Arterial Wall Stress Controls NFAT5 Activity in Vascular Smooth Muscle Cells
Abstract
Background
Nuclear factor of activated T‐cells 5 (NFAT5) has recently been described to control the phenotype of vascular smooth muscle cells (VSMCs). Although an increase in wall stress or stretch (eg, elicited by hypertension) is a prototypic determinant of VSMC activation, the impact of this biomechanical force on the activity of NFAT5 is unknown. This study intended to reveal the function of NFAT5 and to explore potential signal transduction pathways leading to its activation in stretch‐stimulated VSMCs.
Methods and Results
Human arterial VSMCs were exposed to biomechanical stretch and subjected to immunofluorescence and protein‐biochemical analyses. Stretch promoted the translocation of NFAT5 to the nucleus within 24 hours. While the protein abundance of NFAT5 was regulated through activation of c‐Jun N‐terminal kinase under these conditions, its translocation required prior activation of palmitoyltransferases. DNA microarray and ChiP analyses identified the matrix molecule tenascin‐C as a prominent transcriptional target of NFAT5 under these conditions that stimulates migration of VSMCs. Analyses of isolated mouse femoral arteries exposed to hypertensive perfusion conditions verified that NFAT5 translocation to the nucleus is followed by an increase in tenascin‐C abundance in the vessel wall.
Conclusions
Collectively, our data suggest that biomechanical stretch is sufficient to activate NFAT5 both in native and cultured VSMCs where it regulates the expression of tenascin‐C. This may contribute to an improved migratory activity of VSMCs and thus promote maladaptive vascular remodeling processes such as hypertension‐induced arterial stiffening.
Introduction
Phenotypic adaptation of cells is a prerequisite to comply with microenvironmental changes triggered by physiological or pathophysiological factors that may drive the growth of a specific tissue or result in its dysfunction. The vascular system is continuously exposed to a highly variable environment as the biomechanical load defined by blood flow and pressure is steadily altered by lifestyle, age, and physical activity. To tightly regulate these pivotal hemodynamic parameters, vascular smooth muscle cells (VSMCs) located in the media of the arterial vessel wall are able to rapidly respond to subtle changes in blood pressure/flow by contraction or relaxation. Despite this ability, VSMCs are also capable to permanently rearrange the local architecture of the vessel wall in response to chronic changes in blood pressure and/or flow. In this context, it is well documented that hypertension induces arterial remodeling by stimulating the migration and proliferation of VSMCs located in the media,1, 2 which subsequently leads to media hypertrophy and/or hyperplasia.2, 3 By definition, hypertension is characterized by a chronic increase in mean arterial blood pressure that elicits an increase in wall stress—a biomechanical force with major impact on the VSMC phenotype4, 5, 6—and thus a rise in the level of stretch to which these cells are exposed. Prolonged exposure to supra‐physiological levels of transmural pressure or stretch elicits VSMC hypertrophy and/or hyperplasia7, 8 that both require a phenotype change by these cells. As a consequence, VSMCs migrate and proliferate as well as degrade or synthesize components of the extracellular matrix9, 10—a phenotype referred to as “synthetic”.
This shift in VSMC phenotype is orchestrated by the activity of transcription factors controlling the expression of genes promoting the contractile or synthetic state of these cells. For instance, the transcription factor activator protein‐1 (AP‐1) controls their stretch‐induced activation by regulating the expression of many stress response genes, including those associated with a pro‐inflammatory state.11, 12, 13 In contrast, the coactivator myocardin regulates the expression of genes encoding cytoskeletal and contractile proteins in VSMCs such as SM‐α‐actin or calponin through interaction with serum response factor (SRF).14, 15, 16 Recently, nuclear factor of activated T‐cells 5 (NFAT5 or tonicity enhancer binding protein)—a member of the Rel family of transcription factors—was characterized to affect the VSMC phenotype.17 Originally described as a hypertonicity‐responsive transcription factor that orchestrates cellular homeostasis,18 NFAT5 has meanwhile been implicated in regulating the expression of genes associated with migration and proliferation of cells.19, 20, 21 With respect to its function in VSMCs, it has been suggested that NFAT5 regulates the expression of smooth muscle differentiation markers such as SM‐α‐actin depending on the microenvironmental context.17 Moreover, with angiotensin II and platelet‐derived growth factor BB (PDGF‐BB) 2 humoral factors were described that coevally affect the activity and expression of NFAT5 as well as the phenotype of VSMCs.17 As an increase in wall stress or stretch is another well‐known determinant of the VSMC phenotype, we assumed that this biomechanical force affects the activity of NFAT5—a hypothesis that tackles emerging questions in this field of research.22 We further reasoned that under hypertensive conditions NFAT5 may alter the expression of genes that affect the phenotype of VSMCs. Therefore, we have investigated the abundance, localization, and activity of NFAT5 in human arterial VSMCs exposed to biomechanical stretch and in mouse arteries exposed to hypertensive perfusion conditions.
Material and Methods
Cell Culture
Human arterial smooth muscle cells (HUASMCs) were freshly isolated from individual umbilical cords and grown on collagen I‐bonded BioFlex® plates (Flexcell International) with DMEM medium containing 15% FCS, 50 U/mL penicillin, 50 μg/mL streptomycin and fungizone (Invitrogen). The isolation of HUASMCs was approved by the local Ethics Committee (Heidelberg, Germany; reference 336/2005) and conformed to the principles outlined in the Declaration of Helsinki (1997). Biomechanical stretch was typically applied at a frequency of 0.5 Hz and an elongation of 0% to 13% for 24 hours by using a Flexercell FX‐5000 tension system. In addition, cells were exposed to the following reagents: 20 μmol/L SP600125 (SABioscience), 50 μmol/L PD98059 (Biomol), 20 μmol/L SB202190 (Biomol), 13 μmol/L palmostatin B (Merck), and 100 μmol/L 2‐bromopalmitate (Sigma Aldrich). All compounds were dissolved in DMSO. Pure DMSO (0.1%, v/v) was simultaneously applied to the control cells as solvent control. After stretching, the cells were subjected to immunofluorescence analysis.
Perfusion of Isolated Mouse Arteries
All animal experiments were performed with permission from the Regional Council Karlsruhe and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85‐23, revised 1996). Animals were sacrificed by cervical dislocation; the femoral arteries were excised, trimmed of excess adipose tissue, and inserted into the chamber of a myograph (Culture Myograph, DMT). The chambers were placed in an incubator at 37°C and 5% CO2, and the arteries were perfused by applying a constant axial pressure difference (∆P) of 20 mm Hg for up to 24 hours with DMEM medium (Invitrogen) containing 15% FCS. Hypertensive perfusion conditions were mimicked by increasing the mean transmural pressure gradient (∆Ptm) from 60 mm Hg (control conditions) to 110 mm Hg (hypertensive conditions).
Immunocytochemistry
Cells were fixed in ice‐cold methanol for 15 minutes and allowed to dry for 20 minutes. Rehydrated cells were blocked with 0.25% casein and 0.1% BSA for 30 minutes. Cells were incubated with rabbit anti‐NFAT5 antibody 1:100 (Abcam, Cambridge, UK; detects NFAT5 isoforms a to d) at 4°C overnight. After washing, cells were incubated with donkey anti‐rabbit‐Cy3 1:100 (Dianova) for 1 hour and mounted with Mowiol (Calbiochem). Nuclei were visualized by counterstaining the cells with DAPI (Invitrogen). Fluorescence intensity was recorded using an Olympus IX81 confocal microscope (Olympus). Quantitative image analyses were performed using the Olympus Xcellence software.
Immunohistochemistry
Mouse femoral arteries were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Antigens were retrieved by incubating 5‐μm thick rehydrated tissue sections with citrate buffer (pH 6.0) at 100°C for 15 minutes. The sections were consecutively incubated with peroxidase blocking solution for 5 minutes and 0.25% casein and 0.1% BSA for 30 minutes. Thereafter, the sections were incubated with rabbit anti‐NFAT5 antibody 1:50 (Abcam) overnight at 4°C. For visualization, the Dako Envision™ (Dako) and TSA™‐Cy3 system (PerkinElmer) was used according to the manufacturers' instructions. Fluorescence intensity was recorded using an Olympus IX81 confocal microscope (Olympus). Quantitative image analyses were performed using the Olympus Xcellence software.
Transfection With siRNA
HUASMCs were transfected with short interfering RNA directed against NFAT5 (5′‐CCA GTT CCT ACA ATG ATA A‐3′), tenascin‐C (Santa Cruz Biotechnology) or ACTBL2 (Qiagen). As control, commercially available siGENOME Non‐Targeting siRNA (Thermo Scientific) was applied. For each well of a 6‐well plate, 3 μg of siRNA was diluted in Opti‐MEM I (Invitrogen) together with 3 μL of MATra‐si reagent (IBA) to give a final volume of 200 μL. After mixing and incubating for 20 minutes at ambient temperature, the solution was added onto the cells, which had been cultivated in 2 mL Opti‐MEM I prior to the transfection. Cells were then incubated on a magnetic plate (IBA) at 37°C and 5% CO2. After 15 minutes cells were washed and cultured in normal cell medium for a resting period of 48 hours for ACTBL2 and TNC or 72 hours for NFAT5 knockdown.
Analysis of Gene Expression
Total RNA was isolated from the cultured HUASMCs using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. Subsequently, reverse transcription (RT) and polymerase chain reaction (PCR) for the target sequences and 60S ribosomal protein L32 (RPL32) cDNA as an internal standard was performed. Primers based on the following sequences were used for amplification:
NFAT5 for 5′‐AAG AGT GAA GAT GTT ACT CCA ATG GAA G‐3′, rev 5′‐AAA GTC TGT GCT TGT TCT TGT AGT GG‐3′; tenascin‐C for 5′‐TCA TTG TGG GTC CAG ATA CC‐3′, rev 5′‐GGA GTC CAA TTG TGG TGA AG‐3′; retinoic acid receptor beta for 5′‐GGG TAG GAT CCG GAA CGC ATT‐3′, rev 5′‐GAC GAG TTC CTC AGA GCT GG‐3′; junctional adhesion molecule 3 for 5′‐GCA ACC CTC GAC ATG GCG CT‐3′, rev 5′‐ACA GGG GTC ACT GGC TTC ACC T‐3′; RPL32 for 5′‐GTT CAT CCG GCA CCA GTC AG‐3′, rev 5′‐ACG TGC ACA TGA GCT GCC TAC‐3′. CPT1A for 5′‐GAG AGG AGA CAG ACA CCA TC‐3′, rev 5′‐ACT TGT CAA ACC ACC TGT C‐3′. ACTBL2 primers were bought from Qiagen.
Western Blot
HUASMCs were lysed using sample buffer containing 1% Triton X‐100 and 0.1 μmol/L DTT or buffers for preparing nuclear and cytosolic fractions. Protein samples were separated by SDS (10%), blotted onto nitrocellulose membranes and analyzed by chemiluminescence‐based immunodetection according to standard procedures. Primary antibodies: rabbit anti‐NFAT5 1:2000 (Abcam), rabbit anti‐NFAT5 1:500 (Santa Cruz Biotechnology), mouse anti‐β‐actin 1:10 000 (Abcam), anti‐JNK/anti‐phospho (JNK1/2: T183/Y185, JNK3: T221/Y223)‐JNK 1:2000 (R&D systems, Germany), anti‐TNC 1:500 (R&D Systems), anti‐histone H3 (Abcam).
Nuclear Extraction
Nuclear protein extraction was performed according to the following protocol: HUASMCs were lysed using buffer I containing 10 mmol/L HEPES, 10 mmol/L KCl, 1 μmol/L EDTA, 1 μmol/L EGTA, 15% Nonidet, protease and phosphatase inhibitors. After centrifugation (12 000g at 4°C for 15 minutes) the supernatant (cytosolic fraction) was transferred to a new tube and stored or immediately used for Western blotting. The remaining pellet containing the nuclear fraction was dissolved in 40 μL buffer II consisting of 20 mmol/L HEPES, 400 mmol/L NaCl, 0.01 mol/L EDTA, 0.01 mol/L EGTA, 15% Nonidet, and protease and phosphatase inhibitors. Subsequently, this solution was sonicated 2 times for 5 seconds at 50 Watts at 4°C. After centrifugation (12 000g at 4°C for 15 minutes) the supernatant containing the nuclear fraction was transferred to a new tube and stored at −80°C or was immediately used.
Chromatin Immune‐Precipitation (ChIP)
ChIP assay was performed using a ChIP kit (17‐295, Millipore) as described previously.23 In brief, after cross‐linking and cell lysis the chromatin was sheared by sonication (UP50H sonicator) resulting in DNA fragments in the range of 500 to 800 bp. One percent of the diluted cell supernatant was kept as the input material to quantify the DNA content of the samples. The supernatants were immunoprecipitated overnight at 4°C with an antibody against NFAT5 (PA1‐023 from Thermo Scientific Pierce). For a negative control a no‐antibody immunoprecipition was performed in parallel (NAC, no‐antibody control). DNA was isolated using the QiaQuick‐PCR Purification Kit (Qiagen) according to the manufacturer's instructions and used for the subsequent PCR analysis. Amplification of the tenascin‐C promoter fragments (Homo sapiens tenascin‐C, RefSeqGene on chromosome 9, accession number NG_029637) was carried out by conventional PCR adjusting the optimal number of cycles to avoid saturation and visualized by agarose gel electrophoresis. The following primer pair was used (position 31449 to 31592, containing a NFAT5 binding site): 5′‐TCTTGCCTCTTATTGATT‐3′ (for) and 5′‐GACTGAATGCTATACTGA‐3′ (rev). PCR signals obtained from the immunoprecipitated DNA were visualized and analyzed with a GelDoc™ XR molecular imager and the Quantity One 1‐D analysis software (BioRad). Rabbit IgG (#2729 from Cell Signaling Technology) were utilized as IgG control.
Bioinformatic Analysis
About 3512 bp of the promoter sequence upstream of the human tenascin‐C translation start site (Homo sapiens tenascin‐C, RefSeqGene on chromosome 9, accession NG_029637, position 28727 to 32239) were analyzed using the MatInspector software (Genomatrix Software) for potential NFAT5 consensus sequences.
DNA Microarray Analysis
To characterize the stretch‐dependent effects of NFAT5, 3 cell preparations each were stretched for 24 hours (0% to 13% elongation, 0.5 Hz) after siRNA‐mediated knockdown of NFAT5 or transfection with control siRNA. RNA was isolated (see above) and processed for DNA microarray analysis according to manufacturers' instructions:
Gene expression profiling was performed by using the HuGene‐1_0‐st‐v1 array from Affymetrix (High Wycombe). Biotinylated antisense cRNA was then prepared according to the Affymetrix standard labeling protocol. Afterwards, hybridization on the chip was performed in a GeneChip Hybridization oven 640 which was then dyed in a GeneChip Fluidics Station 450 and thereafter scanned with a GeneChip Scanner 3000.
A custom CDF version 14 with Entrez‐based gene definitions was used to annotate the arrays.24 Raw fluorescence intensity values were normalized applying quantile normalization. Differential gene expression analysis was performed with one‐way analysis of variance (ANOVA) using the software package JMP8 Genomics version 4 from SAS (SAS Institute). A false positive rate of a=0.05 with FDR correction was taken as the level of significance.
Collagen Gel Invasion Assay
The spheroid‐based invasion assay was adapted from the spheroid angiogenesis assay as described before.25 In brief, HUASMCs were grown as spheroids (500 cells per spheroid) with methylcellulose‐containing medium in U‐bottom 96‐well plates for 24 hours. Spheroids were then suspended into a collagen‐type‐I‐gel. The gel was aliquotted into a 24‐well plate to polymerize. After 30 minutes, 100 μL FCS‐containing (30%) DMEM medium was added. The cumulative length of VSMCs originating from each spheroid was measured after 24 hours at ×10 magnification. At least 10 spheroids per experimental condition were analyzed.
Cell Migration Assay
After transfection with specific siRNA or control siRNA, the 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 24 hours, the silicon walls were removed and the distance between the cell borders was measured at ×2 magnification. The measurement was repeated after 24 and 48 hours. Optionally, cell culture medium was supplemented for 30 minutes with 0.65 μg/cm² recombinant human TN‐C (R&D systems) following removal of the silicon walls and substituted with new medium to remove excess TN‐C after washing with serum‐free D‐MEM medium.
Statistical Analysis
All results are expressed as means±SD of n individual experiments. Differences between 2 individual experimental groups were analyzed by unpaired Student t test with P< 0.05 considered statistically significant. Differences among 3 or more experimental groups were analyzed by ANOVA, followed by a Tukey multiple comparisons test or repeated measures ANOVA if applicable, with a probability value of P<0.05 considered statistically significant.
Results
Biomechanical Stretch Induces Translocation of NFAT5 to the Nucleus of VSMCs
Changes in osmolarity of the VSMC microenvironment elicit the translocation of NFAT5 to the nucleus (Figure 1). Exposing the cultured HUASMCs to biomechanical stretch for 24 hours produced the same effect (Figure 2). Furthermore, alterations in the overall staining intensity (Figure 2) indicated a change in the expression of NFAT5. Specificity of the antibody was verified by immunofluorescence analyses of VSMCs that had been treated with NFAT5‐specific siRNA (Figure 3).
Stretch‐Induced Expression and Translocation of NFAT5 Depends on JNK Activity
The aforementioned results suggested that biomechanical stretch triggers mechanisms modifying NFAT5 expression and activity in a way that enables it to enter the nucleus. Therefore, we systematically blocked prototypic kinases known to be activated in stretch‐stimulated VSMCs.16 While neither p38 MAP kinase, the ERK1/2 pathway nor the phosphatase calcineurin appeared to play a role in NFAT5 translocation and/or expression (Figure 4), the number of NFAT5‐positive nuclei as well as the overall NFAT5 abundance was markedly decreased in stretch‐stimulated HUASMCs upon blocking the activity of c‐Jun‐N‐terminal kinase (JNK) with SP600125 (Figure 5A through 5E). Correspondingly, we detected that JNK phosphorylation (indicating its activity) is increased right upon exposing the cells to biomechanical stretch (Figure 5F). Further experiments revealed that inhibition of JNK has no impact on the stretch‐induced up‐regulation of NFAT5 mRNA expression (Figure 5G) but diminishes its increased protein abundance under these conditions (Figures 5H and 8D). In this context, we detected a JNK‐dependent change in NFAT5 (serine) phosphorylation in stretch‐stimulated VSMCs at an individual site (S1197) while total serine phosphorylation remained unchanged (Figures 6A through 6C). In fact, phosphorylation of S1197 appears to prevent NFAT5 from entering the nucleus of VSMCs (Figure 6D).
Inhibition of Palmitoyltransferase Activity Blocks Stretch‐Induced Translocation of NFAT5
Considering that osmolarity‐induced NFAT5 activity depends on its palmitoylation,26 we next explored the possibility that stretch‐induced palmitoylation of NFAT5 is a prerequisite for its stretch‐induced nuclear translocation. To this end, we pre‐treated stretch‐stimulated HUASMCs with 2‐bromopalmitate, a general palmitoyltransferase inhibitor, or palmostatin B, an inhibitor of depalmitoylation by blocking acyl protein thioesterase 1. While inhibition of palmitoylation did not affect NFAT5 protein abundance (Figure 7) but attenuated stretch‐induced NFAT5 translocation (Figures 8A through 8C), blocking depalmitoylation had the opposite effect (Figures 8D through 8G). Both compounds had no effect on the localization of NFAT5 in HUASMCs cultured under static conditions (Figure 8H) and did not affect stretch‐induced JNK expression or phosphorylation as evidenced by Western blot analyses (data not shown). Interestingly, biomechanical stretch caused a slower electrophoretic migration speed of the NFAT5 protein band that disappeared upon treatment with 2‐bromopalmitate (Figure 9A). In line with these observations, click chemistry techniques suggest that NFAT5 is palmitoylated in stretch‐stimulated HUASMCs (Figure 9B).
Tenascin‐C is a Transcriptional Target of NFAT5 in Stretch‐activated VSMCs
To assess the impact of NFAT5 on the VSMC phenotype, its expression was silenced by siRNA (Figure 3). By comparing cDNA microarray data acquired by comparison of mRNA isolated from stretch‐stimulated HUASMCs with and without prior knockdown of NFAT5 (Figure 10), we identified about 2000 differently regulated gene products (Figure 11A and Table 1). NFAT5‐dependent expression of selected gene products was verified by RT‐PCR (Figure 11B). With tenascin‐C (a matrix molecule) we next focused on a gene product whose expression has been linked to cardiovascular diseases such as intimal hyperplasia, pulmonary artery hypertension, or atherosclerosis27 and appears to control VSMC differentiation.28 Subsequent in silico analyses of the first 3512 bp of the promoter sequence upstream of the transcription start site of the human tenascin‐C gene revealed seven putative NFAT5 binding sites (Table 2). Exemplary ChIP analysis of binding site no.1 (819 to 837) confirmed a stretch‐mediated binding of NFAT5 to the tenascin‐C promoter (Figure 11C). Likewise, TNC expression was blocked by decoy oligo‐deoxynucleotides mimicking the NFAT5 binding site (Figure 12) and upon inhibiting the activity of JNK with SP600125 (Figure 13). Moreover, stretch‐induced NFAT5 expression and nuclear translocation coincided with the mRNA expression of TNC (Figure 14).
| Gene Name | Symbol | Regulation of Gene Expression (x‐Fold vs Control) |
|---|---|---|
| Actin, beta‐like 2 | ACTBL2 | −5.5x |
| Retinoic acid receptor, beta | RARB | −3.5x |
| Neutral cholesterol ester hydrolase 1 | NCEH1 | −3.3x |
| Transmembrane protein 2 | TMEM2 | −3.0x |
| Annexin A3 | ANXA3 | −2.8x |
| Annexin A10 | ANXA10 | −2.7x |
| Leukemia inhibitory factor receptor alpha | LIFR | −2.7x |
| Lipase A, lysosomal acid, cholesterol esterase | LIPA | −2.6x |
| Aldehyde dehydrogenase 1 family, member A1 | ALDH1A1 | −2.6x |
| Sulfotransferase family, cytosolic, 1B, member 1 | SULT1B1 | −2.6x |
| Lipase, endothelial | LIPG | −2.6x |
| Potassium voltage‐gated channel, member 4 | KCNE4 | −2.6x |
| Inositol polyphosphate‐4‐phosphatase, type II, 105 kDa | INPP4B | −2.6x |
| Speckle‐type POZ protein‐like | SPOPL | −2.5x |
| Phosphoglucomutase 2‐like 1 | PGM2L1 | −2.4x |
| Nucleolar and coiled‐body phosphoprotein 1 | NOLC1 | −2.4x |
| Chromosome 1 open reading frame 27 | C1orf27 | −2.4x |
| Coiled‐coil domain containing 23 | CCDC23 | −2.4x |
| Polo‐like kinase 2 | PLK2 | −2.3x |
| Tetraspanin 13 | TSPAN13 | −2.3x |
| Tenascin C | TNC | −2.3x |
| DEAD (Asp‐Glu‐Ala‐Asp) box polypeptide 18 | DDX18 | −2.2x |
| Phosphatidylcholine transfer protein | PCTP | −2.2x |
| Dual specificity phosphatase 4 | DUSP4 | −2.2x |
| KIAA1324‐like | KIAA1324L | −2.1x |
| Translocated promoter region (to activated MET oncogene) | TPR | −2.1x |
| Sulfotransferase family 1E, estrogen‐preferring, member 1 | SULT1E1 | −2.1x |
| Deoxynucleotidyltransferase, terminal, interacting protein 2 | DNTTIP2 | −2.1x |
| Arylacetamide deacetylase (esterase) | AADAC | −2.1x |
| Natriuretic peptide receptor C/guanylate cyclase C | NPR3 | −2.1x |
| Aldo‐keto reductase family 1, member C2 | AKR1C2 | −2.1x |
| T‐box 18 | TBX18 | −2.1x |
| Serine/threonine kinase 4 | STK4 | −2.0x |
| Suppressor of zeste 12 homolog (Drosophila) | SUZ12 | −2.0x |
| GLE1 RNA export mediator homolog (yeast) | GLE1 | −2.0x |
| Fibroblast growth factor receptor substrate 2 | FRS2 | −2.0x |
| General transcription factor IIE, polypeptide 1, alpha 56 kDa | GTF2E1 | −2.0x |
| Nuclear factor of activated T‐cells 5, tonicity‐responsive | NFAT5 | −2.0x |
| Bone morphogenetic protein 6 | BMP6 | −2.0x |
| General transcription factor IIIC, polypeptide 3, 102 kDa | GTF3C3 | −2.0x |
| Plasminogen activator, urokinase | PLAU | 2.0x |
| Transmembrane protein 45A | TMEM45A | 2.0x |
| Prenylcysteine oxidase 1 | PCYOX1 | 2.0x |
| CD9 molecule | CD9 | 2.0x |
| Protease, serine, 12 (neurotrypsin, motopsin) | PRSS12 | 2.0x |
| Family with sequence similarity 8, member A1 | FAM8A1 | 2.0x |
| Dynamin 1‐like | DNM1L | 2.0x |
| Argininosuccinate synthase 1 | ASS1 | 2.0x |
| Tumor protein D52‐like 1 | TPD52L1 | 2.0x |
| ATPase, class I, type 8B, member 2 | ATP8B2 | 2.0x |
| T‐complex 11 (mouse)‐like 2 | TCP11L2 | 2.0x |
| Claudin 11 | CLDN11 | 2.0x |
| Carnitine palmitoyltransferase 1A | CPT1A | 2.0x |
| CUB domain containing protein 1 | CDCP1 | 2.0x |
| Inositol monophosphatase domain containing 1 | IMPAD1 | 2.0x |
| Perilipin 2 | PLIN2 | 2.0x |
| Leiomodin 1 (smooth muscle) | LMOD1 | 2.0x |
| Heparan sulfate 2‐O‐sulfotransferase 1 | HS2ST1 | 2.0x |
| Calcium channel, voltage‐dependent, beta 3 subunit | CACNB3 | 2.0x |
| STE20‐related kinase adaptor beta | STRADB | 2.1x |
| Chromosome 13 open reading frame 31 | C13orf31 | 2.1x |
| ATPase, class V, type 10D | ATP10D | 2.1x |
| Chromosome 5 open reading frame 32 | C5orf32 | 2.1x |
| Transducer of ERBB2, 1 | TOB1 | 2.1x |
| Superoxide dismutase 2, mitochondrial | SOD2 | 2.1x |
| Armadillo repeat containing, X‐linked 6 | ARMCX6 | 2.1x |
| Keratin 34 | KRT34 | 2.1x |
| BCL2/adenovirus E1B 19 kDa interacting protein 3‐like | BNIP3L | 2.1x |
| PX domain containing serine/threonine kinase | PXK | 2.1x |
| PDZ and LIM domain 3 | PDLIM3 | 2.1x |
| Solute carrier family 37, member 3 | SLC37A3 | 2.1x |
| Transducin (beta)‐like 1 X‐linked receptor 1 | TBL1XR1 | 2.1x |
| Zinc finger protein 286A | ZNF286A | 2.1x |
| Interleukin‐1 receptor‐associated kinase 4 | IRAK4 | 2.1x |
| Platelet‐derived growth factor receptor‐like | PDGFRL | 2.2x |
| Chromosome 9 open reading frame 6 | C9orf6 | 2.2x |
| Myosin ID | MYO1D | 2.2x |
| Metallothionein 1G | MT1G | 2.2x |
| Keratin 19 | KRT19 | 2.2x |
| Calcium/calmodulin‐dependent protein kinase II delta | CAMK2D | 2.2x |
| Nudix‐type motif 21 | NUDT21 | 2.2x |
| Leucine rich repeat containing 8 family, member A | LRRC8A | 2.2x |
| ADAM metallopeptidase domain 12 | ADAM12 | 2.2x |
| Lysyl oxidase‐like 4 | LOXL4 | 2.2x |
| C1q and tumor necrosis factor related protein 1 | C1QTNF1 | 2.2x |
| Dynein, light chain, Tctex‐type 3 | DYNLT3 | 2.2x |
| NMD3 homolog (Saccharomyces cerevisiae) | NMD3 | 2.2x |
| Reversion‐inducing‐cysteine‐rich protein with kazal motifs | RECK | 2.3x |
| v‐raf‐1 murine leukemia viral oncogene homolog 1 | RAF1 | 2.3x |
| Cytochrome P450, family 1, subfamily B, polypeptide 1 | CYP1B1 | 2.3x |
| Zinc finger E‐box binding homeobox 2 | ZEB2 | 2.3x |
| A kinase (PRKA) anchor protein 12 | AKAP12 | 2.3x |
| Sarcoglycan, epsilon | SGCE | 2.3x |
| Cyclin‐dependent kinase 8 | CDK8 | 2.3x |
| DnaJ (Hsp40) homolog, subfamily B, member 6 | DNAJB6 | 2.3x |
| Small nucleolar RNA, C/D box 116‐24 | SNORD116‐24 | 2.3x |
| Fatty acid desaturase 1 | FADS1 | 2.4x |
| Solute carrier family 2, member 10 | SLC2A10 | 2.4x |
| Midkine (neurite growth‐promoting factor 2) | MDK | 2.4x |
| cAMP responsive element binding protein 3‐like 1 | CREB3L1 | 2.4x |
| Solute carrier family 39 (zinc transporter), member 6 | SLC39A6 | 2.4x |
| Surfeit 4 | SURF4 | 2.4x |
| Ring finger protein 149 | RNF149 | 2.4x |
| Transmembrane protein 106C | TMEM106C | 2.4x |
| Synaptotagmin XI | SYT11 | 2.4x |
| Cyclin G1 | CCNG1 | 2.5x |
| Ankyrin repeat domain 13A | ANKRD13A | 2.5x |
| Intercellular adhesion molecule 1 | ICAM1 | 2.5x |
| Transmembrane protein 87B | TMEM87B | 2.5x |
| DDB1 and CUL4 associated factor 12 | DCAF12 | 2.6x |
| Family with sequence similarity 198, member B | FAM198B | 2.6x |
| Family with sequence similarity 171, member B | FAM171B | 2.6x |
| Solute carrier family 35, member B1 | SLC35B1 | 2.7x |
| Sterol O‐acyltransferase 1 | SOAT1 | 2.7x |
| ABI family, member 3 (NESH) binding protein | ABI3BP | 2.7x |
| Hypothetical LOC554202 | LOC554202 | 2.7x |
| Amyloid beta (A4) precursor‐like protein 2 | APLP2 | 2.8x |
| Junctional adhesion molecule 3 | JAM3 | 2.8x |
| WD repeat domain, phosphoinositide interacting 1 | WIPI1 | 2.9x |
| Male‐enhanced antigen 1 | MEA1 | 2.9x |
| Stearoyl‐CoA desaturase 5 | SCD5 | 3.2x |
| Malectin | MLEC | 3.3x |
| Transmembrane and coiled‐coil domains 1 | TMCO1 | 4.4x |
The indicated values represent the degree of up‐ or down‐regulation of gene products in stretch‐stimulated (24 hours) HUASMCs transfected with control or NFAT5‐specific siRNA (P<0.005 for all gene products listed, with highlighted p‐values as follows: RARB P=0.00212; ACTBL2 P=0.000754; TNC=0.0005; JAM3 P=0.002799; CPT1A P=0.00006699; n=3). Gene products printed in bold have been verified by at least one other method of detection. The NFAT5 knockdown efficiency (see Figure 6) of each sample was separately verified by RT‐PCR analysis. HUASMC indicates human arterial smooth muscle cells; NFAT5, nuclear factor of activated T‐cells 5; PCR, polymerase chain reaction; RT, reverse transcription.
| Start | End | Core Similarity | Matrix Similarity | Sequence | |
|---|---|---|---|---|---|
| Matrix | |||||
| V$NFAT5.01 | 819 | 837 | 1 | 0.83 | tttGGAAataaatcaagga |
| V$NFAT5.02 | 2016 | 2034 | 1 | 0.917 | tctGGAAagattttatcct |
| V$NFAT5.02 | 2122 | 2140 | 1 | 0.891 | caaGGAAaattgaactggg |
| V$NFAT5.02 | 2688 | 2706 | 1 | 0.914 | gatGGAAatttaagtcatt |
| V$NFAT5.02 | 2928 | 2946 | 1 | 0.924 | ttgGGAAaaatccattata |
| V$NFAT5.02 | 3075 | 3093 | 1 | 0.911 | tctGGAAaggttttgactc |
| V$NFAT5.02 | 3472 | 3490 | 1 | 0.915 | cagGGAAacttacttgaga |
| Primer | |||||
| NFAT5 ChIP sense | 651 | 669 | |||
| NFAT5 ChIP antisense | 789 | 807 | |||
There are 7 putative NFAT5 binding sites in the human tenascin‐C promotor as evidenced by in silico analysis of the first 3512 bp of the promoter sequence upstream of the transcription start site (C). The maximum core similarity (core similarity 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 similarity).ChIP indicates chromatin immune‐precipitation; NFAT5, nuclear factor of activated T‐cells 5.
With tenascin‐C, we identified an NFAT5 target gene whose expression is upregulated about twofold in stretch‐stimulated HUASMCs (Figure 15A). To evaluate the potential function of this matrix molecule in VSMC migration during hypertension‐associated remodeling processes, HUASMC spheroids were seeded in collagen gels supplemented with tenascin‐C. Subsequent examination of the gels after 24 hours revealed that tenascin‐C stimulates the migratory capacity of VSMC in this 3D culture system (Figures 15B through 15D). However, proliferation of VSMCs seeded onto tenascin‐C‐coated culture plastic surfaces (ie, 2D culture system) was not altered (data not shown). Likewise, siRNA‐mediated knockdown of tenascin‐C (Figure 16) partially inhibited VSMC migration (Figures 15E through 15G). A similar failure in VSMC migration was observed upon NFAT5 knockdown, which was, however, in part rescued by covering the migration surface with recombinant tenascin‐C (Figure 15H).
Hypertensive Perfusion Conditions Induce NFAT5 Translocation and Tenascin‐C Expression in Mouse Femoral Arteries
The increase in stretch of VSMCs is a result of the rise in the transmural pressure difference and thus a consequence of hypertension. To prove the relevance of our findings in this context, mouse femoral arteries were isolated and exposed to physiological or supraphysiological (hypertensive) pressure levels for 24 hours. Detection of NFAT5 in the media by immunofluorescence analyses unveiled a stretch/hypertension‐induced translocation of NFAT5 to the nucleus (Figures 17A through 17C). As could be expected from the aforementioned results, the abundance and mRNA expression of tenascin‐C increased under these conditions (Figures 17D through 17G). Similar results were obtained in rabbit carotid arteries perfused under hypertensive conditions for 24 hours (Figure 18).
Discussion
An increase in wall stress or biomechanical stretch is a consequence of hypertension and a potent driver for arterial remodeling7, 29, 30, 31 causing thickening of the arterial vessel wall and eventually its stiffening and malfunction.2, 32 These detrimental morphological and functional adaptations are preceded by a phenotypic switch of the VSMCs in the media that is controlled by transcription factors such as AP‐1 and SRF.12, 14 Recently, NFAT5 was discovered to modulate the phenotype of these cells upon angiotensin II or PDGF‐BB stimulation.17 In fact, hypertonicity was originally described as the prototypic mechanism of NFAT5 activation. This microenvironmental stimulus induces phosphorylation of the carboxy‐terminal transactivation domain of NFAT5, which subsequently results in its nuclear translocation and thus activation.18, 33, 34, 35, 36 Likewise, angiotensin II (a G‐protein‐coupled receptor agonist that elicits VSMC contraction) stimulates the entry of NFAT5 into the nucleus without altering its protein or mRNA expression.17 By expanding these findings, our study identified biomechanical stretch as a novel regulatory determinant of NFAT5 activity in VSMCs, which increases its mRNA expression, elicits its transient translocation to the nucleus, and like angiotensin II increases its protein abundance. In this context, it is reasonable to assume that AP‐1 controls the mRNA expression of NFAT‐5 in stretch‐stimulated VSMCs as it is a crucial regulator of stretch‐dependent gene expression and may control NFAT5 expression through binding to multiple AP‐1 binding sites located in the NFAT5 promotor.
Despite the implication of different kinases in the control of NFAT5 activity,19, 22 p38 MAP kinase and ERK1/2‐dependent signaling—prototypic stretch‐activated kinase pathways—were not involved in the mechanism that drives the translocation of NFAT5 to the nucleus of the stretched VSMCs. Although biomechanical stretch elicits the release of intracellular calcium37 and the calcium/calcineurin signaling cascade activates NFAT5 in T‐cells,38 a corresponding mechanism does not seem to be involved in the stretch‐dependent control of NFAT5 activity in VSMCs. Interestingly, JNK activity appears to be a prerequisite for this process to occur as it regulates the protein abundance but not mRNA of NFAT5 in stretch‐stimulated VSMCs. Furthermore, it is likely that this kinase phosphorylates NFAT5 at Ser‐1197 under these conditions while no changes were observed in total serine phosphorylation upon the chosen experimental conditions. The role of this specific serine phosphorylation is still unclear and may contribute to the protein stability of NFAT5 under these conditions. However, as S1197‐phosphorylated NFAT5 does not enter the nucleus of stretch‐stimulated VSMCs this modification may restrict nuclear translocation of this transcription factor and thus tightly controls its dynamics in biomechanically stressed VSMCs. Interestingly, phosphorylation of other members of the NFAT family (NFAT1‐4) is usually associated with their localization in the cytosol.39, 40 Activation of these transcription factors is realized by calcineurin, a serine/threonine phosphatase that controls their dephosphorylation and thus their ability to enter the nucleus. In contrast to NFAT1‐4, NFAT5 does not cooperate with Fos/Jun to bind to DNA and its nuclear translocation is independent of calcineurin.34 Nevertheless, phosphorylation of individual NFAT5 sites such as S1197 appear to be controlled by JNK and prevent a distinct portion of NFAT5 from entering the nucleus—a mechanism resembling a basic principle to arrest other members of the NFAT family in the cytoplasm.
The orchestration of NFAT5 activity becomes even more complex when considering that nucleocytoplasmic trafficking of NFAT5 may also be influenced by palmitoylation and/or myristoylation as has been observed upon changes in osmolarity.26 Along these data, we found that palmitoylation of NFAT5 is required to enter the nucleus in stretch‐stimulated VSMCs and is enhanced further upon inhibition of depalmitoylation. Palmitoylation is a highly relevant and reversible post‐translational modification of proteins regulating their activity, localization, and trafficking. The human genome encodes 23 protein palmitoyl acyltransferases that are all capable to link palmitate to cysteine residues acting as putative palmitoylation sites in many proteins such as Src family of kinases, G‐protein‐coupled receptors, Ras GTPases as well as NFAT5.26 Due to the hydrophobic characteristic of lipid anchors, palmitoylation may explain the slower electrophoretic migration speed of proteins26, 41 as was observed for NFAT5 in protein lysates of stretch‐stimulated HUASMCs. Of note, the JNK target c‐Jun as a subunit of the transcription factor AP‐1 controls the expression of carnitine palmitoyltransferase42 which is upregulated in stretch‐stimulated VSMCs and may therefore promote the generation of palmitoylated NFAT5 in VSMCs exposed to biomechanical stretch.
As a ubiquitously expressed transcription factor, NFAT5 regulates the context‐dependent expression of gene products in many different cell types. With smooth muscle α‐actin NFAT5 appears to govern the expression of a protein of the contractile apparatus in angiotensin II‐stimulated VSMCs17 that is also controlled by myocardin.14, 43 According to the fact that biomechanical stretch or an increase in wall stress is associated with the loss of contractile marker gene expression and favors activation of VSMC,16 we identified inter alia tenascin‐C as a stretch‐dependent NFAT5 target that stimulates migration of VSMCs and therefore promotes their activity. Tenascin‐C is a glycoprotein of the extracellular matrix whose expression and synthesis is correlated with proliferation of VSMCs and amplifies the mitogenic response to FGF‐2.44 Likewise, tenascin‐C has been shown to mediate VSMC migration induced by PDGF‐BB,45 which may explain why knockdown of NFAT5 in these cells inhibits their PDGF‐BB‐induced migration.17 Furthermore, tenascin‐C expression has often been associated with detrimental vascular remodeling processes that are linked to vascular injury or an increase in wall stress.46, 47, 48 Most notably, progression of pulmonary hypertension is accompanied by an increased abundance of tenascin‐C in the vessel wall and repression of tenascin‐C expression under these conditions triggers apoptosis of VSMCs and limits vascular thickening.49, 50 In this context, tenascin‐C may act as a survival factor that context‐dependently adjusts growth of VSMCs by providing an anti‐apoptotic scaffold. The close relationship of tenascin‐C to vascular diseases suggests that the medicinal control of NFAT5 activity may be an interesting therapeutic option.
In summary, our findings indicate that (1) biomechanical stretch raises protein abundance and elicits translocation of NFAT5 to the nucleus in both cultured and native arterial smooth muscle cells, (2) palmitoylation of NFAT5 is a prerequisite for its nuclear translocation, and (3) NFAT5 directly upregulates the expression of tenascin‐C—a gene product that orchestrates the migration of VSMCs and thus contributes to the activation of VSMCs under conditions of hypertension. The stretch‐induced translocation of NFAT5 therefore constitutes a novel regulatory mechanism underlying this phenomenon during stretch or wall stress‐induced maladaptive remodeling processes that occur in the early phases of hypertension or atherosclerosis.
Acknowledgments
The authors would like to acknowledge the excellent technical assistance of Gudrun Scheib, Maria Harlacher, Ender Serbest, and Franziska Mohr.
Footnotes
†
Scherer and Pfisterer contributed equally to this work.
‡
Marco Cattaruzza is deceased.
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© 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
History
Received: 9 December 2013
Accepted: 27 December 2013
Published online: 10 March 2014
Published in print: 24 March 2014
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Deutsche Forschungsgemeinschaft
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB TR 23, project sections C5 and C6).
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