Estrogen Signaling on Mitochondrial Dynamics in Human Airway Smooth Muscle Cells
Authors: Jesme Christy , Bhallamudi Sangeeta , Pabelick Christina M , Prakash Y S , Sathish Venkatachalem 
Published: June 2021

BackgroundThe pathophysiology of asthma involves inflammation and altered structure and function of airway smooth muscle (ASM). There is emerging evidence that asthma affects adult women disproportionately with more severity than men. Also, our recent findings suggest that differential estrogen receptors (ER), ERα and ERβ expression and function are present in asthmatic ASM cells. Due to high oxygen environment in the lungs and the presence of inflammation, asthmatic ASM cells undergo oxidative stress. Mitochondrial dysfunction due to this oxidative stress plays a crucial role in the bioenergetics pathways involved in asthma. In this study, we hypothesize that differential ER signaling affects mitochondrial dynamics (fission/fusion) in ASM, which subsequently affects reactive oxygen species (ROS) generation and intracellular Ca2+ ([Ca2+]i) buffering capacity of mitochondria.MethodsHuman ASM cells were enzymatically dissociated from lung samples of non‐asthmatics undergoing thoracic surgery at Mayo Clinic. Cells were grown onto 8‐well labtek plates and treated with pro‐inflammatory cytokine, TNFα (20 mg/mL) in the presence or absence of 1 nM 17β‐estradiol (E2) which is a nonselective ER agonist, 10 nM PPT (ERα agonist) or 10 nM WAY (ERβ agonist) for 24h. For morphology studies, the cells were loaded with Mitotracker Green and then imaged on the Bio‐Tek Lionheart FX Live Cell Imager. Form factor and aspect ratio are used as parameters for determining fission vs fusion in mitochondria and these were calculated using MATLAB. Western blot and qPCR techniques were performed to access the protein and gene expression of the fission and fusion proteins Dynamin Related Protein1 (Drp1) and Mitofusion2 (Mfn2). ROS measurement was performed by loading cells with MitoSOX Red and their fluorescence intensity was recorded on the Live Cell Imager.ResultsIn mitotracker loaded ASM cells, ERβ agonist WAY was effective in increasing the form factor and aspect ratio as compared to vehicle. In addition, ERβ was effective in reducing TNFα induced reduction in form factor and aspect ratio. However, both E2 and ERα agonist, PPT, did not show any changes in form factor and aspect ratio of TNFα exposed ASM cells. Western analysis and gene expression studies showed higher expression of Drp1 and lower expression of Mfn2 in TNFα treated cells. Pre‐treatment with ERβ‐agonist reversed the altered expression of Drp1 and Mfn2 in TNFα treated cells. Using MitoSOX Red, it was observed that ERβ agonist WAY treated cells showed lower ROS generation as compared to TNFα.ConclusionOur data suggests a novel estrogen signaling in ASM cells for maintaining the structure of mitochondria. ERβ is effective in ameliorating the inflammation due to TNFα and hence preserves the morphology of mitochondria during inflammation. Further studies will confirm the subsequent alterations in mitochondrial ROS generation and [Ca2+]i buffering capacity due to this altered mitochondrial morphology.Support or Funding InformationSupported by the UGSRF fellowship by APS (Jesme) and NIH grants R01 HL123494, (Venkatachalem), and R01 HL 088029 (Prakash)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.