Localization and Expression of Nuclear Factor of Activated T-Cells 5 in Myoblasts Exposed to Pro-inflammatory Cytokines or Hyperosmolar Stress and in Biopsies from Myositis Patients

Abstract

Aims: Regeneration in skeletal muscle relies on regulated myoblast migration and differentiation, in which the transcription factor nuclear factor of activated T-cells 5 (NFAT5) participates. Impaired muscle regeneration and chronic inflammation are prevalent in myositis. Little is known about the impact of inflammation on NFAT5 localization and expression in this group of diseases. The goal of this study was to investigate NFAT5 physiology in unaffected myoblasts exposed to cytokine or hyperosmolar stress and in myositis.Methods: NFAT5 intracellular localization and expression were studied in vitro using a cell culture model of myositis. Myoblasts were exposed to DMEM solutions enriched with pro-inflammatory cytokines IFN-γ with IL-1β or hyperosmolar DMEM obtained by NaCl supplementation. NFAT5 localization was visualized using immunohistochemistry (IHC) and Western blotting (WB) in fractionated cell lysates. NFAT5 expression was assessed by WB and RT-qPCR. In vivo localization and expression of NFAT5 were studied in muscle biopsies of patients diagnosed with polymyositis (n = 6), dermatomyositis (n = 10), inclusion body myositis (n = 11) and were compared to NFAT5 localization and expression in non-myopathic controls (n = 13). Muscle biopsies were studied by means of quantitative IHC and WB of total protein extracts.Results: In unaffected myoblasts, hyperosmolar stress ensues in NFAT5 nuclear translocation and increased NFAT5 mRNA and protein expression. In contrast, pro-inflammatory cytokines did not lead to NFAT5 nuclear translocation nor increased expression. Cytokines IL-1β with IFN-γ induced colocalization of NFAT5 with histone deacetylase 6 (HDAC6), involved in cell motility. In muscle biopsies from dermatomyositis and polymyositis patients, NFAT5 colocalized with HDAC6, while in IBM, this was often absent.Conclusions: Our data suggest impaired NFAT5 localization and expression in unaffected myoblasts in response to inflammation. This disturbed myogenic NFAT5 physiology could possibly explain deleterious effects on muscle regeneration in myositis.

Abnormal NFAT5 Physiology in Duchenne Muscular Dystrophy Fibroblasts as a Putative Explanation for the Permanent Fibrosis Formation in Duchenne Muscular Dystrophy

“…Cytokine supplementation slightly increased DMEM’s osmolarity to 128 mM (333 mOsm/L). Therefore, to study the effect of cytokines, the control DMEM medium was supplemented with 18 mM NaCl (“DMEM18”) [ 31 ].…”

“…NFAT5 antibody selectivity for WB was assessed using siRNA NFAT5 (h): sc-43968 (Santa Cruz Biotechnology, Dallas, TX, USA) in DMDFibro, as described earlier and included in Supplementary Materials (Figure S2) . Fractionated WB were performed, as thoroughly described in an earlier work [ 31 ].…”

“…Staining and specificity tests were performed as described previously [ 31 ]. Primary antibody was used for one hour followed by a secondary antibody labelled with AlexaFluor-488 (green) or AlexaFluor-555 (red) (Invitrogen, Waltham, MA, USA) also for one hour.…”

Potential Role of Gene Regulator NFAT5 in the Pathogenesis of Diabetes Mellitus

“…Cells from the renal medulla are physiologically exposed to a hypertonic environment with osmolality values of approximately 1000 mOsm [ 55 ]. In contrast, primary cells such as human limbal epithelial cells exposed to 305-500 mOsm [ 56 ], myoblasts to 280-443 mOsm [ 57 ], human aortic endothelial cells to 285–460 mOsm [ 58 ], and human keratinocytes to 450–600 mOsm [ 59 ] are not physiologically exposed to a hypertonic environment and show a lower threshold (<600 mOsm) for NFAT5 activation. Exposure to moderately elevated glucose (26–51 mOsm/L) can increase the expression and nuclear translocation of NFAT5 [ 4 ].…”

Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis

“…We recently were the first to describe upregulation of the osmolyte accumulators SLC5A3, SLC6A6 and AKR1B1 in muscle tissues from myositis patients ( 24 ), and described NFAT5 expression in myoblasts in culture ( 25 ), yet the precise role osmolyte pathways play in disease mechanisms remained unexplored. With this study, we aim to substantiate and functionally connect these biomarkers with muscle inflammation.…”