Ambhore, Nilesh Sudhakar

The incidence, severity, and mortality of ongoing coronavirus infectious disease 19 (COVID-19) is greater in men compared with women, but the underlying factors contributing to this sex difference are still being explored. In the current study, using primary isolated human airway smooth muscle (ASM) cells from normal males versus females as a model, we explored the effect of estrogen versus testosterone in modulating the expression of angiotensin converting enzyme 2 (ACE2), a cell entry point for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using confocal imaging, we found that ACE2 is expressed in human ASM. Furthermore, Western analysis of ASM cell lysates showed significantly lower ACE2 expression in females compared with males at baseline. In addition, ASM cells exposed to estrogen and testosterone for 24 h showed that testosterone significantly upregulates ACE2 expression in both males and females, whereas estrogen downregulates ACE2, albeit not significant compared with vehicle. These intrinsic and sex steroids induced differences may help explain sex differences in COVID-19.

Purpose Epidemiological evidences suggest higher incidence and severity of asthma in pre‐menopausal women and aging men, indicating the role of sex steroids, especially estrogen. Our recent studies demonstrated that airway smooth muscle (ASM) cells from both male and female expresses estrogen receptors (ERα and β) and these receptors differentially regulate intracellular calcium ([Ca 2+ ] i ) which is a key role player in contraction. The degree of force for contraction can also be altered in a Ca 2+ ‐independent manner via RhoA. Both Ca 2+ ‐dependent and Ca 2+ ‐independent pathways in ASM thus together contribute towards the airway contractility. In this study, we hypothesized that the ERs differentially regulate cyclic adenosine monophosphate (cAMP) and RhoA mechanisms, thereby affecting the overall contractility of human ASM cells. Methods Asthmatic and non‐asthmatic primary human ASM cells (isolated from lung tissue, Mayo Clinic, IRB‐approved) were seeded and grown to 70% confluence. After serum‐deprivation, cells were exposed to 1nM 17β‐estradiol (E 2 ), 10nM PPT (ERα agonist) or WAY (ERβ agonist) for 24hr. IBMX (10μM) was used as phosphodiesterase inhibitor and Isoproterol (100nM for 10min) served as a positive control group for estimation of cAMP. In parallel studies, phosphorylation of myosin light chain (MLC 20 ) and myosin phosphatase targeting subunit‐1 (MYPT1) was assessed through Western analyses. Activated RhoA and cAMP measurements were done through ELISA kits. Cell contraction was accessed through traction force microscopy (TFM) using softgel plates coated with collagen and fluorescent beads. Upon treatments, phase‐contrast and fluorescent images were captured before, during and after 10μM histamine exposure and net traction force was calculated using advanced software. Results Prolonged (24h) activation of ERβ significantly increased the baseline cAMP levels whereas ERα activation decreased it. This effect was more profound in asthmatic ASM cells compared to non‐asthmatics. Furthermore, ERβ activation lowered the active RhoA levels in non‐asthmatic human ASM cells. The phosphorylation of MLC 20 and MYPT1 was significantly attenuated with ERβ activation compared to vehicle. TFM studies demonstrated reduced traction force in non‐asthmatic ASM cells treated with ERβ agonist compared to the vehicle, whereas ERα activation did not show any significant changes. Conclusion Our findings highlight the crucial role of divergent ER signaling in regulating Ca 2+ ‐dependent and Ca 2+ ‐independent pathways, which together govern the contractile function of ASM. Particularly, ERβ activation (and not ERα) mediates ASM relaxation by increasing the cAMP, reducing active RhoA and decreasing phosphorylation of MLC 20 /MYPT1. Thus, ERβ activation might serve as a novel target to manage the increased ASM contractility in asthma. Support or Funding Information Acknowledgement Supported by NIH grants R01‐HL0123494 (Venkatachalem), R01‐HL 088029 (Prakash) and R01‐HL142061 (Pabelick, Prakash).

Altered airway smooth muscle (ASM) mass and extracellular matrix (ECM) deposition in airways are characteristic features of remodeling in asthma. Increased ECM production modulates ASM cell proliferation and leads to airway remodeling. Our previous studies showed that ASM from patients with asthma exhibited increased expression of estrogen receptor (ER)‐β, which upon activation down‐regulated ASM proliferation, implicating an important role for estrogen signaling in airway physiology. There is no current information on the effect of differential ER activation on ECM production. In this study, we evaluated the effect of ER‐α vs. ER‐β activation on ECM production, deposition, and underlying pathways. Primary human ASM cells isolated from asthmatics and nonasthmatics were treated with E 2 , an ER‐α agonist [propylpyrazoletriol (PPT)], and an ER‐β agonist [WAY‐200070 (WAY)] with TNF‐α or platelet‐derived growth factor (PDGF) followed by evaluation of ECM production and deposition. Expression of proteins and genes corresponding to ECM were measured using Western blotting and quantitative RT‐PCR with subsequent matrix metalloproteinase (MMP) activity. Molecular mechanisms of ER activation in regulating ECM were evaluated by luciferase reporter assays for activator protein 1 (AP‐1) and NF‐κB. TNF‐α or PDGF significantly ( P < 0.001) increased ECM deposition and MMP activity in human ASM cells, which was significantly reduced with WAY treatment but not with PPT. Furthermore, TNF‐α‐ or PDGF‐induced ECM gene expression in ASM cells was significantly reduced with WAY ( P < 0.001). Moreover, WAY significantly down‐regulated the activation of NF‐κB( P < 0.001) and AP‐1 ( P < 0.01, P < 0.05) in ASM cells from asthmatics and nonasthmatics. Overall, we demonstrate differential ER signaling in controlling ECM production and deposition. Activation of ER‐β diminishes ECM deposition via suppressing the NF‐κB pathway activity and might serve as a novel target to blunt airway remodeling.—Ambhore, N. S., Kalidhindi, R. S. R., Pabelick, C. M., Hawse, J. R., Prakash, Y. S., Sathish, V. Differential estrogen‐receptor activation regulates extracellular matrix deposition in human airway smooth muscle remodeling via NF‐κB pathway. FASEB J. 33, 13935‐13950 (2019). www.fasebj.org

Abstract Evidence suggests that airway hyperresponsiveness (AHR) is a characteristic feature of asthma. Epidemiological studies have confirmed that the severity of asthma is greater in women, suggesting a critical role of female sex steroid hormones (especially estrogen). Very few in vivo studies have examined the role of sex steroid hormones in asthma, and the sequence of events that occur through differential activation of estrogen receptors (ERs) remains to be determined in asthmatic airways. Our recent in vitro findings indicated that ERβ had increased expression in asthmatic airway smooth muscle (ASM), and that its activation by an ERβ-specific agonist downregulated airway remodeling. In this study, we translated the in vitro findings to a murine asthma model and examined the differential role of ER activation in modulating lung mechanics. C57BL/6J male, female, and ovariectomized mice were exposed to mixed allergen (MA) and subcutaneously implanted with sustained-release pellets of placebo, an ERα agonist (4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol [PPT]), and/or an ERβ agonist (WAY-200070). We then evaluated the effects of these treatments on airway mechanics, biochemical, molecular, and histological parameters. Mice exposed to MA showed a significant increase in airway resistance, elastance, and tissue damping, and a decrease in compliance; pronounced effects were observed in females. Compared with PPT, WAY treatment significantly reversed the MA-induced changes. The increased mRNA/protein expression of ERα, ERβ, and remodeling genes observed in MA-treated mice was significantly reversed in WAY-treated mice. This novel study indicates that activation of ERβ signaling downregulates AHR and airway remodeling, and is a promising target in the development of treatments for asthma.