TonEBP/NFAT5 regulates ACTBL2 expression in biomechanically activated vascular smooth muscle cells

abstract: ...Cytoskeletal reorganization and migration are critical responses which enable vascular smooth muscle cells (VSMCs) cells to evade, compensate, or adapt to alterations in biomechanical stress. An increase in wall stress or biomechanical stretch as it is elicited by arterial hypertension promotes their reorganization in the vessel wall which may lead to arterial stiffening and contractile dysfunction. This adaptive remodeling process is dependent on and driven by subtle phenotype changes including those controlling the cytoskeletal architecture and motility of VSMCs. Recently, it has been reported that the transcription factor nuclear factor of activated T-cells 5 (TonEBP/NFAT5) controls critical aspects of the VSMC phenotype and is activated by biomechanical stretch. We therefore hypothesized that NFAT5 controls the expression of gene products orchestrating cytoskeletal reorganization in stretch-stimulated VSMCs. Automated immunofluorescence and Western blot analyses revealed that biomechanical stretch enhances the expression and nuclear translocation of NFAT5 in VSMCs. Subsequent in silico analyses suggested that this transcription factor binds to the promotor region of ACTBL2 encoding kappa-actin which was shown to be abundantly expressed in VSMCs upon exposure to biomechanical stretch. Furthermore, ACTBL2 expression was inhibited in these cells upon siRNA-mediated knockdown of NFAT5. Kappa-actin appeared to be aligned with stress fibers under static culture conditions, dispersed in lamellipodia and supported VSMC migration as its knockdown diminishes lateral migration of these cells. In summary, our findings delineated biomechanical stretch as a determinant of NFAT5 expression and nuclear translocation controlling the expression of the cytoskeletal protein ACTBL2. This response may orchestrate the migratory activity of VSMCs and thus promote maladaptive rearrangement of the arterial vessel wall during hypertension.

...Front Physiol Front Physiol Front. Physiol. Frontiers in Physiology 1664-042X Frontiers Media S.A. 25520667 4253659 10.3389/fphys.2014.00467 Physiology Original Research Article TonEBP/NFAT5 regulates ACTBL2 expression in biomechanically activated vascular smooth muscle cells Hdebeck Maren Scherer Clemens Wagner Andreas H. Hecker Markus Korff Thomas * Division of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, University of Heidelberg Heidelberg, Germany Edited by

...-stimulated VSMCs. Automated immunofluorescence and Western blot analyses revealed that biomechanical stretch enhances the expression and nuclear translocation of NFAT5 in VSMCs. Subsequent in silico analyses suggested that this transcription factor binds to the promotor region of ACTBL2 encoding kappa-actin which was shown to be abundantly expressed in VSMCs upon exposure to biomechanical stretch. Furthermore, ACTBL2 expression was inhibited in these cells upon siRNA-mediated knockdown of NFAT5. Kappa-actin appeared to be aligned with stress fibers under static culture conditions, dispersed in lamellipodia and supported VSMC migration as its knockdown diminishes lateral migration of these cells. In summary, our findings delineated biomechanical stretch as a determinant of NFAT5 expression and nuclear translocation controlling the expression of the cytoskeletal protein ACTBL2. This response may orchestrate the migratory activity of VSMCs and thus promote maladaptive rearrangement of the arterial

... the control cells as solvent control. Immunocytochemistry Cells were fixed in ice-cold methanol for 15 min and allowed to dry for 20 min. Rehydrated cells were blocked with 0.25% casein and 0.1% BSA for 30 min. Cells were incubated with rabbit anti-NFAT5 antibody 1:100 (sc-13035, Santa Cruz, CA) or anti-ACTBL2 antibody 1:100 (abcam Cambridge, UK) at 4C overnight. After washing, cells were incubated with donkey anti-rabbit-Cy3 1:100 (Dianova, Hamburg, Germany) for 1 h and mounted with Mowiol

... for a resting period of 48 h for NFAT5 knockdown. Analysis of gene expression Total RNA was isolated from the cultured HUASMCs using the peqGOLD Total RNA Kit (Peqlab) according to the manufacturer's instructions. Subsequently, reverse transcription (RT) and quantitative polymerase chain reaction (qPCR) for ACTBL2 and 60S ribosomal protein L32 (RPL32) cDNA as an internal standard was performed. ACTBL2 primers were bought from Qiagen and primers for RPL32 were used for amplification based on the

... anti-ACTBL2 (-actin) 1:1000 (abcam), mouse anti- -actin 1:10,000 (abcam), rabbit anti-histone H3 1:1000 (abcam), rabbit anti-alpha-Tubulin (Cell Signaling) 1:1000. Nuclear extraction Nuclear protein extraction was performed according to the following protocol: HUASMCs were lysed using buffer I containing 10 mM HEPES, 10 mM KCl, 1 M EDTA, 1 M EGTA, 15% Nonidet, protease and phosphatase inhibitors. After centrifugation (12,000 g at 4C for 15 min) the supernatant (cytosolic fraction) was transferred

... immediately used. Bioinformatic analysis About 3203 bp of the promoter sequence upstream of the human ACTBL2 translation start site (Homo sapiens ACTBL2, accession NG_029637, position 28727 to 32239) were analyzed using the MatInspector software (Genomatrix Software, Munich Germany) for potential NFAT5 consensus sequences. Cell migration assay Cultured HUASMCs were seeded into 24-well culture plates with 4 mm thick and 10 mm long silicon walls in the center of each well, after transfection with specific

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Gene: ACTBL2 - actin, beta-like 2 - (NCBI)

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