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• W2000267199 abstract "G protein-coupled receptor kinase 5 (GRK5) plays a key role in cardiac signaling regulation, and its expression is increased in heart failure. Recently, increased expression of GRK5 in the myocardium of mice has been shown to be detrimental in the setting of pressure-overload hypertrophy. The ubiquitous nuclear transcription factor κB (NF-κB) is involved in the regulation of numerous genes in various tissues, and activation of NF-κB has been shown to be associated with heart disease. Here, we investigated the role of NF-κB signaling in the regulation of the GRK5 gene and expression of this kinase in cardiomyocytes. First, in analyzing the 5′-flanking DNA of GRK5, the presence of a potential NF-κB binding site was observed in the promoter region. Phorbol myristate acetate, a known stimulator of NF-κB, increased the levels of GRK5 in myocytes whereas treatment of cells with N-acetyl cysteine, a known inhibitor of NF-κB, or with SC 514, an inhibitor of IκB kinase 2 decreased GRK5. Utilizing EMSA or ChIP assays, we found that both p50 and p65 NF-κB could interact with the promoter of GRK5 following myocytes NF-κB activation. Importantly, short interfering RNA (siRNA)-mediated loss of p65 in myocytes decreased the stimulated increased levels of GRK5 mRNA and protein. Finally, adenovirus-mediated overexpression of a dominant-negative IκBα in myocytes inhibited the levels of GRK5. Taken together, our study demonstrates that NF-κB plays a critical role in the regulation of GRK5 transcription in myocytes and that this may translate to the significant expression changes seen in heart disease. G protein-coupled receptor kinase 5 (GRK5) plays a key role in cardiac signaling regulation, and its expression is increased in heart failure. Recently, increased expression of GRK5 in the myocardium of mice has been shown to be detrimental in the setting of pressure-overload hypertrophy. The ubiquitous nuclear transcription factor κB (NF-κB) is involved in the regulation of numerous genes in various tissues, and activation of NF-κB has been shown to be associated with heart disease. Here, we investigated the role of NF-κB signaling in the regulation of the GRK5 gene and expression of this kinase in cardiomyocytes. First, in analyzing the 5′-flanking DNA of GRK5, the presence of a potential NF-κB binding site was observed in the promoter region. Phorbol myristate acetate, a known stimulator of NF-κB, increased the levels of GRK5 in myocytes whereas treatment of cells with N-acetyl cysteine, a known inhibitor of NF-κB, or with SC 514, an inhibitor of IκB kinase 2 decreased GRK5. Utilizing EMSA or ChIP assays, we found that both p50 and p65 NF-κB could interact with the promoter of GRK5 following myocytes NF-κB activation. Importantly, short interfering RNA (siRNA)-mediated loss of p65 in myocytes decreased the stimulated increased levels of GRK5 mRNA and protein. Finally, adenovirus-mediated overexpression of a dominant-negative IκBα in myocytes inhibited the levels of GRK5. Taken together, our study demonstrates that NF-κB plays a critical role in the regulation of GRK5 transcription in myocytes and that this may translate to the significant expression changes seen in heart disease. G protein-coupled receptors (GPCRs) 4The abbreviations used are: GPCRG protein-coupled receptorGRKG protein-coupled receptor kinaseHFheart failureHDAChistone deacetylaseNRVMneonatal rat ventricular myocitePMAphorbol 12-myristate 13-acetateNACN-acetyl cysteinesiRNAshort interfering RNANEnuclear extract(s)ROSreactive oxygen speciesAdadenovirus. , such as β-adrenergic receptors (βARs), play crucial signaling and functional roles in the heart. GPCRs undergo nodal regulation following agonist activation triggered by phosphorylation via family of kinases such as GPCR kinases (GRKs) (1Rockman H.A. Koch W.J. Lefkowitz R.J. Seven transmembrane-spanning receptors and heart function.Nature. 2002; 415: 206-212Crossref PubMed Scopus (783) Google Scholar, 2Brinks H. Koch W.J. Targeting G protein-coupled receptor kinases (GRKs) in heart failure.Drug Discov. Today Dis. Mech. 2010; 7: e129-e134Crossref PubMed Scopus (18) Google Scholar). GRK phosphorylation of activated receptors triggers a process of desensitization that involves the loss of the G protein signal through β-arrestin binding to the phosphorylated receptor (1Rockman H.A. Koch W.J. Lefkowitz R.J. Seven transmembrane-spanning receptors and heart function.Nature. 2002; 415: 206-212Crossref PubMed Scopus (783) Google Scholar). Seven GRKs have been identified, and they all have distinct tissue distribution and subcellular localization, and undergo specific regulatory actions (2Brinks H. Koch W.J. Targeting G protein-coupled receptor kinases (GRKs) in heart failure.Drug Discov. Today Dis. Mech. 2010; 7: e129-e134Crossref PubMed Scopus (18) Google Scholar, 3Premont R.T. Gainetdinov R.R. Physiological roles of G protein-coupled receptor kinases and arrestins.Annu. Rev. Physiol. 2007; 69: 511-534Crossref PubMed Scopus (399) Google Scholar). For example, GRK2 and 3 are cytosolic kinases that actively translocate to the plasma membrane to target GPCRs and do so by binding to the βγ subunits of dissociated heterotrimeric G proteins, whereas GRK4–6 are primarily associated with the membrane at all times (3Premont R.T. Gainetdinov R.R. Physiological roles of G protein-coupled receptor kinases and arrestins.Annu. Rev. Physiol. 2007; 69: 511-534Crossref PubMed Scopus (399) Google Scholar). In addition, nuclear localization and export signal sequences have been described for GRK4–6, as these kinases can be found in the nucleus of cells (4Johnson L.R. Scott M.G. Pitcher J.A. G protein-coupled receptor kinase 5 contains a DNA-binding nuclear localization sequence.Mol. Cell. Biol. 2004; 24: 10169-10179Crossref PubMed Scopus (87) Google Scholar, 5Martini J.S. Raake P. Vinge L.E. DeGeorge Jr., B.R. DeGeorge Jr., B. Chuprun J.K. Harris D.M. Gao E. Eckhart A.D. Pitcher J.A. Koch W.J. Uncovering G protein-coupled receptor kinase-5 as a histone deacetylase kinase in the nucleus of cardiomyocytes.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 12457-12462Crossref PubMed Scopus (175) Google Scholar, 6Yi X.P. Gerdes A.M. Li F. Myocyte redistribution of GRK2 and GRK5 in hypertensive, heart failure-prone rats.Hypertension. 2002; 39: 1058-1063Crossref PubMed Scopus (69) Google Scholar). G protein-coupled receptor G protein-coupled receptor kinase heart failure histone deacetylase neonatal rat ventricular myocite phorbol 12-myristate 13-acetate N-acetyl cysteine short interfering RNA nuclear extract(s) reactive oxygen species adenovirus. Although expressed in several tissues, GRK2 and GRK5 are the most abundant GRKs found in myocytes, and interestingly, both have been found to be up-regulated in conditions of compromised myocardial function, including human heart failure (HF) (7Zhang Y. Matkovich S.J. Duan X. Gold J.I. Koch W.J. Dorn 2nd, G.W. Nuclear effects of G-protein receptor kinase 5 on histone deacetylase 5-regulated gene transcription in heart failure.Circ. Heart Fail. 2011; 4: 659-668Crossref PubMed Scopus (40) Google Scholar). Using several animal models, enhanced activity of both GRK2 and GRK5 have been shown to be pathological to the heart (1Rockman H.A. Koch W.J. Lefkowitz R.J. Seven transmembrane-spanning receptors and heart function.Nature. 2002; 415: 206-212Crossref PubMed Scopus (783) Google Scholar, 2Brinks H. Koch W.J. Targeting G protein-coupled receptor kinases (GRKs) in heart failure.Drug Discov. Today Dis. Mech. 2010; 7: e129-e134Crossref PubMed Scopus (18) Google Scholar, 8Huang Z.M. Gold J.L. Koch W.J. G protein-coupled receptor kinases in normal and failing myocardium.Front Biosci. 2011; 17: 3047-3060Google Scholar), including our recent report where nuclear GRK5 activity in myocytes was found to promote maladaptive cardiac hypertrophy in mice after pressure overload (5Martini J.S. Raake P. Vinge L.E. DeGeorge Jr., B.R. DeGeorge Jr., B. Chuprun J.K. Harris D.M. Gao E. Eckhart A.D. Pitcher J.A. Koch W.J. Uncovering G protein-coupled receptor kinase-5 as a histone deacetylase kinase in the nucleus of cardiomyocytes.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 12457-12462Crossref PubMed Scopus (175) Google Scholar). Because enhanced expression of GRKs appears to be a critical component to detrimental cardiac signaling and function, it is important to uncover specific regulators of GRK gene expression in myocytes to potentially block untoward increases in levels and activity. This is an area of GRK biology that has not been well studied and is of particular importance to GRK5 because it can act in a GRK-independent manner in the nucleus of myocytes (5Martini J.S. Raake P. Vinge L.E. DeGeorge Jr., B.R. DeGeorge Jr., B. Chuprun J.K. Harris D.M. Gao E. Eckhart A.D. Pitcher J.A. Koch W.J. Uncovering G protein-coupled receptor kinase-5 as a histone deacetylase kinase in the nucleus of cardiomyocytes.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 12457-12462Crossref PubMed Scopus (175) Google Scholar). This occurs because of a novel nuclear activity of GRK5 acting as a class II histone deacetylase (HDAC) kinase, and its up-regulation causes enhanced nuclear activity of GRK5 phosphorylating HDAC5 and its disinhibition of hypertrophic and maladaptive gene transcription (5Martini J.S. Raake P. Vinge L.E. DeGeorge Jr., B.R. DeGeorge Jr., B. Chuprun J.K. Harris D.M. Gao E. Eckhart A.D. Pitcher J.A. Koch W.J. Uncovering G protein-coupled receptor kinase-5 as a histone deacetylase kinase in the nucleus of cardiomyocytes.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 12457-12462Crossref PubMed Scopus (175) Google Scholar). Nuclear transcription factor κB (NF-κB) is a ubiquitous transcription factor that has been shown to regulate gene expression in a variety of cell types in the cardiovascular system, including cardiomyocytes (9Gordon J.W. Shaw J.A. Kirshenbaum L.A. Multiple facets of NF-κB in the heart. To be or not to NF-κB.Circ. Res. 2011; 108: 1122-1132Crossref PubMed Scopus (415) Google Scholar). The NF-κB-dependent transcriptional network is thought to have great significance to cardiovascular disease (10Jones W.K. Brown M. Wilhide M. He S. Ren X. NF-κB in cardiovascular disease. Diverse and specific effects of a “general” transcription factor?.Cardiovasc. Toxicol. 2005; 5: 183-202Crossref PubMed Scopus (71) Google Scholar) although its activation has been shown to be both beneficial and detrimental to cardiac function. The dynamic roles of NF-κB in different cardiovascular pathologies may be due to its ability to regulate a wide range of genes. NF-κB may play a key role in the pathophysiology of myocardial ischemia/reperfusion injury, atherosclerosis, and HF (11Valen G. Yan Z.Q. Hansson G.K. Nuclear factor κ-B and the heart.J. Am. Coll. Cardiol. 2001; 38: 307-314Crossref PubMed Scopus (402) Google Scholar). In HF, NF-κB may play a detrimental role by up-regulating the expression of TNF-α and other proinflammatory molecules but may also have beneficial roles for the resolution of inflammation and in tissue remodeling because it regulates several antiapoptotic genes (9Gordon J.W. Shaw J.A. Kirshenbaum L.A. Multiple facets of NF-κB in the heart. To be or not to NF-κB.Circ. Res. 2011; 108: 1122-1132Crossref PubMed Scopus (415) Google Scholar, 12Lawrence T. Gilroy D.W. Colville-Nash P.R. Willoughby D.A. Possible new role for NF-κB in the resolution of inflammation.Nat. Med. 2001; 7: 1291-1297Crossref PubMed Scopus (669) Google Scholar). NF-κB is activated by multiple stimuli, including those present in ischemic and hypertrophic myocardium (9Gordon J.W. Shaw J.A. Kirshenbaum L.A. Multiple facets of NF-κB in the heart. To be or not to NF-κB.Circ. Res. 2011; 108: 1122-1132Crossref PubMed Scopus (415) Google Scholar, 10Jones W.K. Brown M. Wilhide M. He S. Ren X. NF-κB in cardiovascular disease. Diverse and specific effects of a “general” transcription factor?.Cardiovasc. Toxicol. 2005; 5: 183-202Crossref PubMed Scopus (71) Google Scholar). TNF-α is a potent stimulator of NF-κB activity, and it can also be activated in cardiomyocytes by specific GPCR agonists, especially those that induce hypertrophy (9Gordon J.W. Shaw J.A. Kirshenbaum L.A. Multiple facets of NF-κB in the heart. To be or not to NF-κB.Circ. Res. 2011; 108: 1122-1132Crossref PubMed Scopus (415) Google Scholar, 13Higuchi Y. Otsu K. Nishida K. Hirotani S. Nakayama H. Yamaguchi O. Matsumura Y. Ueno H. Tada M. Hori M. Involvement of reactive oxygen species-mediated NF-κ B activation in TNF-α-induced cardiomyocyte hypertrophy.J. Mol. Cell Cardiol. 2002; 34: 233-240Abstract Full Text PDF PubMed Scopus (165) Google Scholar), which could have a direct influence on nuclear GRK5 activity. Interestingly, GRK5 has been shown to interact with and influence NF-κB signaling (14Patial S. Luo J. Porter K.J. Benovic J.L. Parameswaran N. G-protein-coupled-receptor kinases mediate TNFα-induced NFκB signalling via direct interaction with and phosphorylation of IκBα.Biochem. J. 2010; 425: 169-178Crossref Scopus (86) Google Scholar, 15Patial S. Shahi S. Saini Y. Lee T. Packiriswamy N. Appledorn D.M. Lapres J.J. Amalfitano A. Parameswaran N. G-protein-coupled receptor kinase 5 mediates lipopolysaccharide-induced NFκB activation in primary macrophages and modulates inflammation in vivo in mice.J. Cell. Physiol. 2011; 226: 1323-1333Crossref PubMed Scopus (44) Google Scholar, 16Sorriento D. Ciccarelli M. Santulli G. Campanile A. Altobelli G.G. Cimini V. Galasso G. Astone D. Piscione F. Pastore L. Trimarco B. Iaccarino G. The G-protein-coupled receptor kinase 5 inhibits NFκB transcriptional activity by inducing nuclear accumulation of IκB α.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 17818-17823Crossref PubMed Scopus (92) Google Scholar, 17Sorriento D. Santulli G. Fusco A. Anastasio A. Trimarco B. Iaccarino G. Intracardiac injection of AdGRK5-NT reduces left ventricular hypertrophy by inhibiting NF-κB-dependent hypertrophic gene expression.Hypertension. 2010; 56: 696-704Crossref PubMed Scopus (82) Google Scholar), and thus, there may be a link between these two systems. In light of this and the role NF-κB has as a potential critical mediator of cardiac hypertrophy, we investigated whether this transcription factor could specifically regulate GRK5 gene expression in cardiomyocytes. Our data presented below unveils novel aspects of the GRK5 promoter region and specific regulation of this gene by activation of NF-κB signaling in cardiac myocytes promoting stress-induced GRK5 up-regulation. Ventricular cardiomyocytes from 1- to 2-day-old rat neonatal hearts (NRVMs) were prepared, as we have published recently (18Brinks H. Boucher M. Gao E. Chuprun J.K. Pesant S. Raake P.W. Huang Z.M. Wang X. Qiu G. Gumpert A. Harris D.M. Eckhart A.D. Most P. Koch W.J. Level of G protein-coupled receptor kinase-2 determines myocardial ischemia/reperfusion injury via pro- and anti-apoptotic mechanisms.Circ. Res. 2010; 107: 1140-1149Crossref PubMed Scopus (107) Google Scholar). Myocytes were cultured in Ham's F-10 supplemented with penicillin/streptomycin (100 units/ml) and 5% FBS at 37 °C in 5% CO2 humidified atmosphere for 2–3 days. NRVMs were cultured for 24 h in complete medium consisting of 85% Ham's F-10, 10% heat-inactivated horse serum, and 5% FBS at 37 °C in the presence of 5% CO2 and 95% ambient air followed by maintenance in 5% FBS containing medium for another 24 h. After 2 days, cells were maintained in serum-free medium in the presence or absence of PMA or NAC or SC 514 or in combination for 24 h. In some experiments, myocytes were treated with p65 siRNA (in the presence or absence of PMA) or adenoviruses expressing the dominant negative form of IκBα or GFP. The cells were scraped from the dishes using ice-cold phosphate-buffered saline containing protease inhibitor mixture (1 tablet/10 ml) (Roche) and lysed using lysis buffer. Protein concentrations were determined by a Pierce BCA protein assay kit. Proteins (10 μg) were separated on NuPAGE Novex 4–20% bis-tris gels (Invitrogen) and transferred to nitrocellulose membranes. The membranes were then analyzed by immunoblotting using specific antibodies to GRK5, p65, phosphorylated p65 (P-p65), p50, IκBα, β-actin, and GAPDH. Antibodies were obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, and Cell Signaling Technology, Inc. Nuclear extracts (NE) were prepared from NRVMs as described previously (19Lee K.A. Bindereif A. Green M.R. A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing.Gene Anal. Tech. 1988; 5: 22-31Crossref PubMed Scopus (394) Google Scholar). Protein concentrations were determined by a Pierce BCA protein assay kit. Double-stranded oligonucleotides corresponding to the NF-κB (NF-κB is underlined) and flanking sequences (sense, 5′-GAG CGT GGG GAT CCC GGG AGT C-3′ and antisense, 5′-GAC TCC CGG GAT CCC CAC GCT C-3′) in the GRK5 gene were used in the EMSA. The oligos were end-labeled using IRdye 700 and used as probes (Integrated DNA Technologies, Inc.). Nuclear proteins (7 μg) were incubated with IRdye-labeled NF-κB oligonucleotide in the dark for 30 min at room temperature in reaction buffer (20 mm Hepes (pH 7.6), 75 mm KCl, 0.2 mm EDTA, 20% glycerol) and 1 μg of poly(dI-dC)-poly(dI-dC) (Li-Cor) as a nonspecific competitor. For the antibody-mediated super shift assay, 1 μg of p50 or p65 was incubated in reaction mixture for 30 min followed by incubation with an IRdye-labeled probe for another 30 min. Protein-DNA complexes were separated from the free probe on a 4% non-denaturing polyacrylamide gel. The image was visualized and scanned by an Odyssey infrared imaging system. NRVMs were cultured for 24 h in complete medium and then changed to 5% FBS containing medium for another 24 h. Cells in 5% medium were then maintained in serum-free medium in the presence or absence of PMA for 24 h. ChIP was performed as described in detail previously (20Islam K.N. Mendelson C.R. Permissive effects of oxygen on cyclic AMP and interleukin-1 stimulation of surfactant protein A gene expression are mediated by epigenetic mechanisms.Mol. Cell. Biol. 2006; 26: 2901-2912Crossref PubMed Scopus (50) Google Scholar). The soluble chromatin (500 μl) was incubated with antibodies for p50 and p65 (Santa Cruz) at 4 °C overnight. Two aliquots were reserved as “controls:” one incubated without antibody and the other with non-immune IgG. Immune complexes were isolated using protein A/G Plus-agarose beads and collected by centrifugation. Cross-linking of the immunoprecipitated chromatin complexes and “input controls” (5% of the total soluble chromatin) were reversed by heating the samples at 65 °C for 4 h, followed by incubation with proteinase K, DNA purification by phenolchloroform extraction, and precipitation in EtOH at −20 °C. Samples and input controls were diluted in TE buffer just prior to PCR. Real-time QPCR was carried out using the following primers to amplify 112 bp of the rGRK5 5′-flanking region surrounding the NF-κB (forward, 5′-TTTGGTTATAATGAAAATCGTAGGG-3′; reverse, 5′ GTCTACGCGAACCAGAATGC-3′). NRVMs were cultured for 24 h in complete medium and then changed to 5% FBS containing medium for another 24 h. Cells in 5% medium were then maintained in serum-free medium in the presence or absence of PMA or p65 siRNA or in combination for 24 h. Total RNA was isolated from these cells by the one-step method described previously (21Islam K.N. Mendelson C.R. Glucocorticoid/glucocorticoid receptor inhibition of surfactant protein-A (SP-A) gene expression in lung type II cells is mediated by repressive changes in histone modification at the SP-A promoter.Mol. Endocrinol. 2008; 22: 585-596Crossref PubMed Scopus (50) Google Scholar) (TRIzol, Invitrogen). RNA was treated with DNase to remove any contaminating DNA, and 1 μg was reverse-transcribed using the isoscript cDNA synthesis kit from Bio-Rad. Validated primer sets directed against GRK5 (forward, 5′-CAA GGA GCT GAA TGT GTT CGG AC-3′; reverse, 5′-GCT GCT TCC AGT GGA GTT TGA AT-3′) and p65 (forward, 5′-CAA GTG CCT TAA TAG CAG GGC AAA-3′; reverse, 5′-AGA GCT AGA AAG AGC AAG AGT CCA AT-3′) along with the constitutively expressed 18S rRNA (forward, 5′-ACC GCA GCT AGG AAT AAT GGA-3′; reverse, 5′-GCC TCA GTT CCG AAA ACC A-3′) were used for quantitative PCR amplification. The Bio-Rad detection system (MyIQ) was employed using the DNA binding dye SYBR Green for the quantitative detection of PCR products. The cycle threshold was set at a level where the exponential increase in PCR amplification was approximately parallel between all samples. Data were normalized by analyzing the ratio of the target cDNA concentrations to that of 18 S rRNA. We have compared expression of 18S rRNA to that of GAPDH and have found both to be unaffected by PMA or p65 siRNA treatment. NRVMs were cultured for 24 h in complete medium and then changed to 5% FBS containing medium for another 24 h. Cells in 5% medium were then maintained in serum-free medium in the presence or absence of p65 siRNA (30 nm) for 24 h using Hiperfect as transfecting reagent from Qiagen. The media were changed and followed by incubation for another 24 h. Cells were treated with or without 20 nm PMA for 24 h. After harvesting the cells total lysates and RNA were prepared. Lysates were used to determine proteins levels by immunoblot analysis. cDNAs obtained from RNA were analyzed by real-time QPCR using the primers for GRK5, p65, and 18S. NRVMs were cultured for 24 h in complete medium and then changed to 5% FBS-containing medium for another 24 h. Cells in 5% medium were then maintained in serum-free medium and were infected with recombinant adenoviruses as described previously (22Peppel K. Zhang L. Orman E.S. Hagen P.O. Amalfitano A. Brian L. Freedman N.J. Activation of vascular smooth muscle cells by TNF and PDGF: overlapping and complementary signal transduction mechanisms.Cardiovasc. Res. 2005; 65: 674-682Crossref PubMed Scopus (54) Google Scholar). Briefly, the cells were incubated overnight with recombinant adenoviruses expressing either dominant-negative IκBα (Ad-dm-IκBα) (multiplicity of infection = 100, 200) or with GFP (Ad-GFP) (multiplicity of infection = 100, 200). The next day, media were removed and changed to serum-free media and incubated another 48 h. Cells were harvested and lysed followed by immunoblot analysis. All values in the text and figures are presented as mean ± S.E. from at least three independent experiments from given n sizes. Statistical significance between two groups was determined using the two-tailed Student's t test. p values of < 0.05 were considered significant. In characterizing the 5′-flanking region of the GRK5 gene we found a consensus sequence for a putative binding site for NF-κB. This region is conserved between the rat, mouse, and human GRK5 genes and lies ∼800 bp from the start of transcription (Fig. 1A). To examine whether this putative site is active, we tested whether the p50 or p65 subunits of NF-κB could bind to the GRK5 gene promoter. We first analyzed binding of nuclear proteins from neonatal rat ventricular myocytes (NRVMs) to an NF-κB consensus oligonucleotide using EMSA. As shown, nuclear extracts from myocytes had positive binding to the NF-κB oligonucleotide (Fig. 1B, lane 2). To determine whether the binding of NF-κB is specific, we then utilized competitive EMSA and antibody-mediated super shift EMSA in myocyte nuclear extracts. In this experiment, the non-labeled NF-κB consensus oligonucleotide competed with labeled NF-κB oligonucleotides (Fig. 1B, lane 5). Moreover, antibodies to NF-κB proteins, p50 and p65, added alone reduced or abolished the intensity of the band of NF-κB-nuclear protein complex (Fig. 1B, lanes 3 and 4). These results indicate the presence of p50 and p65 in the GRK5 promoter complex. PMA is a specific activator of PKC (23Vijayan K. Szotek E.L. Martin J.L. Samarel A.M. Protein kinase C-alpha-induced hypertrophy of neonatal rat ventricular myocytes.Am. J. Physiol. 2004; 287: H2777-H2789Crossref PubMed Scopus (42) Google Scholar), which can activate NF-κB through PKC as well as through the induced formation of reactive oxygen species (ROS) (24Lee H.B. Yu M.R. Song J.S. Ha H. Reactive oxygen species amplify protein kinase C signaling in high glucose-induced fibronectin expression by human peritoneal mesothelial cells.Kidney Int. 2004; 65: 1170-1179Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). PMA is a well established hypertrophic agent in myocytes and, accordingly, was tested to determine whether it could induce GRK5 expression in NRVMs. Using RT-PCR methods we determined the effects of PMA on mRNA expression of GRK5 in myocytes. As shown in Fig. 2A, 20 nm of PMA induced the expression of GRK5 within 30 min, and this induction remained for over 6 h. Of note, p65 increased in a parallel fashion after myocytes were treated with this hypertrophic agent (Fig. 2B). In light of stimulatory effects on GRK5 mRNA expression as well as p65, we determined the effects of PMA on NF-κB and GRK5 protein as well as the DNA binding activity of NF-κB to the GRK5 promoter. Protein levels for NF-κB and GRK5 were analyzed by immunoblotting, and binding activity of NF-κB was analyzed by EMSA using nuclear proteins from myocytes treated with PMA or control media for 24 h. As shown in Fig. 2, C and D, protein levels of GRK5, p50, and p65 were markedly increased by the treatment of myocytes with PMA. In the EMSA analysis, PMA-treated myocytes showed increased NF-κB binding activity to GRK5 promoter as compared with untreated cells (Fig. 2E). Because PMA increases the expression of both GRK5 and NF-κB in myocytes, presumably due at least in part to increased binding of NF-κB to the GRK5 promoter we carried out ChIP to analyze the effects of PMA on in vivo binding of p50 and p65 to the GRK5 promoter. NRVMs were cultured in the presence or absence of PMA for 24 h and nuclear DNA was purified from lysate samples immunoprecipitated using with anti-p50 or anti-p65 sera. Subsequently, quantitative PCR was used to amplify a 112 bp genomic region surrounding the NF-κB site within the 5′-flanking region of GRK5 promoter (Fig. 3A). As shown in Fig. 3B and C, PMA stimulated the in vivo recruitment of p50 and p65 to the GRK5 promoter. NAC is known inhibitor of NF-κB and it can also act as an antioxidant by scavenging ROS. To determine the effect of NAC for both proteins levels and DNA-binding activity of NF-κB in myocytes, an EMSA was carried out. We found that NAC decreased NF-κB DNA binding activity (Fig. 4A). Further, NAC also decreased GRK5 and p65 protein levels when myocytes were treated with PMA and also basally (Fig. 4, B and C). Lowering the levels of both proteins as well as DNA binding activity by NAC indicates the involvement of ROS and NF-κB for induction of GRK5 expression. In addition to NAC effect on GRK5 expression, SC 514, a selective inhibitor of IκB kinase 2 (IKK2) (also known as inhibitor of NF-κB mediated gene expression or inhibitor of NF-κB activation) has been tested on PMA-induced GRK5 expression in myocytes. To determine the effect of SC 514, cells were treated with PMA or SC 514 or in combination. EMSA was carried out to determine NF-κB DNA binding activity, and immunoblot analysis was used to analyze GRK5 protein levels. As can be seen in Fig. 4D, SC 514 markedly decreased the NF-κB DNA binding activity in control cell as well as cells treated with PMA. Interestingly, the levels of GRK5 were also significantly decreased by SC 514 in the presence or absence of PMA (Fig. 4, E and F), suggesting that activation of NF-κB plays an important role in GRK5 expression. The data presented thus far suggest that NF-κB proteins act to mediate induction of GRK5 expression in myocytes. To directly demonstrate the mechanistic action of NF-κB, we treated NRVMs with siRNA against rat p65. After transfection cells were treated with or without PMA. As can be seen, cells treated with p65 siRNA showed significant reduction of p65 levels compared with levels found in cells treated with control siRNA (Fig. 5A). Importantly, the loss of p65 translated to a lowering of GRK5 protein in myocytes (Fig. 5A) that was also seen at the mRNA level (B). PMA treatment was not able to induce either mRNA or proteins levels of GRK5 in p65 siRNA transfected cells (Fig. 5, A and B). The immunoblot analysis in Fig. 5A showed profound phosphorylation of p65 by PMA treatment. Silencing of p65 prevented p65 phosphorylation evoked by PMA. Thus, it appears that levels of p65 directly determine GRK5 expression levels in myocytes both under basal and stimulated conditions. To further confirm the involvement of NF-κB in the regulation of GRK5 in myocytes, cells were infected with recombinant adenoviruses expressing a dominant-negative form of IκBα (Ad-dm-IκBα) or GFP (Ad-GFP) as a control. The dominant-negative isoform of IκB contains mutations in the IκB kinase phosphorylation sites (Ser-34, Ser-36) and is resistant to degradation by the ubiquitin/proteasome pathway (22Peppel K. Zhang L. Orman E.S. Hagen P.O. Amalfitano A. Brian L. Freedman N.J. Activation of vascular smooth muscle cells by TNF and PDGF: overlapping and complementary signal transduction mechanisms.Cardiovasc. Res. 2005; 65: 674-682Crossref PubMed Scopus (54) Google Scholar). This enables the overexpressed proteins to sequester p65/p50 complexes within the cell, thereby blocking activation of NF-κB. Overexpression of dm-IκBα was confirmed by immunoblotting (Fig. 5C, center panel). As also shown in Fig. 5C (top panel), this dominant-negative IκB inhibited GRK5 expression, as levels are significantly lower than in myocytes expressing GFP (D). These findings again suggest a role of endogenous NF-κB proteins in the regulation of GRK5 gene expression in myocytes. GRKs are recognized as critical regulators of GPCR signaling on the b" @default.
• W2000267199 created "2016-06-24" @default.
• W2000267199 creator A5030932376 @default.
• W2000267199 creator A5075648082 @default.
• W2000267199 date "2012-04-01" @default.
• W2000267199 modified "2023-10-11" @default.
• W2000267199 title "Involvement of Nuclear Factor κB (NF-κB) Signaling Pathway in Regulation of Cardiac G Protein-coupled Receptor Kinase 5 (GRK5) Expression" @default.
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