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

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

Complex fibroblast response to glucocorticoids may underlie variability of clinical efficacy in the vocal folds

“…We, therefore, sought to elucidate potential interactions between TGF-β and GC signaling to provide insight into clinical variability with the ultimate goal of optimized therapeutic efficacy. Previously, our laboratory immunolocalized the GC receptor (GR) in the vocal fold mucosa in vivo 12 and in our human vocal fold fibroblast cell line with an emphasis on three major serine phosphorylation sites within the N-terminal region of the receptor involved in transcriptional regulation (Ser 203 , Ser 211 , and Ser 226 ) 11 . In response to dexamethasone, Ser 211 localized predominately to the nucleus 11,13,14 .…”

The Antiproliferative and Antifibrotic Effects of Cisplatin on Primary Human Vocal Fold Fibroblasts

“…Previous articles have demonstrated an antifibrotic effect of pirfenidone in which the level of p-Smad2/3 in the nucleus faded with pirfenidone treatment [11]. Mukudai et al [20] demonstrated that the mechanistic effect of steroids on vocal fold repair involves significantly altered Smad3 and Smad7 mRNA expression after steroid administration. Thus, this new discovery indicates that cisplatin may be an ideal antifibrotic agent for local application in patients diagnosed with laryngeal cancer who require CO 2 microsurgery.…”

“…HVFFs were incubated with cisplatin at different concen-(For figure see next page.) trations (10,20,40,80, and 160 μΜ; Fig. 2a).…”

The Role of Cytokines in Modulating Vocal Fold Fibrosis: A Contemporary Review

“…Conversely, Smad7 is a competitive inhibitor of Smad activation and is therefore thought to be antifibrotic. 31 Specifically, Smad3 is considered a master regulator of fibrosis. 32 Molecular signaling offers a new strategy to intervene in scar formation.…”