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A1234 - Estrogen Receptor Beta Signaling Negatively Regulates PDGF Induced Human Airway Smooth Muscle Proliferation
Author Block: N. S. Ambhore1, R. Katragadda1, R. Kalidhindi1, M. Thompson2, C. M. Pabelick2, Y. Prakash2, S. Venkatachalem1; 1Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND, United States, 2Department of Anesthesiology and Perioperative Medicine, Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, United States.
Rational: Airway smooth muscle (ASM) cell hyperplasia driven by persistent inflammation is a hallmark feature of remodelling in disorders such as asthma. Sex steroid signaling (particularly estrogen) in the lung 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 cells; however, very limited data are available regarding molecular mechanisms of ER signaling in human ASM cell proliferation, or of differential roles for ERα vs. ERβ isoforms. In this study, we evaluated the effect of specific ERα and ERβ modulators on platelet-derived growth factor (PDGF)-induced ASM proliferation and the mechanisms involved. Methods: Asthmatic and non-asthmatic primary human ASM cells isolated from surgical lung resections were cultured in DMEM-F12 medium (Mayo IRB approved). Cells were seeded in culture plates and at ~80% confluency, cells were treated with PDGF, 17β-estradiol (E2), ERα-agonist (PPT), and/or ERβ-agonist (WAY-200070) and proliferation rates measured using CyQuant and MTT assays followed by flow cytometry for cell cycle analysis. Gene expression of cell cycle related antigens (PCNA and Ki67) and C/EBP were measured by Quantstudio real-time RT-PCR and Western analysis. Expression of cell signaling proteins such as p-ERK, p-AKT and p-p38 were measured using both normal and asthmatic human ASM cells. Results: CyQuant and MTT assays showed that PDGF (2ng/ml) significantly increased ASM proliferation in non-asthmatic and asthmatic samples. Treatment with ERα-agonist showed no significant effect on PDGFinduced proliferation, whereas ERβ agonist interestingly suppressed proliferation via inhibition of ERK1/2, AKT and p38 signaling. Significant increases in gene expression of PCNA, Ki67 and C/EBP were observed in PDGF treated asthmatic and non-asthmatic ASM cell lysates. Pre-treatment with ERβ-agonist showed significantly lower expression of these markers. Furthermore, ERβ-agonist inhibited PDGF activated PCNA, C/EBP, cyclinD1 and cyclin-E. Conclusion: Overall, we demonstrate estrogen receptor isoform specific signalling in the context of ASM proliferation. Activation of ERβ can diminish remodelling in human ASM by inhibiting pro-proliferative signalling pathways, and may thus point to a novel tact for blunting airway remodeling.