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TonEBP/NFAT5 Regulates ACTBL2 Expression in Biomechanically Activated Vascular Smooth Muscle Cells


TonEBP/NFAT5 Regulates ACTBL2 Expression in Biomechanically Activated Vascular Smooth Muscle Cells

Maren Hödebeck et al. Front Physiol.


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.

Keywords: NFAT5; biomechanical stretch; hypertension; migration; smooth muscle cells.


Figure 1
Figure 1
Biomechanical stretch induces nuclear translocation of Nuclear factor of activated T cells 5 (NFAT5). HUASMCs were exposed to biomechanical stretch for 24 h. Subsequent Western blot analyses of nuclear and cytosolic protein fractions showed an increase of nuclear NFAT5 abundance compared to control conditions (A). Automated immunofluorescence analyses (TissueGnostics/TissueQuest) revealed a significant increase of NFAT5 positive nuclei (B, ***p < 0.001 vs. control, n = 3) and NFAT5-specific nuclear/cytoplasmic fluorescence intensity (B, ***p < 0.001 vs. control, n = 3) in stretch-stimulated VSMCs which is also demonstrated by corresponding scattergramms. The threshold was set according to the basal NFAT5 fluorescence intensity under control conditions (scale bar: 100 μm).
Figure 2
Figure 2
ACTBL2 is a transcriptional target of NFAT5. In silico promoter analysis of the human ACTBL2 promoter revealed six putative NFAT5 binding sites (A). The first 3203 bp of the promoter sequence upstream of the transcription start site were analyzed. The maximum core similarity (Core sim.) of 1.0 is only reached when the highest conserved bases of a matrix are exactly matched by the sequence (cf. capitals in the sequence). A good match to the matrix has a similarity of >0.80 (Matrix sim.). Quantitative PCR (qPCR) of stretch-stimulated HUASMCs treated with NFAT5-specific siRNA (siNFAT5) showed a significant decrease in ACTBL2 mRNA expression compared to stretch-stimulated control siRNA(siScramble)-transfected cells (B, **p < 0.01 vs. control siRNA, n = 3 scale bar: 20 μm). SiRNA-mediated knockdown of NFAT5 (C, *p < 0.05 vs. siScramble) decreased κ-actin (C, *p < 0.05 vs. siScramble) in stretch-stimulated VSMCs as evidenced by immunofluorescence analyses (scale bar: 100 μm).
Figure 3
Figure 3
ACTBL2 expression in stretch-stimulated SMCs is dependent on the Carnitine palmitoyltransferase family 1 (CPT1). Immunofluorescence analyses indicated a significant increase of κ-actin abundance in HUASMCs upon stretch stimulation (A, ***p < 0.001 vs. control, n = 3; scale bar: 20 μm) which was confirmed by Western blot analyses (B, ***p < 0.001 vs. control, n = 3). Etomoxir, a specific CPT1-inhibitor (40 μM) reduced the stretch-induced NFAT5 translocation which was determined by analyzing nuclear protein extracts (C, ***p < 0.001 control DMSO vs. stretch DMSO, *p < 0.05 stretch DMSO vs. stretch Etomoxir, n = 5). Quantitative real-time PCR revealed a decline in ACTBL2 mRNA expression in stretch-stimulated Etomoxir-treated HUASMCs (D, *p < 0.05 vs. stretch DMSO, n = 3). Stretch-dependent (24 h) increase in κ-actin protein abundance is attenuated upon treatment with Etomoxir as evidenced by immunofluorescence detection (E, ***p < 0.001 vs. static DMSO, ##p < 0.01 vs. stretch DMSO; bar graphs represent the mean (±SD) fluorescence intensity of κ-actin in ten microscopic fields of view; representative images are shown on the right, scale bar: 100 μm).
Figure 4
Figure 4
κ-actin modulated VSMC migration. κ-actin is localized in stress fibers of resting cells (A) and dispersed in protrusions of migrating cells (B,C) as evidenced by contrast enhancing immunofluorescence detection. ACTBL2 knockdown efficiency was verified by RT-PCR and immunofluorescence analysis 2 days after transfection (D, **p < 0.01 and ***p < 0.001 vs. siScramble, n = 3) and did not affect spreading or viability of the HUASMCs (E, scale bar: 100 μm). In contrast to control, planar cell migration is inhibited in HUASMCs after ACTBL2 silencing (F,G, ***p < 0.001 vs. siScramble, n = 6, scale bar: 50 μm).

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