Estrogen receptors differentially regulate intracellular calcium handling in human nonasthmatic and asthmatic airway smooth muscle cells

Abstract

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 ([Ca2+]i) levels in ASM cells. However, the role of long‐term ER signaling in regulating [Ca2+]i of ASM is unknown, especially during inflammation or in asthma. Accordingly, we hypothesized that estrogen receptor isoforms have differential roles in [Ca2+]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 (E2), 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 [Ca2+]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 [Ca2+]i response to BK, His, Ach as compared to vehicle. Conversely E2 and ERα agonists showed no change or a slight increase in [Ca2+]i response compared to vehicle. The effectiveness of ERβ in reducing the [Ca2+]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 [Ca2+]i response, while E2 and ERα agonists did not draw out any notable changes. To understand the mechanistic basis of the ER specific signaling in [Ca2+]i regulation, we measured L‐type calcium channel activity (using L‐type channel blocker nifedipine, 1μM) with KCl. ERβ agonist reduced KCl induced [Ca2+]i increase, which was comparable to the effect of nifedipine. [Ca2+]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 [Ca2+]i handling in ASM. Specifically ERβ activation is more effective in reducing [Ca2+]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...

Sex steroids skew ACE2 expression in human airway: a contributing factor to sex differences in COVID-19?

“…Perhaps more interestingly, Tes exposure significantly upregulated ACE2 expression in human ASM cells. Here, it is important to note that E 2 and Tes concentrations used in our study are physiological ( 4 , 5 , 7 , 22 , 38 , 39 ). Furthermore, we have previously reported that both male and female human ASM expresses estrogen receptor isoforms (alpha and beta) as well as the androgen receptor ( 6 , 22 ).…”

“…Serum-deprived human ASM cells were exposed to vehicle, E 2 [1 nM ( 5 , 7 )], and Tes (10 nM ( 22 ),) in DMEM/F12 (FBS free) for 24 h followed by protein collection for Western analyses ( 4 , 5 , 22 ).…”

Role of Estrogen Receptors α and β in a Murine Model of Asthma: Exacerbated Airway Hyperresponsiveness and Remodeling in ERβ Knockout Mice

“…Furthermore, we have also shown that ERb activation inhibits PDGF induced proliferation in primary human ASM cells via AKT/ERK/p38 pathways (Ambhore et al, 2018). Many studies have shown the impact of ER signaling on asthma in vitro (Townsend et al, 2010;Townsend et al, 2012b;Martin et al, 2015;Sathish et al, 2015a;Aravamudan et al, 2017;Ambhore et al, 2018;Ambhore et al, 2019b;Bhallamudi et al, 2019); however, very few studies have explored the role of estrogen in asthma in vivo (Carey et al, 2007a;Carey et al, 2007c;Riffo-Vasquez et al, 2007;Dimitropoulou et al, 2009;Jia et al, 2011). Few studies reported down-regulated AHR upon administration of estrogen in females and OVX mice; however, the receptorspecific role of estrogen has not been explored (Riffo-Vasquez et al, 2007;Matsubara et al, 2008;Dimitropoulou et al, 2009).…”

“…Incidence of asthma is more common in pre-pubescent boys and adult women and the severity of asthma is increased during pregnancy (de Marco et al, 2000;Caracta, 2003;Carey et al, 2007b;Bonds and Midoro-Horiuti, 2013). In this context, multiple studies have explored and suggested a role for sex hormones in airway biology, especially estrogen (Keselman and Heller, 2015;Sathish et al, 2015a;Ambhore et al, 2018;Ambhore et al, 2019a;Ambhore et al, 2019b;Bhallamudi et al, 2019;Fuentes et al, 2019;Fuentes and Silveyra, 2019a). However, there is still a debate on the contradicting role of estrogen as few studies suggest its role in reducing inflammation (Myers and Sherman, 1994;Lieberman et al, 1995;Haggerty et al, 2003), while others suggest estrogen to induce AHR and inflammation (Riffo-Vasquez et al, 2007;Sakazaki et al, 2008).…”

“…Estrogen primarily acts through estrogen receptors (ER) ERa and ERb, which are widely considered to be nuclear receptors (Altucci and Gronemeyer, 2001). Multiple studies suggest both ERa and ERb signal through different cell signaling pathways eliciting different effects on cellular functions (Kuiper et al, 1996;Clarke et al, 2003;Heldring et al, 2007;Edvardsson et al, 2011;Williams and Lin, 2013;Aravamudan et al, 2017;Ambhore et al, 2018;Ambhore et al, 2019b;Bhallamudi et al, 2019;Fuentes and Silveyra, 2019a). ER's are expressed on a wide array of cells and our group has recently showed differential ER expression in human ASM cells, which is upregulated during inflammation/ asthma (especially ERb) (Aravamudan et al, 2017).…”