influences SOCE by regulating STIM1 and Orai1 expression and function. Methods: Asthmatic and non-asthmatic primary human airway smooth muscle cells isolated from surgical lung resections were cultured in DMEM-F12 (Mayo IRB approved). ASM cells were treated with 1nM E2, 10nM propyl pyrazole triol (PPT, ERα agonist) or WAY-200070 (WAY, ERβ agonist) in the presence vs. absence of pro-inflammatory cytokines (TNFα, 20ng/mL or IL-13, 50ng/mL) for 24 h followed by imaging using the calcium indicator Fluo-3 dye on an Olympus FV500 fluorescent microscope. Standard SOCE protocol was followed while imaging. Expression of STIM1, Orai1 and TMEM66 were determined using Western analysis and qRT-PCR. Results: SOCE was significantly increased in human ASM cells treated with TNFα or IL-13. Asthmatic human ASM cells showed increased SOCE compared to non-asthmatics. Furthermore, cells treated with PPT alone showed increased SOCE while Western analysis found increased expression of STIM1 and Orai1. Conversely, WAY-treated cells showed significant decreases in SOCE, corroborated by Western data showing decreased STIM1 and Orai1. In the presence TNFα or IL-13, WAY significantly reduced SOCE particularly in asthmatic but also in non-asthmatic human ASM cells while PPT did not elicit any notable changes compared to TNFα or IL-13 treatment alone. This was further confirmed by Western analysis of STIM1, Orai1 and TMEM66 expression and FRET of their interactions. Conclusions: In human ASM, estrogen receptors ERα and ERβ regulate expression of STIM1 and Orai1, which are key proteins in SOCE. ERα increases SOCE via increased STIM1 and Orai1 whereas ERβ down-regulates their expression decreasing SOCE. Thus, differential estrogen receptor effects may be critical in the overall effect of estrogens on intracellular calcium regulation in the airway. Conversely, targeting ERβ may be a novel mechanism to blunt airway hyper responsiveness in inflammation.