Estrogen Receptor Signaling and Intracellular Calcium Regulation in Human Airway Smooth Muscle

다국어 초록 (Multilingual Abstract)

Bronchial asthma is a chronic inflammation of the airways that is characterized by airway remodeling, hyperactive responsiveness and acute bronchoconstriction of airway smooth muscle (ASM) cells. Clinical findings suggest that there is a higher incidence of asthma in pre‐menopausal women and aging men, strongly indicating the role of sex steroids in modulating the airway tone. A major sex steroid, estrogen mediates its role through estrogen receptors (ER) ‐ ERα and ERβ. Our recent study demonstrated that these receptors are expressed in both male and female ASM cells and their expression is upregulated in the presence of inflammation/asthma. However, the role of ER signaling in asthma or genomic mechanisms in intracellular Ca2+ ([Ca2+]i ) regulation in human ASM are unknown. In this study, we evaluated the genomic effect of specific ERα and ERβ modulators on the [Ca2+]i in human ASM cells.
Human ASM cells were enzymatically dissociated from tissue obtained incidental to lung surgery from Mayo Clinic (IRB approved). Cells were plated onto 8‐well Lab‐Tek plates and grown to 70% confluence in a 5% CO2 humidified incubator. Cells were exposed to pro‐inflammatory cytokines TNFα (20ng/ml) or IL‐13 (50ng/ml) in the presence and absence of 1nM 17β‐estradiol (E2), 10nM PPT (ERα agonist) or WAY (ERβ agonist) for 24h. Cells were washed with HBSS and incubated with 3 μm Fluo‐3 for 1h for calcium imaging. The [Ca2+]i levels are measured from the intensity change observed following exposure to 10μM histamine under both physiological (2 mM) and zero extracellular calcium using Biotek LFX Fluorescence Imager.
In fluo‐3 loaded ASM cells, 24h exposure to WAY (and to a lesser extent PPT and E2) was effective in reducing [Ca2+]i levels. TNFα and IL‐13 significantly increased [Ca2+]i compared to vehicle. In the presence of TNFα or IL‐13, WAY significantly reduced [Ca2+]i in ASM cells, while PPT did not draw out any notable changes compared to TNFα or IL‐13 treatment alone. Addition of nifedipine (1μM, L‐type calcium channel blocker) resulted in a ~40% decrease in [Ca2+]i compared to control. This nifedipine blunting effect was similar in PPT treated group and to a higher extend to the WAY on [Ca2+]i responses to histamine, suggesting that ER effects were mediated via L‐type calcium channels. To verify the ER effect on sarcoplasmic reticulum (SR) Ca2+ reuptake, extracellular Ca2+ was removed by perfusion with zero Ca2+ HBSS, and Ca2+ influx was non‐specifically blocked with 1 mM LaCl3. Under these conditions, the [Ca2+]i responses were significantly reduced in the presence of WAY. Further, we examined effects of ER signaling on [Ca2+]i sequestration to SR (SERCA activity). In both control ASM cells as well as in those exposed to cytokines, WAY increased the rate of fall of [Ca2+]i responses to histamine, suggesting greater sequestration of [Ca2+]i.
Overall, our data highlights that both ERα and ERβ specific agonist were able to reduce [Ca2+]i in the inflamed ASM via receptor‐mediated genomic action. Specifically, ERβ activation is more effective in the presence of inflammation.
Support or Funding Information
Supported by NIH grants R01 HL123494 and R01 HL123494‐02S1 (Venkatachalem)
This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.