Abstract 439: Biomechanical Stimulation Promotes Translocation of Nuclear Factor of Activated T-cells 5 Into The Nucleus of Vascular Smooth Muscle Cells

Abstract

Chronic alterations in the biomechanical stimulation of vascular smooth muscle cells (VSMC) as experienced during hypertension lead to changes in VSMC phenotype and function and further enable the structural remodeling of the vessel wall. In this context, we recently reported that an increase in wall stress or biomechanical stretch is sufficient to activate nuclear factor of activated T-cells 5 (NFAT5). This transcription factor promotes the expression of gene products such as tenascin-C and κ-actin, both involved in VSMC migration. Based on these findings, we hypothesized that biomechanical stretch elicits NFAT5 mRNA expression and induces biochemical modifications of NFAT5 on the post-translational level, a prerequisite for its entry into the nucleus and transcriptional activity. To scrutinize this hypothesis, human arterial VSMC were exposed to biomechanical stretch (13%, 0.5 Hz) and subjected to detailed mRNA expression analyses. While a ~3-fold reduction in NFAT5 splice variant 1 (isoform A) mRNA expression was observed in stretch-stimulated VSMC as compared to the static controls (n=3, p<0.05), splice variant 3 (isoform C) mRNA levels were induced ~1.8-fold (n=3, p<0.05). Overexpression of corresponding Flag-tagged NFAT5 proteins in VSMC and subsequent immunofluorescence as well as biochemical analyses revealed that isoform A was primarily located in the cytoplasm of static and stretch-stimulated VSMC while isoform C was preferentially localized in the nucleus under baseline conditions and further accumulated in the nucleus upon biomechanical stimulation (n=3, p<0.05). Nuclear translocation of isoform C was amplified for phosphorylation-deficient mutants generated by exchanging serine to alanine at position 1197 even under static culture conditions while a phosphomimetic mutation at this residue (serine to glutamate) inhibited NFAT5c nuclear translocation (n=3, p<0.05). Collectively, our findings indicate that exposure of VSMC to biomechanical stretch triggers the expression of NFAT5 isoform C and controls its entry into the nucleus via phosphorylation at S-1197. Current investigations are focusing on the impact of NFAT5 on hypertensive remodeling utilizing inducible smooth muscle cell-specific NFAT5-deficient mice.