Mol. Nutr. Food Res. 2015, 59, 221–230
221
DOI 10.1002/mnfr.201400279
RESEARCH ARTICLE
Sulforaphane suppresses cardiac hypertrophy by inhibiting GATA4/GATA6 expression and MAPK signaling pathways Hae Jin Kee1 , Gwi Ran Kim1 , In Kyeom Kim2 and Myung Ho Jeong1∗ 1 2
Cardiovascular Convergence Research Center, Chonnam National University Hospital, Gwangju, South Korea Department of Pharmacology, Cardiovascular Research Institute, Kyungpook National University School of Medicine, Daegu, South Korea
Scope: Sulforaphane (SFN) is a naturally occurring isothiocynate compound found in cruciferous vegetables. Here, we report the effect of SFN on cardiac hypertrophy and propose an underlying mechanism. Methods and results: SFN suppresses cardiomyocyte hypertrophy induced by hypertrophic stimuli in vitro and in vivo. SFN suppresses the expression of fetal genes, including atrial natriuretic peptide, brain natriuretic peptide, and beta myosin heavy chain. We used an siRNA technique and atrial natriuretic peptide promoter with mutated GATA binding sites to demonstrate that SFN mediates cardiac hypertrophy by modulating transcription factors GATA4/6. Conclusion: These results suggest that SFN has the potential to prevent cardiac hypertrophy by downregulating GATA4/6 and mitogen-activated protein kinase signaling pathways.
Received: April 26, 2014 Revised: October 5, 2014 Accepted: October 15, 2014
Keywords: Cardiac hypertrophy / GATA binding factor 4 / GATA binding factor 6 / Mitogenactivated protein kinase signaling / Sulforaphane
1
Introduction
Cardiac hypertrophy occurs in various diseases, including hypertension, aortic stenosis, myocardial infarction, and valve diseases. However, with sustained stress signals, a compensated heart may enter a pathological decompensated state, resulting in the development of heart failure. Cardiac hypertrophy is associated with multiple signaling pathways mediated by signaling factors such as mitogen-activated protein kinases (MAPKs), phosphatidylinositol-3-kinase (PI3K/Akt), nuclear factor-B (NF-B), and nuclear factor of activated T cells (NFAT) pathways [1–3]. Correspondence: Dr. Hae Jin Kee, Cardiovascular Convergence Research Center, Chonnam National University Hospital, 42 Jebong-ro, Dong-gu, Gwangju 501-757, South Korea E-mail: [email protected] Fax: +82-62-228-4227 Abbreviations: ANP, atrial natriuretic peptide; -MHC, beta myosin heavy chain; BNP, brain natriuretic peptide; ERK, extracellular signal regulated kinase; ET-1, endothelin-1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GATA6, GATA binding factor 6; GATA4, GATA binding factor 4; ISP, isoproterenol; JNK, c-Jun N-terminal kinase; MEF2C, myocyte enhancer factor 2C; MUT, mutant; p38, p38 mitogen-activated protein kinase; PE, phenylephrine; SFN, sulforaphane; siGATA6, GATA6 siRNA; siGATA4, GATA4 siRNA C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Transcription factors GATA4 (GATA binding factor 4) and GATA6 (GATA binding factor 6) are key regulators of cardiac genes in normal cardiac development, as well as in pathological hypertrophy [4, 5]. GATA binding sites are required for in vivo activation of cardiac promoters during cardiac hypertrophy. A number of other cardiac-specific transcription factors, including myocyte-enhancer factor 2 (MEF2), serum response factor (SRF), and homeobox protein Nkx2.5 are involved in cardiac development and hypertrophy [6, 7]. Sulforaphane (SFN) is a phytochemical found in cruciferous vegetables such as broccoli, cauliflower, and cabbages [8]. SFN is known to be an inducer of nuclear factor erythroid 2-related factor 2 (Nrf2) [9]. Recent studies show that SFN prevents growth of various types of cancer [10], vascular restenosis [11], inflammation [12], obesity [13], and even neurodegenerative diseases [14]. However, the effect of SFN and the mechanism underlying its action on cardiac hypertrophy remain unknown. In this study, we investigated the effect of SFN and the related molecular mechanism in cultured cardiomyocytes and a mouse disease model. Our findings demonstrate that SFN acts as a novel negative regulator of cardiac hypertrophy by ∗ Additional corresponding author: E-mail: [email protected]
Dr.
Myung
Ho
Jeong
www.mnf-journal.com
222
H. J. Kee et al.
downregulating transcription factors GATA4/6 and inhibiting MAPK signaling pathway.
2
Materials and Methods
2.1 Reagents and antibodies Phenylephrine (PE, P6126), endothelin-1 (ET-1, E7764), isoproterenol (ISP, I5627), and SFN (S4441) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Anti-GAPDH (GAPDH is glyceraldehyde 3-phosphate dehydrogenase, sc-32233), anti-homeobox protein Nkx2.5 (sc-12514), and brain natriuretic peptide (anti-BNP, sc-271185) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); antibody raised against atrial natriuretic peptide (antiANP) was obtained from Meridian Life Science (Memphis, TN, USA); anti-GATA4, anti-GATA6 (ab39123), and myocyte enhancer factor 2C (MEF2C, ab64644) antibodies were obtained from Abcam (Cambridge, UK); and anti-sarcomere ␣-actinin (A7811) antibody was obtained from Sigma-Aldrich Co. p-MAPK Family Ab Sampler kit was purchased from Cell Signaling Technology (Danvers, MA, USA).
2.2 Cell cultures of cardiomyocytes, H9c2, and 293T cells Cardiomyocytes from 2-day-old Sprague-Dawley rats were prepared as described previously [15]. Briefly, heart tissue was enzymatically dissociated with collagenase and the cells were isolated using Percoll gradients. Cells were pre-plated on tissue culture dishes to remove contaminating fibroblasts. Cells were plated on gelatin-coated culture dishes and cultured in DMEM with 10% FBS. The growth medium was changed to DMEM without 10% FBS and incubated for 24 h. After 24 h of serum starvation, cells were pretreated with SFN for 2 h and then co-incubated with 100 M PE or 100 nM ET-1. H9c2 cells were maintained in DMEM with 10% FBS. HEK 293 cells were obtained from the Seoul Korean Cell Line Bank (Seoul, Korea) and were maintained in DMEM containing 10% FBS. 2.3 Measurement of cell viability The cardiomyocytes were plated onto 24-well plates and treated with increasing dose of SFN for 24 h. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide assay. 2.4 Transient transfection and luciferase assay For the promoter mapping study, we used 5 serial deletion ANP promoter constructs (−3003, −638, −366, −189, −105, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Mol. Nutr. Food Res. 2015, 59, 221–230
and −64 bp). The −189 ANP promoter with a mutated GATAbinding site was generated using a site-directed mutagenesis kit (Stratagene, Cedar Creek, TX, USA). The pGL3–3003 ANP promoter, pGL3–1000 BNP promoter, and pCMV--galactosidase were transfected with Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA, USA) or PolyFect reagent (Qiagen, Venlo, The Netherlands) according to the manufacturer’s instructions. Cell lysates were prepared and luciferase activity was measured with a luciferase assay kit (Promega, Madison, WI, USA).
2.5 GATA4 and GATA6 siRNA Cardiomyocytes were transfected with siRNA against rat GATA4 (100 nM, sc-270093) and rat GATA6 (100 nM, sc-270094) using RNAiMAX (Invitrogen) reagent. Nontargeting siRNA (sicontrol) was used as a negative control. Transfected cells were serum-starved overnight and incubated with 100 M PE or 100 nM ET-1 for 24 h. For promoter study, H9c2 cells were transfected with −638 ANP luciferase plasmid and pCMV -galactosidase in serum-free DMEM using Lipofectamine and Plus Reagent (Invitrogen). Cells were transfected with 100 nM of control siRNA or GATA4/6 siRNA using RNAiMAX reagent (Invitrogen) 12 h after transfection and subsequently treated with vehicle or SFN (10 M) for 24 h. Luciferase activity was determined by luciferase assay kit (Promega).
2.6 [3 H]Leucine incorporation assay [3 H]Leucine incorporation assay was performed as described previously [16]. Briefly, cardiomyocytes were incubated in serum-free DMEM medium for 24 h. The cells were pretreated with SFN (10 M) and then costimulated with 100 M PE or 100 nM ET-1 for 24 h before the addition of 1.0 Ci/mL [3 H]leucine for another 12 h. After removing the medium, cells were fixed with 10% trichloroacetic acid and then solubilized with 0.25 N NaOH. The radioactivity was measured in a liquid scintillation counter (Winspectral 1414, Wallac, Turku, Finland).
2.7 Western blot analysis Western blots were performed as described previously [17]. Cell lysates were prepared with RIPA buffer (150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM NaF, 10 mM sodium vanadate, and 1 mM PMSF) containing protease inhibitors. Proteins were separated on 8–12% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were probed with previously listed antibodies and developed using Immobilon Western Detection Reagents (Millipore, Billerica, MA, USA). www.mnf-journal.com
223
Mol. Nutr. Food Res. 2015, 59, 221–230
2.8 Real-time RT-PCR Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies) and 1 g of RNA was used for the reversetranscription reaction with TOPscript RT DryMIX (Enzynomics, Daejeon, South Korea). Quantification of mRNA levels was done with the SYBR Green PCR kit (Applied Biosystems, Inc.). PCR was performed using the following oligonucleotide primers: for ANP, sense, 5 -GCTCGAGCAGATCGCAAAAG-3 , and antisense, 5 -GAGTGGGAGAGGTAAGGCCT-3 ; for BNP, sense, 5 - GACGGGCTGAGGTTGTTTTA-3 , and antisense, 5 -ACTGTGGCAAGTTTGTGCTG-3 ; for β-MHC (beta myosin heavy chain), sense, 5 - CCTCGCAATATCA AGGGAAA-3 , and antisense, 5 -TACAGGTGCATCAGCTC CAG-3 ; for Gapdh, sense, 5 -TGCACCACCAACTGCTTAG3 , and antisense, 5 -GATGCAGGGATGATGTTC-3 .
2.9 Cell size and stress-fiber formation Cardiomyocytes were plated onto 8-well chambers and fluorescent immunocytochemistry was performed as described previously [18]. Cardiomyocytes were pretreated for 2 h with vehicle or 10 M SFN, followed by co-incubation with 100 M PE or 100 nM ET-1 for 24 h. Following fixation with 4% paraformaldehyde, cells were permeabilized and visualized by staining with Alexa 488 phalloidin (Invitrogen). To examine the stress-fiber formation, cells were incubated with primary antibody against sarcomeric ␣-actinin (1:200, Sigma) and the cell size was measured using NIS Elements Software (Nikon, Tokyo, Japan).
means ± SEM. A p-value of
221
DOI 10.1002/mnfr.201400279
RESEARCH ARTICLE
Sulforaphane suppresses cardiac hypertrophy by inhibiting GATA4/GATA6 expression and MAPK signaling pathways Hae Jin Kee1 , Gwi Ran Kim1 , In Kyeom Kim2 and Myung Ho Jeong1∗ 1 2
Cardiovascular Convergence Research Center, Chonnam National University Hospital, Gwangju, South Korea Department of Pharmacology, Cardiovascular Research Institute, Kyungpook National University School of Medicine, Daegu, South Korea
Scope: Sulforaphane (SFN) is a naturally occurring isothiocynate compound found in cruciferous vegetables. Here, we report the effect of SFN on cardiac hypertrophy and propose an underlying mechanism. Methods and results: SFN suppresses cardiomyocyte hypertrophy induced by hypertrophic stimuli in vitro and in vivo. SFN suppresses the expression of fetal genes, including atrial natriuretic peptide, brain natriuretic peptide, and beta myosin heavy chain. We used an siRNA technique and atrial natriuretic peptide promoter with mutated GATA binding sites to demonstrate that SFN mediates cardiac hypertrophy by modulating transcription factors GATA4/6. Conclusion: These results suggest that SFN has the potential to prevent cardiac hypertrophy by downregulating GATA4/6 and mitogen-activated protein kinase signaling pathways.
Received: April 26, 2014 Revised: October 5, 2014 Accepted: October 15, 2014
Keywords: Cardiac hypertrophy / GATA binding factor 4 / GATA binding factor 6 / Mitogenactivated protein kinase signaling / Sulforaphane
1
Introduction
Cardiac hypertrophy occurs in various diseases, including hypertension, aortic stenosis, myocardial infarction, and valve diseases. However, with sustained stress signals, a compensated heart may enter a pathological decompensated state, resulting in the development of heart failure. Cardiac hypertrophy is associated with multiple signaling pathways mediated by signaling factors such as mitogen-activated protein kinases (MAPKs), phosphatidylinositol-3-kinase (PI3K/Akt), nuclear factor-B (NF-B), and nuclear factor of activated T cells (NFAT) pathways [1–3]. Correspondence: Dr. Hae Jin Kee, Cardiovascular Convergence Research Center, Chonnam National University Hospital, 42 Jebong-ro, Dong-gu, Gwangju 501-757, South Korea E-mail: [email protected] Fax: +82-62-228-4227 Abbreviations: ANP, atrial natriuretic peptide; -MHC, beta myosin heavy chain; BNP, brain natriuretic peptide; ERK, extracellular signal regulated kinase; ET-1, endothelin-1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GATA6, GATA binding factor 6; GATA4, GATA binding factor 4; ISP, isoproterenol; JNK, c-Jun N-terminal kinase; MEF2C, myocyte enhancer factor 2C; MUT, mutant; p38, p38 mitogen-activated protein kinase; PE, phenylephrine; SFN, sulforaphane; siGATA6, GATA6 siRNA; siGATA4, GATA4 siRNA C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Transcription factors GATA4 (GATA binding factor 4) and GATA6 (GATA binding factor 6) are key regulators of cardiac genes in normal cardiac development, as well as in pathological hypertrophy [4, 5]. GATA binding sites are required for in vivo activation of cardiac promoters during cardiac hypertrophy. A number of other cardiac-specific transcription factors, including myocyte-enhancer factor 2 (MEF2), serum response factor (SRF), and homeobox protein Nkx2.5 are involved in cardiac development and hypertrophy [6, 7]. Sulforaphane (SFN) is a phytochemical found in cruciferous vegetables such as broccoli, cauliflower, and cabbages [8]. SFN is known to be an inducer of nuclear factor erythroid 2-related factor 2 (Nrf2) [9]. Recent studies show that SFN prevents growth of various types of cancer [10], vascular restenosis [11], inflammation [12], obesity [13], and even neurodegenerative diseases [14]. However, the effect of SFN and the mechanism underlying its action on cardiac hypertrophy remain unknown. In this study, we investigated the effect of SFN and the related molecular mechanism in cultured cardiomyocytes and a mouse disease model. Our findings demonstrate that SFN acts as a novel negative regulator of cardiac hypertrophy by ∗ Additional corresponding author: E-mail: [email protected]
Dr.
Myung
Ho
Jeong
www.mnf-journal.com
222
H. J. Kee et al.
downregulating transcription factors GATA4/6 and inhibiting MAPK signaling pathway.
2
Materials and Methods
2.1 Reagents and antibodies Phenylephrine (PE, P6126), endothelin-1 (ET-1, E7764), isoproterenol (ISP, I5627), and SFN (S4441) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Anti-GAPDH (GAPDH is glyceraldehyde 3-phosphate dehydrogenase, sc-32233), anti-homeobox protein Nkx2.5 (sc-12514), and brain natriuretic peptide (anti-BNP, sc-271185) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); antibody raised against atrial natriuretic peptide (antiANP) was obtained from Meridian Life Science (Memphis, TN, USA); anti-GATA4, anti-GATA6 (ab39123), and myocyte enhancer factor 2C (MEF2C, ab64644) antibodies were obtained from Abcam (Cambridge, UK); and anti-sarcomere ␣-actinin (A7811) antibody was obtained from Sigma-Aldrich Co. p-MAPK Family Ab Sampler kit was purchased from Cell Signaling Technology (Danvers, MA, USA).
2.2 Cell cultures of cardiomyocytes, H9c2, and 293T cells Cardiomyocytes from 2-day-old Sprague-Dawley rats were prepared as described previously [15]. Briefly, heart tissue was enzymatically dissociated with collagenase and the cells were isolated using Percoll gradients. Cells were pre-plated on tissue culture dishes to remove contaminating fibroblasts. Cells were plated on gelatin-coated culture dishes and cultured in DMEM with 10% FBS. The growth medium was changed to DMEM without 10% FBS and incubated for 24 h. After 24 h of serum starvation, cells were pretreated with SFN for 2 h and then co-incubated with 100 M PE or 100 nM ET-1. H9c2 cells were maintained in DMEM with 10% FBS. HEK 293 cells were obtained from the Seoul Korean Cell Line Bank (Seoul, Korea) and were maintained in DMEM containing 10% FBS. 2.3 Measurement of cell viability The cardiomyocytes were plated onto 24-well plates and treated with increasing dose of SFN for 24 h. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide assay. 2.4 Transient transfection and luciferase assay For the promoter mapping study, we used 5 serial deletion ANP promoter constructs (−3003, −638, −366, −189, −105, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Mol. Nutr. Food Res. 2015, 59, 221–230
and −64 bp). The −189 ANP promoter with a mutated GATAbinding site was generated using a site-directed mutagenesis kit (Stratagene, Cedar Creek, TX, USA). The pGL3–3003 ANP promoter, pGL3–1000 BNP promoter, and pCMV--galactosidase were transfected with Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA, USA) or PolyFect reagent (Qiagen, Venlo, The Netherlands) according to the manufacturer’s instructions. Cell lysates were prepared and luciferase activity was measured with a luciferase assay kit (Promega, Madison, WI, USA).
2.5 GATA4 and GATA6 siRNA Cardiomyocytes were transfected with siRNA against rat GATA4 (100 nM, sc-270093) and rat GATA6 (100 nM, sc-270094) using RNAiMAX (Invitrogen) reagent. Nontargeting siRNA (sicontrol) was used as a negative control. Transfected cells were serum-starved overnight and incubated with 100 M PE or 100 nM ET-1 for 24 h. For promoter study, H9c2 cells were transfected with −638 ANP luciferase plasmid and pCMV -galactosidase in serum-free DMEM using Lipofectamine and Plus Reagent (Invitrogen). Cells were transfected with 100 nM of control siRNA or GATA4/6 siRNA using RNAiMAX reagent (Invitrogen) 12 h after transfection and subsequently treated with vehicle or SFN (10 M) for 24 h. Luciferase activity was determined by luciferase assay kit (Promega).
2.6 [3 H]Leucine incorporation assay [3 H]Leucine incorporation assay was performed as described previously [16]. Briefly, cardiomyocytes were incubated in serum-free DMEM medium for 24 h. The cells were pretreated with SFN (10 M) and then costimulated with 100 M PE or 100 nM ET-1 for 24 h before the addition of 1.0 Ci/mL [3 H]leucine for another 12 h. After removing the medium, cells were fixed with 10% trichloroacetic acid and then solubilized with 0.25 N NaOH. The radioactivity was measured in a liquid scintillation counter (Winspectral 1414, Wallac, Turku, Finland).
2.7 Western blot analysis Western blots were performed as described previously [17]. Cell lysates were prepared with RIPA buffer (150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM NaF, 10 mM sodium vanadate, and 1 mM PMSF) containing protease inhibitors. Proteins were separated on 8–12% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were probed with previously listed antibodies and developed using Immobilon Western Detection Reagents (Millipore, Billerica, MA, USA). www.mnf-journal.com
223
Mol. Nutr. Food Res. 2015, 59, 221–230
2.8 Real-time RT-PCR Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies) and 1 g of RNA was used for the reversetranscription reaction with TOPscript RT DryMIX (Enzynomics, Daejeon, South Korea). Quantification of mRNA levels was done with the SYBR Green PCR kit (Applied Biosystems, Inc.). PCR was performed using the following oligonucleotide primers: for ANP, sense, 5 -GCTCGAGCAGATCGCAAAAG-3 , and antisense, 5 -GAGTGGGAGAGGTAAGGCCT-3 ; for BNP, sense, 5 - GACGGGCTGAGGTTGTTTTA-3 , and antisense, 5 -ACTGTGGCAAGTTTGTGCTG-3 ; for β-MHC (beta myosin heavy chain), sense, 5 - CCTCGCAATATCA AGGGAAA-3 , and antisense, 5 -TACAGGTGCATCAGCTC CAG-3 ; for Gapdh, sense, 5 -TGCACCACCAACTGCTTAG3 , and antisense, 5 -GATGCAGGGATGATGTTC-3 .
2.9 Cell size and stress-fiber formation Cardiomyocytes were plated onto 8-well chambers and fluorescent immunocytochemistry was performed as described previously [18]. Cardiomyocytes were pretreated for 2 h with vehicle or 10 M SFN, followed by co-incubation with 100 M PE or 100 nM ET-1 for 24 h. Following fixation with 4% paraformaldehyde, cells were permeabilized and visualized by staining with Alexa 488 phalloidin (Invitrogen). To examine the stress-fiber formation, cells were incubated with primary antibody against sarcomeric ␣-actinin (1:200, Sigma) and the cell size was measured using NIS Elements Software (Nikon, Tokyo, Japan).
means ± SEM. A p-value of
