Cross-talk between calcineurin/NFAT and Jak/STAT signalling induces cardioprotective alphaB-crystallin gene expression in response to hypertrophic stimuli

Abstract

Among the stress proteins that are up-regulated in the heart due to imposed biomechanical stress, alphaB-crystallin (CryAB) is the most abundant and pivotal in rendering protection against stress-induced cell damage. Cardiomyocyte-specific expression of the CryAB gene was shown to be dependent upon an intact alphaBE4 cis-element located in the CryAB enhancer. To date, there is no evidence on the identity of regulatory proteins and associated signalling molecules that control CryAB expression in cardiomyocytes. In this study, we define a mechanism by which the calcineurin/NFAT and Jak/STAT pathways regulate CryAB gene expression in response to a hypertrophic agonist endothelin-1 (En-1), in hypertrophic hearts of mice with pressure overload (TAC) and in heart-targeted calcineurin over-expressing mice (MHC-CnA). We observed that in response to various hypertrophic stimuli the transcription factors NFAT, Nished and STAT3 form a dynamic ternary complex and interact with the alphaBE4 promoter element of the CryAB gene. Both dominant negative NFAT and AG490, an inhibitor of the Jak2 phosphorylation, inhibited CryAB gene transcription in transient transfection assays. AG490 was also effective in blocking the nuclear translocation of NFAT and STAT3 in cardiomyocytes treated with En-1. We observed a marked increase in CryAB gene expression in MHC-CnA mouse hearts accompanied with increased phosphorylation of STAT3. We conclude that hypertrophy-dependent CryAB gene expression can be attributed to a functional linkage between the Jak/STAT and calcineurin/NFAT signalling pathways, each of which are otherwise known to be involved independently in the deleterious outcome in cardiac hypertrophy.

Fig 1

αBE4 enhancer activity. (A) Comparison of sequences of cardiac specific elements, IRE in the MLC2v and the αBE4 in the CryAB gene. The IRE and the αBE4 sequences contain a NFAT binding site, shown in red, 5′ to the Nished binding element, shown in blue. (B) Schematic representation of WT (CryAB-Luc) and mutant (mutBE4-Luc) gene constructs. Numbers denote the position of 5′ and 3′ ends of the gene relative to the transcription initiation site. HL-1 cardiomyocytes were transfected with CryAB-Luc or mutBE4-Luc plasmids. (C) HL-1 cells were co-transfected with CryAB-Luc or mutBE4-Luc plasmids along with parental vector (pcDNAV5) or Nished expression plasmid (pcDNAV5-Nished). Luciferase activities are expressed relative to the mean value derived from cells transfected with CryAB-Luc plasmids (B). In cells co-transfected with pcDNAV5-Nished, luciferase activities are expressed relative to the mean value derived from cells cotransfected with parental vector (pcDNAV5) and CryAB-Luc or mutBE4-luc plasmids. Renilla luciferase activity was used for normalization of transfection efficiency. Experiments have been repeated at least three times. Data are mean ± SE. *P < 0.05 and **P < 0.01. (D) Nuclear extracts from heart tissue of WT mice were incubated with a ³²P-labeled oligonucleotide encompassing the αBE4 sequence and analyzed by gel mobility shift assay. Fifty-fold of unlabeled αBE4 DNA was used as a specific competitor. Preincubation of cardiac nuclear extracts with anti-Nished antibody shows a supershift indicated by arrow (n= 3).

Fig 2

αBE4 enhancer activity in response to En-1 treatment. (A) Serum-starved HL-1 cells were treated with either different concentrations of En-1 (25, 50, 100 nM) for 12, 24 and 48 hrs before collection or vehicle (water). Total RNA levels were examined by Northern blotting with a CryAB cDNA probe. 18S ribosomal RNA was used as a loading control, (lower panel). Quantification of time and concentration dependent effect of En-1 on CryAB mRNA levels in HL-1 cells (n= 2). (B) HL-1 cells were cotransfected with an expression plasmid encoding Nished or parental vector where indicated along with CryAB-Luc, or mutBE4-Luc plasmids. After preincubation in serum-free medium cells were treated with 50 nM En-1. Luciferase activities in cells treated with En-1 are expressed to the mean value obtained from cells exposed to vehicle (water). In cells co-transfected with pcDNAV5-Nished (Nished) treated with En-1, luciferase activities are expressed relative to the mean value derived from cells cotransfected with pcDNAV5 (parental vector) and CryAB-Luc or mutBE4-luc plasmids and treated with En-1. Renilla luciferase expression was used for normalization. Experiments have been repeated at least three times. Data are mean ± SE. **P < 0.01.

Fig 3

αBE4 enhancer activity in response to pressure-overload hypertrophy. Mice were subjected to transverse aortic constriction (TAC). (A) Semi-quantitative RT-PCR was performed with primers from the coding region of CryAB, ANF and GAPDH genes. Quantification of the CryAB mRNA level relative to the GAPDH mRNA level is shown in the lower panel. Data shown are representative of two separate experiments, with three individual mice per experiment. (B) Nuclear extracts were prepared from hearts of sham (C) and TAC mice. GMSA was performed with radiolabeled αBE4 oligonucleotide. Competition with 50× molar excess of unlabeled αBE4 (S) and nonspecific (NS) oligonucleotides is shown. Preincubation of cardiac nuclear extracts with anti-Nished antibody shows a supershift, indicated by arrow. Data shown are one representative of two separate experiments, with two mice per experiment.

Fig 4

Calcineurin and NFAT are necessary for constitutive and En-1 inducible CryAB gene transcription. HL-1 cells were transfected either with CryAB-Luc (A), or with mutBE4-Luc (B). Reporter constructs were cotransfected with an expression plasmid encoding either cain or pECE-flag (parental vector) (A), or either dominant negative NFAT (dnNFAT) plasmid or pcDNA3. (parental vector) (B). Cells were treated with 50 nM En-1 or vehicle (water). Renilla luciferase expression was used for normalization. Luciferase activities in cells cotransfected with cain or dnNFAT are expressed relative to the mean value obtained from cells cotransfected with parental vector and treated with the vehicle only. Cells cotransfected with cain or dnNFAT and exposed to En-1 versus cotransfected with parental vector exposed to En-1. Experiments have been repeated at least three times. Data are mean ± SE. *P < 0.05.

Fig 5

MHC-CnA mice have enhanced levels of CryAB mRNA and induced αBE-4/protein complex formation. (A) Real time PCR was done for quantification of CryAB mRNA in the hearts of WT C57BL/6 (WT) (n= 7) and transgenic MHC-CnA (CN) (n= 7) mice. β-actin was used for normalization. Data are mean ± SE. *P < 0.05. (B) Nuclear extracts were prepared from hearts of WT and MHC-CnA (CN) littermates. Gel mobility assays were performed with radiolabeled αBE4 or mutated αBE4 oligonucleotides. Binding reactions were performed in the presence of anti-Nished antibody, 50x molar excess unlabeled αBE4 (S) and nonspecific (NS) oligonucleotides. (C) Preincubation of cardiac nuclear extracts isolated from MHC-CnA mice (CN) with anti-NFATc3 (c3) and c4 (c4) antibodies were shown. Data shown are representative of three separate experiments, with two individual mice per experiment. (D) Chromatin isolated from WT and MHC-CnA mice (CN) hearts was subjected to ChIP. The chromatin DNA was immunoprecipitated with antibody against NFATc4, c3, STAT3 and acetylated histone H3 and analyzed by semiquantitative PCR with primers flanking the αBE4 sequence. A non-specific IgG was used as a negative control for immunoprecipitation and antibody alone (Ab), without lysate, served as a control for cross-contamination. Input DNA represents 10% of total chromatin used in each reaction. Primers specific to GAPDH were used before (input) and after immunoprecipitation as a control to monitor immunoprecipitation specificity. Data shown are representative of three separate experiments, with two individual mice per experiment.

Fig 6

Interaction between the αBE4 binding proteins is induced in MHC-CnA mice. (A) Cardiac nuclear protein extracts from WT and MHC-CnA (CN) mice were subjected to SDS-PAGE fractionation followed by western blot with anti-phospho-Stat3 (Tyr705) antibody (that detects only Stat3 phosphorylated at tyrosine 705), anti-NFATc3, anti-NFATc4 and anti-Nished antibodies (n= 2). Lamin B was used as a loading control. Quantification of protein levels in the (WT) and MHC-CnA (CN) mice. (B) Cardiac cell lysates from WT and MHC-CnA (CN) mice were immunoprecipitated (IP) with anti-STAT3 antibody and western blotted (WB) with anti-NFATc4, anti-NFATc3 and anti-STAT3 antibodies. Data shown represent one of two separate experiments, with two individual mice per experiment.

Fig 7

Jak2 regulates the transcriptional activity of the CryAB promoter in cardiomyocytes. (A) HL-1 cells were co-transfected with CryAB-Luc plasmid along with parental vector (pcDNAV5) or pTel-Jak2. Cells were serum-starved and treated with vehicle (DMSO) or 50 μM AG490 for 24 hrs. Luciferase activities in cells treated with AG490 and co-transfected with Tel-Jak are expressed relative to the mean value of the co-transfected cells exposed to the vehicle. Renilla luciferase expression was used for normalization. Experiments have been repeated at least three times. Data are mean ± SE. *P < 0.05 and **P < 0.01. (B) Serum-starved HL-1 cells were treated for 5, 10 and 30 min. with either En-1 (50 nM) or vehicle (water) and subjected to ChIP assays. Soluble chromatin from cross-linked cells was immunoprecipitated with indicated antibodies. A non-specific IgG was used as a negative control for immunoprecipitation and the antibody alone (Ab), without lysate, served as a control for cross-contamination. Input DNA represents 10% of total chromatin used in each reaction. DNA was amplified performed with primers flanking the αBE4 sequence. Primers specific to GAPDH were used before (input) and after immunoprecipitation as a control to monitor immunoprecipitation specificity. Data shown are one representative of three separate experiments.

Fig 8

Immunocytochemical localization of STAT3 and NFATc3 in En-1 induced and AG490 treated rat primary cardiomyocytes. Cultured neonatal primary cardiomyocytes were treated with vehicle (DMSO) (left panel), En-1 (50 nM) (middle panel), or En-1 (50 nM) with AG490 (5 μM) (right panel), then immunostained for sub-cellular localization of STAT3 and NFATc3. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (Dapi) and merged patterns are shown in the bottom panel. All images were obtained at 40× magnification.