Shigeyuki Mukudai1, Nao Hiwatashi1, Renjie Bing1, Michael Garabedian2, Ryan C Branski1. 1. From the Department of Otolaryngology-Head and Neck Surgery , NYU Voice Center, New York, New York, U.S.A. 2. the Department of Microbiology , New York University School of Medicine, New York, New York, U.S.A.
Abstract
OBJECTIVES/HYPOTHESIS: Direct glucocorticoid (GC) injection for vocal fold (VF) scarring has evolved as a therapeutic strategy, but the mechanisms underlying the antifibrotic effects remain unclear. GCs act via the glucocorticoid receptor (GR), which is phosphorylated at multiple serine residues in a hormone-dependent manner to affect bioactivity. We hypothesize that GCs regulate SMAD signaling via GR phosphorylation in vocal fold fibroblasts (VFFs). STUDY DESIGN: In vitro. METHODS: Human VFFs were treated with dexamethasone (DM; 10-5 -10-7 M) ± transforming growth factor (TGF)-β1 (10 ng/mL). RU486 (10-6 M) was employed to isolate the regulatory effects of GR. Total GR, Ser211 , and Ser203 phosphorylation was examined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunocytochemistry. Quantitative polymerase chain reaction was employed to determine GR-mediated effects of DM on genes related to fibrosis. RESULTS: Total GR and Ser211 phosphorylation was observed predominantly in the nucleus 1 hour after DM administration. DM decreased total GR expression, but Ser203 and Ser211 phosphorylation increased. RU486 limited the effects of DM. SMAD3 and SMAD7 mRNA expression significantly decreased 4 hours after DM administration (P < 0.05); this response was negated by RU486. COL1A1 remained unchanged, and ACTA2 significantly increased following 24 hours of DM treatment (P < 0.05). CONCLUSION: DM regulated TGF-β1 signaling via altered SMAD3 and SMAD7 expression. This response was associated with altered GR phosphorylation. These findings provide insight into the mechanisms of steroidal effects on vocal fold repair; ultimately, we seek to enhance therapeutic strategies for these challenging patients. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E187-E193, 2019.
OBJECTIVES/HYPOTHESIS: Direct glucocorticoid (GC) injection for vocal fold (VF) scarring has evolved as a therapeutic strategy, but the mechanisms underlying the antifibrotic effects remain unclear. GCs act via the glucocorticoid receptor (GR), which is phosphorylated at multiple serine residues in a hormone-dependent manner to affect bioactivity. We hypothesize that GCs regulate SMAD signaling via GR phosphorylation in vocal fold fibroblasts (VFFs). STUDY DESIGN: In vitro. METHODS:Human VFFs were treated with dexamethasone (DM; 10-5 -10-7 M) ± transforming growth factor (TGF)-β1 (10 ng/mL). RU486 (10-6 M) was employed to isolate the regulatory effects of GR. Total GR, Ser211 , and Ser203 phosphorylation was examined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunocytochemistry. Quantitative polymerase chain reaction was employed to determine GR-mediated effects of DM on genes related to fibrosis. RESULTS: Total GR and Ser211 phosphorylation was observed predominantly in the nucleus 1 hour after DM administration. DM decreased total GR expression, but Ser203 and Ser211 phosphorylation increased. RU486 limited the effects of DM. SMAD3 and SMAD7 mRNA expression significantly decreased 4 hours after DM administration (P < 0.05); this response was negated by RU486. COL1A1 remained unchanged, and ACTA2 significantly increased following 24 hours of DM treatment (P < 0.05). CONCLUSION:DM regulated TGF-β1 signaling via altered SMAD3 and SMAD7 expression. This response was associated with altered GR phosphorylation. These findings provide insight into the mechanisms of steroidal effects on vocal fold repair; ultimately, we seek to enhance therapeutic strategies for these challenging patients. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E187-E193, 2019.
Authors: Camilo Mora-Navarro; Andreea Badileanu; Ana M Gracioso Martins; Emily W Ozpinar; Lewis Gaffney; Ian Huntress; Erin Harrell; Jeffrey R Enders; Xinxia Peng; Ryan C Branski; Donald O Freytes Journal: ACS Biomater Sci Eng Date: 2020-02-11
Authors: Ryosuke Nakamura; Renjie Bing; Carina P Doyle; Michael J Garabedian; Ryan C Branski Journal: Exp Cell Res Date: 2021-06-02 Impact factor: 4.145