Connell, Jennifer

Purpose Asthma prevalence has been shown higher in female as compared to male, strongly indicating the involvement of sex steroids in modulating airway hyperreactivity. Airway smooth muscle (ASM) cells are major structural cells of the airway that determine the airway tone. Our recent study demonstrated the presence of estrogen receptors (ERα and ERβ) in ASM cells and that both ER's are significantly upregulated during asthma and/or inflammation with pronounced ERβ expression. We previously reported that estrogen acutely reduces agonist‐induced intracellular calcium ([Ca 2+ ] i ) levels in ASM cells. However, the role of long‐term ER signaling in regulating [Ca 2+ ] i of ASM is unknown, especially during inflammation or in asthma. Accordingly, we hypothesized that estrogen receptor isoforms have differential roles in [Ca 2+ ] i regulation in ASM cells, with enhanced functionality during inflammation. Methods Human ASM cells were enzymatically dissociated from tissues obtained incidental to lung surgery (Mayo Clinic). Asthmatic and non‐asthmatic ASM cells were exposed to pro‐inflammatory cytokines TNFα (20ng/ml) or IL‐13 (50ng/ml) in the presence or absence of 17β‐estradiol (E 2 ), various ERα (PPT, THC) or ERβ (WAY, FERB, DPN) agonists and antagonists (MPP,PHTPP) for 24h. Cells were incubated with calcium indicator dye Fura‐2 AM and [Ca 2+ ] i responses in these cells were measured using various bronchoconstrictors such as bradykinin (BK,10nM), acetylcholine (Ach,1μM), histamine (His,10μM) and KCl (100μM) under Olympus epifluorescence imaging system. Results In both asthmatic and non‐asthmatic human ASM cells, ERβ agonists reduced [Ca 2+ ] i response to BK, His, Ach as compared to vehicle. Conversely E 2 and ERα agonists showed no change or a slight increase in [Ca 2+ ] i response compared to vehicle. The effectiveness of ERβ in reducing the [Ca 2+ ] i in human ASM cells was further confirmed by ERβ siRNA and ERβ overexpression studies. In the presence of inflammation induced by TNFα or IL‐13, ERβ agonists significantly reduced [Ca 2+ ] i response, while E 2 and ERα agonists did not draw out any notable changes. To understand the mechanistic basis of the ER specific signaling in [Ca 2+ ] i regulation, we measured L‐type calcium channel activity (using L‐type channel blocker nifedipine, 1μM) with KCl. ERβ agonist reduced KCl induced [Ca 2+ ] i increase, which was comparable to the effect of nifedipine. [Ca 2+ ] i reuptake pump (SERCA) which sequesters the cytosolic calcium into sarcoplasmic reticulum showed increased expression and activity in ASM cells with ERβ activation during inflammation. Conclusion Overall, our data highlights the presence of divergent ER signaling in [Ca 2+ ] i handling in ASM. Specifically ERβ activation is more effective in reducing [Ca 2+ ] i during inflammation, through both L‐type channel inhibition and increased SERCA activity. These varied signaling mechanisms could play a crucial role in smooth muscle contractility and tone in asthma. Support or Funding Information Supported by NIH grants R01 HL123494 (Venkatachalem), and R01 HL088029 (Prakash). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Purpose 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 Ca 2+ ([Ca 2+ ] i ) regulation in human ASM are unknown. In this study, we evaluated the genomic effect of specific ERα and ERβ modulators on the [Ca 2+ ] i in human ASM cells. Methods 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% CO 2 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 (E 2 ), 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 [Ca 2+ ] 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. Results In fluo‐3 loaded ASM cells, 24h exposure to WAY (and to a lesser extent PPT and E 2 ) was effective in reducing [Ca 2+ ] i levels. TNFα and IL‐13 significantly increased [Ca 2+ ] i compared to vehicle. In the presence of TNFα or IL‐13, WAY significantly reduced [Ca 2+ ] 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 [Ca 2+ ] i compared to control. This nifedipine blunting effect was similar in PPT treated group and to a higher extend to the WAY on [Ca 2+ ] i responses to histamine, suggesting that ER effects were mediated via L‐type calcium channels. To verify the ER effect on sarcoplasmic reticulum (SR) Ca 2+ reuptake, extracellular Ca 2+ was removed by perfusion with zero Ca 2+ HBSS, and Ca 2+ influx was non‐specifically blocked with 1 mM LaCl 3. Under these conditions, the [Ca 2+ ] i responses were significantly reduced in the presence of WAY. Further, we examined effects of ER signaling on [Ca 2+ ] 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 [Ca 2+ ] i responses to histamine, suggesting greater sequestration of [Ca 2+ ] i. Conclusions Overall, our data highlights that both ERα and ERβ specific agonist were able to reduce [Ca 2+ ] 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 .

Asthma is defined as chronic inflammation of the airways and is characterized by airway remodeling, hyperresponsiveness, and acute bronchoconstriction of airway smooth muscle (ASM) cells. Clinical findings suggest a higher incidence and severity of asthma in adult women, indicating a concrete role of sex steroids in modulating the airway tone. Estrogen, a major female sex steroid mediates its role through estrogen receptors (ER) ERα and ERβ, which are shown to be expressed in human ASM, and their expression is upregulated in lung inflammation and asthma. Previous studies suggested rapid, nongenomic signaling of estrogen via ERs reduces intracellular calcium ([Ca2+]i), thereby promoting relaxation of ASM. However, long-term ER activation on [Ca2+]iregulation in human ASM during inflammation or in asthma is still not known. In Fura-2-loaded nonasthmatic and asthmatic human ASM cells, we found that prolonged (24 h) exposure to ERα agonist (PPT) increased [Ca2+]iresponse to histamine, whereas ERβ activation (WAY) led to decreased [Ca2+] compared with vehicle. This was further confirmed by ER overexpression and knockdown studies using various bronchoconstrictor agents. Interestingly, ERβ activation was more effective than 17β-estradiol in reducing [Ca2+]iresponses in the presence of TNF-α or IL-13, while no observable changes were noticed with PPT in the presence of either cytokine. The [Ca2+]i-reducing effects of ERβ were mediated partially via L-type calcium channel inhibition and increased Ca2+sequestration by sarcoplasmic reticulum.Overall, these data highlight the differential signaling of ERα and ERβ in ASM during inflammation. Specific ERβ activation reduces [Ca2+]iin the inflamed ASM cells and is likely to play a crucial role in regulating ASM contractility, thereby relaxing airways.