Airway smooth muscle (ASM) cell hyperplasia driven by persistent inflammation is a hallmark feature of remodeling in asthma. Sex steroid signaling in the lungs is of considerable interest, given epidemiological data showing more asthma in pre-menopausal women and aging men. Our previous studies demonstrated that estrogen receptor (ER) expression increases in asthmatic human ASM; however, very limited data are available regarding differential roles of ERα vs. ERβ isoforms in human ASM cell proliferation. In this study, we evaluated the effect of selective ERα and ERβ modulators on platelet-derived growth factor (PDGF)-stimulated ASM proliferation and the mechanisms involved. Asthmatic and non-asthmatic primary human ASM cells were treated with PDGF, 17β-estradiol, ERα-agonist and/or ERβ-agonist and/or G-protein-coupled estrogen receptor 30 (GPR30/GPER) agonist and proliferation was measured using MTT and CyQuant assays followed by cell cycle analysis. Transfection of small interfering RNA (siRNA) ERα and ERβ significantly altered the human ASM proliferation. The specificity of siRNA transfection was confirmed by Western blot analysis. Gene and protein expression of cell cycle-related antigens (PCNA and Ki67) and C/EBP were measured by RT-PCR and Western analysis, along with cell signaling proteins. PDGF significantly increased ASM proliferation in non-asthmatic and asthmatic cells. Treatment with PPT showed no significant effect on PDGF-induced proliferation, whereas WAY interestingly suppressed proliferation via inhibition of ERK1/2, Akt, and p38 signaling. PDGF-induced gene expression of PCNA, Ki67 and C/EBP in human ASM was significantly lower in cells pre-treated with WAY. Furthermore, WAY also inhibited PDGF-activated PCNA, C/EBP, cyclin-D1, and cyclin-E. Overall, we demonstrate ER isoform-specific signaling in the context of ASM proliferation. Activation of ERβ can diminish remodeling in human ASM by inhibiting pro-proliferative signaling pathways, and may point to a novel perception for blunting airway remodeling.

The incidence, severity and mortality of ongoing coronavirus infectious disease-19 (COVID-19) is greater in men compared to 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 vs. females as a model, we explored the effect of estrogen vs. 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 to 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 to vehicle. These intrinsic and sex steroids induced differences may help explain sex differences in COVID-19.

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Epidemiological data suggests increased prevalence of asthma in females than males, suggesting a plausible role for sex-steroids, especially estrogen in the lungs. Estrogen primarily acts through estrogen-receptors (ERa and ERb), which play a differential role in asthma. Our previous studies demonstrated increased expression of ERb in asthmatic human airway smooth muscle (ASM) cells and its activation diminished ASM proliferation in vitro and airway hyperresponsiveness (AHR) in vivo in a mouse (wild-type, WT) model of asthma. In this study, we evaluated the receptor specific effect of circulating endogenous estrogen in regulating AHR and remodeling using ERa and ERb knockout (KO) mice. C57BL/6J WT, ERa KO, and ERb KO mice were challenged intranasally with a mixedallergen (MA) or PBS. Lung function was measured using flexiVent followed by collection of broncho-alveolar lavage fluid for differential leukocyte count (DLC), histology using hematoxylin and eosin (H&E) and Sirius red-fast green (SRFG) and detecting asmooth muscle actin (a-SMA), fibronectin and vimentin expression using immunofluorescence (IF). Resistance (Rrs), elastance (Ers), tissue-damping (G) and tissue-elasticity (H) were significantly increased, whereas compliance (Crs) was significantly decreased in WT, ERa KO, and ERb KO mice (males and females) challenged with MA compared to PBS. Interestingly, ERb KO mice showed declined lung function compared to ERa KO and WT mice at baseline. MA induced AHR, remodeling and immune-cell infiltration was more prominent in females compared to males across all populations, while ERb KO females showed maximum AHR and DLC, except for neutrophil count. Histology using H&E suggests increased smooth muscle mass in airways with recruitment of inflammatory cells, while SRFG staining showed increased collagen deposition in MA challenged ERb KO mice compared to ERa KO and WT mice (males and females), with pronounced effects in ERb KO females. Furthermore, IF studies showed increased expression of a-SMA, fibronectin and vimentin in MA challenged populations compared to PBS, with prominent changes in ERb KO females. This novel study indicates ERb plays a pivotal role

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)-b, 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-a vs. ER-b activation on ECM production, deposition, and underlying pathways. Primary human ASM cells isolated from asthmatics and nonasthmatics were treated with E 2 , an ER-a agonist [propylpyrazoletriol (PPT)], and an ER-b agonist [WAY-200070 (WAY)] with TNF-a 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-kB. TNF-a 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-a-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-kB (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-b diminishes ECM deposition via suppressing the NF-kB pathway activity and might serve as a novel target to blunt airway remodeling.-