Phosphorylation of the glucocorticoid receptor alters SMAD signaling in vocal fold fibroblasts
Portions of these data were accepted for presentation at the American Laryngological Association, Combined Otolaryngology Spring Meetings, National Harbor, Maryland, U.S.A., April 18–20, 2018.
Funding for this work was provided by the National Institute on Deafness and Other Communication Disorders (NIDCD)/the National Institutes of Health (NIH), (RO1 DC013277 and RO1 DC017397, PI-Branski). The authors have no other funding, financial relationships, or conflicts of interest to disclose.
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; 10−5–10−7M) ± transforming growth factor (TGF)-β1 (10 ng/mL). RU486 (10−6M) 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
