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Estrogen Receptors Differentially Regulate the Overall Contractility of Human Airway Smooth Muscle
Author(s) -
Bhallamudi Sangeeta,
Ambhore Nilesh Sudhakar,
Saladi Swetha,
Pabelick Christina M.,
Prakash Y.S.,
Sathish Venkatachalem
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.05418
Subject(s) - rhoa , contractility , endocrinology , medicine , agonist , estrogen receptor , receptor , cyclic adenosine monophosphate , contraction (grammar) , calcium in biology , myosin light chain phosphatase , myosin , estrogen , microbiology and biotechnology , myosin light chain kinase , biology , chemistry , signal transduction , cancer , breast cancer
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).