Phosphorylation of the glucocorticoid receptor alters SMAD signaling in vocal fold fibroblasts

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.