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The dual-specificity mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) inactivates MAP kinases by dephosphorylation. Here we show that the proinflammatory cytokine interleukin (IL)-17A induces adult mouse primary cardiac fibroblast (CF) proliferation and migration via IL-17 receptor A//IL-17 receptor C-dependent MKP-1 suppression, and activation of p38 MAPK and ERK1/2. IL-17A mediated p38 MAPK and ERK1/2 activation is inhibited by MKP-1 overexpression, but prolonged by MKP-1 knockdown. IL-17A induced miR-101 expression via PI3K/Akt, and miR-101 inhibitor reversed MKP-1 down regulation. Importantly, MKP-1 knockdown, pharmacological inhibition of p38 MAPK and ERK1/2, or overexpression of dominant negative MEK1, each markedly attenuated IL-17A-mediated CF proliferation and migration. Similarly, IL-17F and IL-17A/F heterodimer that also signal via IL-17RA/IL-17RC, stimulated CF proliferation and migration. These results indicate that IL-17A stimulates CF proliferation and migration via Akt/miR-101/MKP-1-dependent p38 MAPK and ERK1/2 activation. These studies support a potential role for IL-17 in cardiac fibrosis and adverse myocardial remodeling., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The in vivo role of TNF signaling in the genesis of β-adrenergic receptor (β-AR)-mediated cardiac hypertrophy is unknown. Wild-type (WT), TNF receptor 1 (TNFR1)-/- and TNFR2-/- mice were given isoproterenol (ISO, 12.5 μg/kg/h) or saline (SAL) for 1 or 7 days. In WT mice, 7 days of ISO yielded chamber/myocyte hypertrophy and hyperdynamic function without hypertension or fibrosis. WT ISO hearts exhibited an early (1 day) pro-inflammatory response with significant (p < 0.05) activation of nuclear factor (NF)-κB and activator protein 1 (AP-1) and upregulation of TNF, interleukin (IL)-1β and IL-6, inducible nitric oxide synthase (iNOS) and monocyte chemotactic protein-1 (MCP-1), together with increased anti-inflammatory IL-10. This response diminished markedly by 7 days. As compared with WT ISO mice, TNFR1-/- ISO mice exhibited significantly (p < 0.05) less NF-κB and AP-1 activation, less IL-1β, TNF, iNOS and MCP-1 upregulation, but greater IL-10 at 1 day. However, there were no differences in hypertrophy or contractility at 7 days. In contrast, TNFR2-/- ISO mice exhibited augmented NF-κB and AP-1 activation, increased IL-1β and diminished IL-10 expression at 1 day, and significant exaggeration of hypertrophy and less contractile augmentation at 7 days. Moreover, TNFR2-/- mice exposed to tenfold higher ISO doses displayed significant mortality. TNF signaling contributes to β-AR-mediated cardiac remodeling in vivo in a receptor-specific manner. Unopposed TNFR1 activation is pro-inflammatory, pro-hypertrophic and promotes functional decline. However, co-activation of TNFR2 during β-AR stress is anti-inflammatory and counterbalances these deleterious effects. TNF modulatory strategies that maintain TNFR2 signaling may help prevent the detrimental long-term effects of β-AR stimulation in the heart.

Congestive heart failure and chronic kidney disease are characterized by chronically elevated angiotensin II (Ang II) and muscle wasting. Ang II causes skeletal muscle wasting by reducing appetite and by enhancing catabolism. The serine/threonine kinase AMP-activated protein kinase (AMPK) functions mainly as a sensor of cellular energy status. It is energy sparing and favors ATP generation. We hypothesized that Ang II induces muscle wasting in part by inhibiting AMPK signaling and altering cellular energy balance. Our results show that Ang II infusion in mice reduced gastrocnemius muscle weight by 26% and depleted ATP by 74%. In addition, Ang II upregulated protein phosphatase 2Cα by 2.6-fold and reduced AMPK phosphorylation and signaling in muscle. Importantly, the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside restored AMPK activity to levels of pair-fed controls and reversed Ang II-mediated ATP depletion and muscle wasting. Moreover, 5-aminoimidazole-4-carboxamide ribonucleoside activated Akt and inhibited Ang II-induced increases in E3 ubiquitin ligase expression. These novel results demonstrate critical roles for energy depletion and AMPK inhibition in Ang II-induced skeletal muscle wasting and suggest a therapeutic potential for AMPK activators in diseases characterized by muscle wasting.

IL-18 and the extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN) stimulate the expression of proinflammatory cytokines and MMPs and are elevated in myocardial hypertrophy, remodeling, and failure. Here, we report several novel findings in primary cardiomyocytes treated with IL-18. First, IL-18 activated multiple transcription factors, including NF-κB (p50 and p65), activator protein (AP)-1 (cFos, cJun, and JunD), GATA, CCAAT/enhancer-binding protein, myocyte-specific enhancer-binding factor, interferon regulatory factor-1, p53, and specific protein (Sp)-1. Second, IL-18 induced EMMPRIN expression via myeloid differentiation primary response gene 88/IL-1 receptor-associated kinase/TNF receptor-associated factor-6/JNK-dependent Sp1 activation. Third, IL-18 induced a number of MMP genes, particularly MMP-9, at a rapid rate as well as tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-3 at a slower rate. Finally, the IL-18 induction of MMP-9 was mediated in part via EMMPRIN and through JNK- and ERK-dependent AP-1 activation and p38 MAPK-dependent NF-κB activation. These results suggest that the elevated expression of IL-18 during myocardial injury and inflammation may favor EMMPRIN and MMP induction and extracellular matrix degradation. Therefore, targeting IL-18 or its signaling pathways may be of potential therapeutic benefit in adverse remodeling.

The anthracycline antibiotic doxorubicin (DOX) is a potent cancer chemotherapeutic agent that exerts both acute and chronic cardiotoxicity. Here we show that in adult mouse cardiomyocytes, DOX activates (i) the pro-apoptotic p53, (ii) p38MAPK and JNK, (iii) Bax translocation, (iv) cytochrome c release, and (v) caspase 3. Further, it (vi) inhibits expression of anti-apoptotic Akt, Bcl-2 and Bcl-xL, and (vii) induces internucleosomal degradation and cell death. WNT1-inducible signaling pathway protein-1 (WISP1), a CCN family member and a matricellular protein, inhibits DOX-mediated cardiomyocyte death. WISP1 inhibits DOX-induced p53 activation, p38 MAPK and JNK phosphorylation, Bax translocation to mitochondria, and cytochrome c release into cytoplasm. Additionally, WISP1 reverses DOX-induced suppression of Bcl-2 and Bcl-xL expression and Akt inhibition. The pro-survival effects of WISP1 were recapitulated by the forced expression of mutant p53, wild-type Bcl-2, wild-type Bcl-xL, or constitutively active Akt prior to DOX treatment. WISP1 also induces the pro-survival factor Survivin via PI3K/Akt signaling. Overexpression of wild-type, but not mutant Survivin, blunts DOX cytotoxicity. Further, WISP1 stimulates PI3K-Akt-dependent GSK3beta phosphorylation and beta-catenin nuclear translocation. Importantly, WISP1 induces its own expression. Together, these results provide important insights into the cytoprotective effects of WISP1 in cardiomyocytes, and suggest a potential therapeutic role for WISP1 in DOX-induced cardiotoxicity., (2009 Elsevier Inc. All rights reserved.)

The adipocyte-derived cytokine adiponectin is known to exert anti-inflammatory and anti-apoptotic effects. In patients with atherosclerotic cardiovascular disease, circulating levels of adiponectin correlate inversely with those of the proinflammatory, proapoptotic cytokine interleukin (IL)-18. The opposing actions of IL-18 and adiponectin on both cell survival and inflammation led us to investigate whether adiponectin signaling antagonizes IL-18-mediated endothelial cell death and to identify the underlying molecular mechanisms. Treatment with IL-18 suppressed Akt phosphorylation and its associated kinase activity, induced IkappaB kinase (IKK)-NF-kappaB-dependent PTEN activation, and promoted endothelial cell death. Pretreatment with adiponectin stimulated APPL1-dependent AMPK activation, reversed Akt inhibition in a phosphatidylinositol 3-kinase-dependent manner, blocked IKK-NF-kappaB-PTEN signaling, reduced caspase-3 activity, blocked Bax translocation, and inhibited endothelial cell death. The cytoprotective effect of adiponectin signaling was recapitulated by treatment with the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-riboside. Collectively, these results demonstrated that adiponectin reverses IL-18-mediated endothelial cell death through an AMPK-associated mechanism, which may thus have therapeutic potential for diminishing IL-18-dependent vascular injury and inflammation.

An inverse correlation between the pro-inflammatory cytokine interleukin-18 and the anti-atherogenic adipokine adiponectin has been reported in the chronic pathological conditions obesity, insulin resistance, coronary artery disease, and metabolic syndrome. We investigated whether this relationship is coincidental or has a causal basis. Here we show that interleukin-18 (IL-18) suppresses adiponectin transcription, mRNA expression, and secretion by 3T3-L1 adipocytes. IL-18 suppresses adiponectin promoter-reporter activity, an effect reversed by deletion or mutation of the NFATc4 core DNA-binding site. IL-18 induces NFATc4 phosphorylation (Ser(676)), nuclear translocation, and in vivo DNA binding. IL-18 induces ERK1/2 phosphorylation and enzyme activity, and pretreatment with the MEK inhibitor U0126, ERK1/2 inhibitor PD98059, or small interference RNA targeted to ERK1/2 attenuates ERK1/2 activation and NFATc4 phosphorylation. Finally, inhibition of ERK1/2 or NFATc4 knockdown reverses IL-18-mediated adiponectin suppression. In contrast to its inhibitory effects on adiponectin expression, IL-18 potently stimulates PAI-1 secretion. These data demonstrate for the first time that IL-18 selectively suppresses adiponectin expression via ERK1/2-dependent NFATc4 activation and suggest that the inverse relationship observed between IL-18 and adiponectin in various chronic pathological conditions is causally related. Thus, targeting IL-18 expression may enhance adiponectin expression and mitigate disease progression.

CXCL16 is a transmembrane non-ELR CXC chemokine that signals via CXCR6 to induce aortic smooth muscle cell (ASMC) proliferation. While bacterial lipopolysaccharide (LPS) has been shown to stimulate CXCL16 expression in SMC, its effects on CXCR6 are not known. Here, we demonstrate that LPS upregulates CXCR6 mRNA, protein, and surface expression in human ASMC. Inhibition of TLR4 with neutralizing antibodies or specific siRNA interference blocked LPS-mediated CXCR6 expression. LPS stimulated both AP-1 (c-Fos, c-Jun) and NF-kappaB (p50 and p65) activation, but only inhibition of AP-1 attenuated LPS-induced CXCR6 expression. Using dominant negative expression vectors and siRNA interference, we demonstrate that LPS induces AP-1 activation via MyD88, TRAF6, ERK1/2, and JNK signaling pathways. Furthermore, the flavoprotein inhibitor diphenyleniodonium chloride significantly attenuated LPS-mediated AP-1-dependent CXCR6 expression, as did inhibition of NOX4 NADPH oxidase by siRNA. Finally, CXCR6 knockdown inhibited CXCL16-induced ASMC proliferation. These results demonstrate that LPS-TLR4-NOX4-AP-1 signaling can induce CXCR6 expression in ASMC, and suggest that the CXCL16-CXCR6 axis may be an important proinflammatory pathway in the pathogenesis of atherosclerosis.

The tumor suppressor gene PTEN (phosphatase and tensin homologue deleted on chromosome 10) antagonizes the pro-survival signaling of Akt and promotes cell death. Previously, we demonstrated that IL-18 induced apoptosis in human cardiac microvascular endothelial cells (HCMEC). Here we have investigated the role of PTEN in this response. Our results demonstrate that IL-18 reduced phospho-Akt and bcl-2 levels, stimulated NF-kappaB activation, and induced PTEN-promoter-reporter activity, mRNA expression, and protein levels in HCMEC. IL-18-mediated PTEN transcription was enhanced by ectopic expression of wild type p65, but inhibited by dominant negative (dn) IkappaB-alpha, dnp65, and dnIKKbeta. Furthermore, overexpression of constitutively active Akt and wild type bcl-2 blocked IL-18-mediated cell death. While forced expression of PTEN potentiated, expression of catalytically inactive PTEN attenuated IL-18-mediated cell death. IL-18-induced activation of NF-kappaB and PTEN upregulation were mediated by p38MAPK. Together, these studies demonstrate a novel signal transduction pathway involving p38MAPK-NF-kappaB-PTEN in IL-18-mediated HCMEC death, and identify IL-18 as potential therapeutic target to inhibit or reduce myocardial inflammation and injury.

Endothelial cells are the primary targets of circulating immune and inflammatory mediators. We hypothesize that interleukin-18, a proinflammatory cytokine, induces endothelial cell apoptosis. Human cardiac microvascular endothelial cells (HCMEC) were treated with interleukin (IL) 18. mRNA expression was analyzed by ribonuclease protection assay, protein levels by immunoblotting, and cell death by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis. We also investigated the signal transduction pathways involved in IL-18-mediated cell death. Treatment of HCMEC with IL-18 increases 1) NF-kappaB DNA binding activity; 2) induces kappaB-driven luciferase activity; 3) induces IL-1beta and TNF-alpha expression via NF-kappaB activation; 4) inhibits antiapoptotic Bcl-2 and Bcl-X(L); 5) up-regulates proapoptotic Fas, Fas-L, and Bcl-X(S) expression; 6) induces fas and Fas-L promoter activities via NF-kappaB activation; 7) activates caspases-8, -3, -9, and BID; 8) induces cytochrome c release into the cytoplasm; 9) inhibits FLIP; and 10) induces HCME cell death by apoptosis as seen by increased annexin V staining and increased levels of mono- and oligonucleosomal fragmented DNA. Whereas overexpression of Bcl-2 significantly attenuated IL-18-induced endothelial cell apoptosis, Bcl-2/Bcl-X(L) chimeric phosphorothioated 2'-MOE-modified antisense oligonucleotides potentiated the proapoptotic effects of IL-18. Furthermore, caspase-8, IKK-alpha, and NF-kappaB p65 knockdown or dominant negative IkappaB-alpha and dominant negative IkappaB-beta or kinase dead IKK-beta significantly attenuated IL-18-induced HCME cell death. Effects of IL-18 on cell death are direct and are not mediated by intermediaries such as IL-1beta, tumor necrosis factor-alpha, or interferon-gamma. Taken together, our results indicate that IL-18 activates both intrinsic and extrinsic proapoptotic signaling pathways, induces endothelial cell death, and thereby may play a role in myocardial inflammation and injury.

It is well established that cytokines can induce the production of chemokines, but the role of chemokines in the regulation of cytokine expression has not been fully investigated. Exposure of rat cardiac-derived endothelial cells (CDEC) to lipopolysaccharide-induced CXC chemokine (LIX), and to a lesser extent to KC and MIP-2, activated NF-kappaB and induced kappaB-driven promoter activity. LIX did not activate Oct-1. LIX-induced interleukin-1beta and tumor necrosis factor-alpha promoter activity, and up-regulated mRNA expression. Increased transcription and mRNA stability both contributed to cytokine expression. LIX-mediated cytokine gene transcription was inhibited by interleukin-10. Transient overexpression of kinase-deficient NF-kappaB-inducing kinase (NIK) and IkappaB kinase (IKK), and dominant negative IkappaB significantly inhibited LIX-mediated NF-kappaB activation in rat CDEC. Inhibition of G(i) protein-coupled signal transduction, poly(ADP-ribose) polymerase, phosphatidylinositol 3-kinase, and the 26 S proteasome significantly inhibited LIX-mediated NF-kappaB activation and cytokine gene transcription. Blocking CXCR2 attenuated LIX-mediated kappaB activation and kappaB-driven promoter activity in rat CDEC that express both CXCR1 and -2, and abrogated its activation in mouse CDEC that express only CXCR2. These results indicate that LIX activates NF-kappaB and induces kappaB-responsive proinflammatory cytokines via either CXCR1 or CXCR2, and involved phosphatidylinositol 3-kinase, NIK, IKK, and IkappaB. Thus, in addition to attracting and activating neutrophils, the ELR(+) CXC chemokines amplify the inflammatory cascade, stimulating local production of cytokines that have negative inotropic and proapoptotic effects.

Vascular smooth muscle cell (SMC) migration is an important mechanism in atherogenesis and postangioplasty arterial remodeling. Previously, we demonstrated that the proinflammatory cytokine interleukin (IL)18 is a potent inducer of SMC migration. Since extracellular matrix metalloproteinase inducer (EMMPRIN) stimulates ECM degradation and facilitates cell migration, we investigated whether IL18 and EMMPRIN regulate each other's expression, whether their cross talk induces SMC migration, and whether the phytoalexin resveratrol inhibits IL-18EMMPRIN signaling and SMC migration. Our studies demonstrate that 1) IL-l8 induces EMMPRIN mRNA and protein expressions and stimulates EMMPRIN secretion from human aortic SMCs; 2) IL-18 stimulates EMMPRIN expression via oxidative stress and phosphatidylinositol 3-kinase (PI3K)-Akt-ERK signaling; 3) IL-18-stimulated SMC migration is significantly blunted by EMMPRIN knockdown, EMMPRIN function-blocking antibodies, or adenoviral transduction of mutant EMMPRIN; 4) conversely, EMMPREN stimulates IL-18 expression and secretion via P13K, Akt, and ERK; and 5) resveratrol attenuates IL-18and EMMPRIN-mediated P13K, Akt, and ERK activations; blunts IL-18-mediated oxidative stress; blocks IL-18EMMPRIN cross-regulation; and inhibits SMC migration. Collectively, our results demonstrate that the coexpression and regulation of IL-18 and EMMPRIN in the vessel wall may amplify the inflammatory cascade and promote atherosclerosis and remodeling. Resveratrol, via its antioxidant and anti-inflammatory properties, has the potential to inhibit the progression of atherosclerosis by blocking IL-18 and EMMPRIN cross-regulation and SMC migration. [ABSTRACT FROM AUTHOR]

We investigated the expression of the proinflammatory cytokine interleukin (IL)-17 in cardiac fibroblasts and its induction by high glucose (HG). Our results show that primary mouse cardiac fibroblasts (mCFs) secrete low basal levels of IL- 17 and that HG (25 mM n-glucose) as opposed to low glucose (5 mM n-glucose + 20 mM mannitol) significantly enhances its secretion. HG induces IL- 17 mRNA expression by both transcriptional and posttranscriptional mechanisms. HG induces phosphoinositide 3- kinase [P13K; inhibited by adenoviral (Ad).dominant negative (dn)PI3Kp85I, Akt (inhibited by Ad.dnAktl), and ERK (inhibited by PD-98059) activation and induces IL-17 expression via P13K→Akt→ERK-dependent signaling. Moreover, mCFs express both IL-17 receptors A and C, and although IL-17RA is upregulated, HG fails to modulate IL-17RC expression. Furthermore, IL-17 stim- ulates net collagen production by mCFs. Pretreatment with the phytoalexin resveratrol blocks HG-induced P13K-, Akt-, and ERK-dependent IL-17 expression. These results demonstrate that 1) cardiac fibroblasts express IL-17 and its receptors; 2) HG upregulates IL-17 and IL-17RA, suggesting a positive amplification loop in IL-17 signaling in hyperglycemia; 3) IL- 17 enhances net collagen production; and 4) resveratrol can inhibit these HG-induced changes. Thus, in hyperglycemic conditions, IL- 17 may potentiate myocardial inflammation, injury, and remodeling through autocrine and paracrine mechanisms, and resveratrol has therapeutic potential in ameliorating this effect. [ABSTRACT FROM AUTHOR]

We investigated whether β-adrenergic receptor (β-AR) stimulation induces the expression of interleukin (IL)-18, a proinflammatory cytokine, in myocardium and in cardiac-derived endothelial cells (CDEC) via activation of nuclear factor (NF)-κB. Our results indicate that isoproterenol (ISO) activates NF-κB DNA binding activity, and induces myocardial and systemic elaboration of IL-18 via β2-AR signaling. Furthermore, in CDEC, ISO increased basal and inducible promoter activities, increased IL-18 gene transcription and mRNA stability, and induced IL-18 expression via β2-AR agonism. Signaling required Gi, PI3K, Akt, IKK, and NF-κB. In conclusion, our results indicate for the first time that isoproterenol induces myocardial and systemic elaboration of IL-18 via a β2-AR and NF-κB-dependent mechanism. Similar events may occur in heart failure, a disease state characterized by sustained β-AR activation. [Copyright &y& Elsevier]

Abstract: Both the sympathetic nervous system and the proinflammatory cytokine interleukin-18 (IL-18) play key roles in the pathophysiology of the hypertrophied failing heart. IL-18 binding protein (IL-18BP), a natural inhibitor of IL-18, counters its biological effects. β-AR stimulation induces IL-18 expression, but whether it also regulates IL-18BP is not known. Here we demonstrate that the β-AR agonist isoproterenol (ISO) increases steady state IL-18BP mRNA and protein levels in adult mouse cardiomyocytes in a β2-AR-dependent manner. We cloned mouse Il18bp 5′cis-regulatory region, and identified putative CREB and C/EBPβ transcription factor-binding sites. Forced expression of mutant CREB or C/EBPβ knockdown markedly attenuated ISO-induced Il18bp transcription and deletion or mutation of CREB and C/EBP motifs in the Il18bp promoter reduced ISO-induced promoter-reporter gene activity. ISO induced CREB and C/EBPβ activation in cardiomyocytes via PI3K/Akt and ERK1/2. Importantly, ISO-induced hypertrophy in vitro was dependent on IL-18 induction as it was blunted by IL-18 neutralizing antibodies and forced expression of IL-18BP. Moreover, ISO-induced hypertrophy was markedly attenuated in IL-18 null and IL-18BP transgenic mice. These data support the novel concept that β-AR activation, in addition to inducing cardiomyocyte hypertrophy via IL-18, concomitantly induces a countering effect by stimulating IL-18BP expression, and that ISO-induced cardiomyocyte hypertrophy may result from a net effect of IL-18 and IL-18BP induction. [Copyright &y& Elsevier]

Abstract: Interleukin (IL)-18 is a cardiotropic proinflammatory cytokine chronically elevated in the serum of patients with cardiac hypertrophy (LVH). The purpose of this study was to examine the role of IL-18 in pressure-overload hypertrophy using wild type (WT) and IL-18 −/− (null) mice. Adult male C57Bl/6 mice underwent transaortic constriction (TAC) for 7days or sham surgery. Heart weight/body weight ratios showed blunted hypertrophy in IL-18 null TAC mice compared to WT TAC animals. Microarray analyses indicated differential expression of hypertrophy-related genes in WT versus IL-18 nulls. Northern, Western, and EMSA analyses showed Akt and GATA4 were increased in WT but unchanged in IL-18 null mice. Our results demonstrate blunted hypertrophy with reduced expression of contractile-, hypertrophy-, and remodeling-associated genes following pressure overload in IL-18 null mice, and suggest that IL-18 plays a critical role in the hypertrophic response. [Copyright &y& Elsevier]