Description of a Novel Mechanism Possibly Explaining the Antiproliferative Properties of Glucocorticoids in Duchenne Muscular Dystrophy Fibroblasts Based on Glucocorticoid Receptor GR and NFAT5.

Sandrine Herbelet,Jan L De Bleecker,Boel De Paepe

doi:10.3390/ijms21239225

Abstract

Glucocorticoids are drugs of choice in Duchenne muscular dystrophy (DMD), prolonging patients’ ambulation. Their mode of action at the protein level is not completely understood. In DMD, muscle tissue is replaced by fibrotic tissue produced by fibroblasts, reducing mobility. Nuclear factor of activated T-cells 5 (NFAT5) is involved in fibroblast proliferation. By treating one DMD fibroblast cell culture and one of unaffected skeletal muscle fibroblasts with methylprednisolone (MP) or hydrocortisone (HC) for 24 h or 12 d, the antiproliferative properties of glucocorticoids could be unraveled. NFAT5 localization and expression was explored by immunocytochemistry (ICC), Western blotting (WB) and RT-qPCR. NFAT5 and glucocorticoid receptor (GR) colocalization was measured by ImageJ. GR siRNA was used, evaluating GR’s influence on NFAT5 expression during MP and HC treatment. Cell proliferation was monitored by IncuCyte ZOOM. In DMD fibroblasts, treatment with MP for 24 h induced dots (ICC) positive for NFAT5 and colocalizing with GR. After 12 d of MP or HC in DMD fibroblasts, NFAT5 expression was decreased (RT-qPCR and WB) and growth arrest was observed (Incucyte ZOOM), whereas NFAT5 expression and cell growth remained unchanged in unaffected skeletal muscle fibroblasts. This study may help understand the antiproliferative properties of glucocorticoids in DMD fibroblasts.

Highlights

The major mode of action of glucocorticoids (GCs) was described in 1994 by Ray and Prefontaine [1]: the glucocorticoid receptor GR binds to the master regulator of inflammation nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)
Duchenne muscular dystrophy (DMD) fibroblasts exposed for 24 h to HC and to a lesser extend MP showed Nuclear factor of activated T-cells 5 (NFAT5) colocalizing with GR in round structures located in the perinuclear area and the cytoplasm
Localization of NFAT5-GR complexes in the cytoplasm could occur by masking of the NF-κB nuclear localization signal (NLS), due to the presence of HC or MP induced IκB

Summary

Introduction

The major mode of action of glucocorticoids (GCs) was described in 1994 by Ray and Prefontaine [1]: the glucocorticoid receptor GR binds to the master regulator of inflammation nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). MP is produced as methylprednisolone sodium succinate (MPSS) for better dissolution in plasma. 75% of intravenous (IV) MP binds in plasma to albumin and corticoid binding globulin (CBG), known as transcortin. This results in a nonlinear relationship between free and bound MP and an impact on MP bioavailability [6]. Cell culture media are often supplemented with bovine serum naturally containing albumin and transcortin. Ranges from 0.39 to 1.21 μg/mL total cortisol in plasma and 0.05–0.39 μg/mL of free cortisol in plasma have been measured from time point + 0.5–5 h after IV administration [12]

Methods

Results

Conclusion