Dose‐Dependent Glucocorticoid Regulation of Transcription Factors in Vocal Fold Fibroblasts and Macrophages

Abstract

ObjectiveVariable outcomes of glucocorticoid (GC) therapy for laryngeal disease are putatively due to diverse interactions of the GC receptor (GR) with cell signaling pathways, limited consideration regarding concentration‐dependent effects, and inconsistent selection of GCs. In the current study, we evaluated the concentration‐dependent effects of three frequently administered GCs on transcription factors with an emphasis on the phosphorylation of GR at Ser203 and Ser211 regulating the nuclear translocation of GR. This study provides foundational data regarding the diverse functions of GCs to optimize therapeutic approaches.Study designIn vitro.MethodsHuman vocal fold fibroblasts and THP1‐derived macrophages were treated with different concentrations of dexamethasone, methylprednisolone, and triamcinolone in combination with IFN‐γ, TNF‐α, or IL4. Phosphorylated STAT1, NF‐κB family molecules, and phosphorylated STAT6 were analyzed by Western blotting. Ser211‐phosphorylated GR (S211‐pGR) levels relative to GAPDH and Ser203‐phosphorylated GR (S203‐pGR) were also analyzed.ResultsGCs differentially altered phosphorylated STAT1 and NF‐κB family molecules in different cell types under IFN‐γ and TNF‐α stimuli. GCs did not alter phosphorylated STAT6 in IL4‐treated macrophages. The three GCs were nearly equivalent. A lower concentration of dexamethasone increased S211‐pGR/GAPDH ratios relative to increased S211‐pGR/S203‐pGR ratios regardless of cell type and treatment.ConclusionThe three GCs employed in two cell lines had nearly equivalent effects on transcription factor regulation. Relatively high levels of Ser203‐phosphorylation at low GC concentrations may be related to concentration‐dependent differential effects of GCs in the two cell lines.Level of EvidenceNA Laryngoscope, 133:2704–2711, 2023

A Novel Method for Thyroarytenoid Myofiber Culture

“…7A–C) and S211‐pGR ( U = 4, p < 0.001; Fig. 7D–F) nuclear translocation, consistent with previous work 27–32 …”

“…Total GR and s211‐pGR nuclear translocation was determined via immunolabeling as an indicator of a physiologically relevant response to DM treatment 32,40,41 . Dexamethasone significantly increased tGR and s211‐pGR nuclear translocation in TA myofibers, consistent with previous work 27–32 …”

“…32,40,41 Dexamethasone significantly increased tGR and s211-pGR nuclear translocation in TA myofibers, consistent with previous work. [27][28][29][30][31][32] Collectively, this work confirmed myofiber specificity, viability, and predictable reactivity to clinically relevant pharmacological treatment. This model provides a foundation to investigate TA muscle-specific phenomena in an appropriate micro-environment including myogenic satellite cells and potential signaling/interactions via adherence to basement membrane components.…”

“…Quantification of tGR and s211-pGR nuclear translocation was performed using ImageJ. 27,28 Briefly, nuclear regions within each myofiber were defined by DAPI-positivity. The cytoplasm was defined as DAPI-negative and MHC-positive.…”

High-dose methylprednisolone mediates YAP/TAZ-TEAD in vocal fold fibroblasts with macrophages

“…15,30 Our previous work with indirect co-culture models found brotic and in ammatory responses of VF broblasts triggered by macrophage-derived paracrine signals were promoted and inhibited by 'high' and 'low' concentrations of methylprednisolone, respectively. 31 Based on this nding, we hypothesized minimizing GC concentrations to su ciently inhibit in ammation improves e cacy of GC therapy. However, previous co-culture studies employed a cell culture insert to allow only paracrine signaling.…”