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<p>The pro-fibrotic effects of glucocorticoids may lead to a suboptimal therapeutic response for vocal fold (VF) pathology. Targeting macrophage-fibroblast interactions is an interesting therapeutic strategy; macrophages alter their phenotype to mediate both inflammation and fibrosis. In the current study, we investigated concentration-dependent effects of methylprednisolone on the fibrotic response, with an emphasis on YAP/TAZ-TEAD signaling, and inflammatory gene expression in VF fibroblasts in physical contact with macrophages. We sought to provide foundational data to optimize therapeutic strategies for millions of patients with voice/laryngeal disease-related disability. Following induction of inflammatory (M(IFN/LPS)) and fibrotic (M(TGF)) phenotypes, THP-1-derived macrophages were seeded onto HVOX vocal fold fibroblasts. Cells were co-cultured ± 0.3–3000 nM methylprednisolone ± 3 µM verteporfin, a YAP/TAZ inhibitor. Inflammatory (<i>CXCL10</i>, <i>TNF</i>, <i>PTGS2</i>) and fibrotic genes (<i>ACTA2</i>, <i>CCN2</i>, <i>COL1A1</i>) in fibroblasts were analyzed by real-time polymerase chain reaction after cell sorting. Ser211-phosphorylated glucocorticoid receptor (S211-pGR) was assessed by Western blotting. Nuclear localization of S211-pGR and YAP/TAZ was analyzed by immunocytochemistry. Methylprednisolone decreased <i>TNF</i> and <i>PTGS2</i> in fibroblasts co-cultured with M(IFN/LPS) macrophages and increased <i>ACTA2</i> and <i>CCN2</i> in fibroblasts co-cultured with M(IFN/LPS) and M(TGF). Lower concentrations were required to decrease <i>TNF</i> and <i>PTGS2</i> expression and to increase S211-pGR than to increase <i>ACTA2</i> and <i>CCN2</i> expression and nuclear localization of S211-pGR. Methylprednisolone also increased YAP/TAZ nuclear localization. Verteporfin attenuated upregulation of <i>CCN2</i>, but not <i>PTGS2</i> downregulation. High concentration methylprednisolone induced nuclear localization of S211-pGR and upregulated fibrotic genes mediated by YAP/TAZ activation.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年3月31日

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  • Ryosuke Nakamura,
  • Renjie Bing,
  • Gary J. Gartling,
  • Michael J. Garabedian,
  • Ryan C. Branski

摘要

The pro-fibrotic effects of glucocorticoids may lead to a suboptimal therapeutic response for vocal fold (VF) pathology. Targeting macrophage-fibroblast interactions is an interesting therapeutic strategy; macrophages alter their phenotype to mediate both inflammation and fibrosis. In the current study, we investigated concentration-dependent effects of methylprednisolone on the fibrotic response, with an emphasis on YAP/TAZ-TEAD signaling, and inflammatory gene expression in VF fibroblasts in physical contact with macrophages. We sought to provide foundational data to optimize therapeutic strategies for millions of patients with voice/laryngeal disease-related disability. Following induction of inflammatory (M(IFN/LPS)) and fibrotic (M(TGF)) phenotypes, THP-1-derived macrophages were seeded onto HVOX vocal fold fibroblasts. Cells were co-cultured ± 0.3–3000 nM methylprednisolone ± 3 µM verteporfin, a YAP/TAZ inhibitor. Inflammatory (CXCL10, TNF, PTGS2) and fibrotic genes (ACTA2, CCN2, COL1A1) in fibroblasts were analyzed by real-time polymerase chain reaction after cell sorting. Ser211-phosphorylated glucocorticoid receptor (S211-pGR) was assessed by Western blotting. Nuclear localization of S211-pGR and YAP/TAZ was analyzed by immunocytochemistry. Methylprednisolone decreased TNF and PTGS2 in fibroblasts co-cultured with M(IFN/LPS) macrophages and increased ACTA2 and CCN2 in fibroblasts co-cultured with M(IFN/LPS) and M(TGF). Lower concentrations were required to decrease TNF and PTGS2 expression and to increase S211-pGR than to increase ACTA2 and CCN2 expression and nuclear localization of S211-pGR. Methylprednisolone also increased YAP/TAZ nuclear localization. Verteporfin attenuated upregulation of CCN2, but not PTGS2 downregulation. High concentration methylprednisolone induced nuclear localization of S211-pGR and upregulated fibrotic genes mediated by YAP/TAZ activation.