Goorhouse, Katelyn J.

Introduction Asthma is acutely aggravated and chronically worsened by cigarette smoke (CS), as well as secondhand smoke exposure. In this regard, studies show significant differences between women vs. men in their response to the detrimental effects of CS, suggesting that female sex steroids (particularly estrogen) may be involved. Airway smooth muscle (ASM) cell proliferation and airway remodeling is a key feature of asthma and COPD. Surprisingly, there is little information regarding sex steroid effects on cigarette smoke‐induced changes of airway structure and function. In the current study, we hypothesize that estrogen interferes with CSE exposure‐induced proliferation, thereby contributing to airway smooth muscle remodeling. Methods and Results Human ASM tissues (epithelium‐denuded) and isolated ASM cells were from lung samples of patients with known smoking histories undergoing thoracic surgery for non‐infectious, focal pathology. Exposure of human ASM to cigarette smoke extract (CSE, Kentucky research cigarette 1RF4) 1%CSE or 2%CSE for 24h significantly increased ASM proliferation (proliferation assay: Cell Titer Aueous One, and Cyquant). Exposure of physiological concentrations of 17‐β‐estradiol (E 2, 1nM) did not alter ASM proliferation at baseline. Interestingly, E 2 pre‐treatment (1 h prior to CSE exposure) significantly reduced CSE‐induced human ASM proliferation. This was further confirmed by Western blot analysis for proliferative markers PCNA and Cyclin E. In addition, to explore the mechanistic bases of differential effects of E 2 on ASM proliferation under basal and CSE‐exposed conditions, the estrogen receptor profile in ASM tissue was screened in parallel for patients with and without smoking histories. Human ASM tissue from smokers samples showed substantial increased ERα and β expression compared to non‐smokers. Inhibition of ERα and β prevented the effects of estrogen on CSE‐induced increases in proliferation. Conclusions Collectively, estrogen exposure decreased human ASM proliferation induced by CSE. ER expression and signaling is enhanced in CSE induced inflamed ASM, with a potential beneficial role in reducing remodeling with CS exposure. These results will lay the foundation for examining the role of estrogen signaling in pathogenesis of cigarette smoke‐induced airway remodeling and possible sex based therapeutic targets in smoking‐related diseases. Support or Funding Information Supported by Flight Attendants Medical Research Institute (VS) and NIH R01 grant HL123494 (VS)

The prevalence of asthma is higher in pre‐pubescent and aging males, and in post‐pubertal females, strongly indicating that sex steroids (especially estrogen) may be an important modulator in lung disease. We recently demonstrated that airway smooth muscle (ASM) expresses both alpha and beta forms of the estrogen receptor (ERα and ERβ) in males and females, and that these receptors regulate intracellular [Ca 2+ ] and ASM contractility. Although both ERα and ERβ have multiple splice variants, it is unclear if and how the expression of these variants is modulated under conditions such as chronic inflammation/asthma. In order to test the hypothesis that the differential expression of ERα and ERβ variants contributes to the pathogenesis of asthma, we profiled the expression of various ERα and ERβ genes in asthmatic and inflamed (TNFα‐ or IL‐13‐treated) ASM. Gene expression was assessed at both the mRNA and protein levels in asthmatic ASM cells or non‐asthmatic cells treated with TNFα (20 ng/ml) or IL‐13 (50 ng/ml). We observed marked variation in the expression of ER isoforms in response to inflammatory stimuli, and in non‐asthmatic versus asthmatic ASM. Changes in protein levels of ERα and ERβ corresponded with the observed differential mRNA patterns. Pharmacological studies implicate cytosolic (p42/44 MAPK and PI3 K) and nuclear (NFκB, STAT6, and AP‐1) signaling pathways as putative mechanisms that mediate and/or regulate effects of inflammation on ER expression. We conclude that variations in ASM ER expression profiles occur with inflammation and that ER variants could contribute to estrogen signaling in airway diseases such as asthma. J. Cell. Physiol. 232: 1754–1760, 2017. © 2016 Wiley Periodicals, Inc.