Aldosterone-induced cardiomyocyte growth, and fibroblast migration and proliferation are mediated by TRAF3IP2.

Sustained activation of the Renin-Angiotensin-Aldosterone System (RAAS) contributes to the pathogenesis of heart failure. Aldosterone (Aldo) is known to induce both myocardial hypertrophy and fibrosis through oxidative stress and proinflammatory pathways. Here we have investigated whether Aldo-mediated cardiomycocyte hypertrophy is dependent on TRAF3IP2, an upstream regulator of IKK and JNK. We also investigated whether the pro-mitogenic and pro-migratory effects of Aldo on cardiac fibroblasts are also mediated by TRAF3IP2. Aldo induced both superoxide and hydrogen peroxide in isolated adult mouse cardiomyocytes (CM), and upregulated TRAF3IP2 expression in part via the mineralocorticoid receptor and oxidative stress. Silencing TRAF3IP2 blunted Aldo-induced IKKß, p65, JNK, and c-Jun activation, IL-18, IL-6 and CT-1 upregulation, and cardiomyocyte hypertrophy. In isolated adult mouse cardiac fibroblasts (CF), Aldo stimulated TRAF3IP2-dependent IL-18 and IL-6 production, CTGF, collagen I and III expression, MMP2 activation, and proliferation and migration. These in vitro results suggest that TRAF3IP2 may play a causal role in Aldo-induced adverse cardiac remodeling in vivo, and identify TRAF3IP2 as a potential therapeutic target in hypertensive heart disease.

Pressure overload induces IL-18 and IL-18R expression, but markedly suppresses IL-18BP expression in a rabbit model. IL-18 potentiates TNF-α-induced cardiomyocyte death.

Recurrent or sustained inflammation plays a causal role in the development and progression of left ventricular hypertrophy (LVH) and its transition to failure. Interleukin (IL)-18 is a potent pro-hypertrophic inflammatory cytokine. We report that induction of pressure overload in the rabbit, by constriction of the descending thoracic aorta induces compensatory hypertrophy at 4weeks (mass/volume ratio: 1.7±0.11) and ventricular dilatation indicative of heart failure at 6weeks (mass/volume ratio: 0.7±0.04). In concordance with this, fractional shortening was preserved at 4weeks, but markedly attenuated at 6weeks. We cloned rabbit IL-18, IL-18Rα, IL-18Rß, and IL-18 binding protein (IL-18BP) cDNA, and show that pressure overload, while enhancing IL-18 and IL-18R expression in hypertrophied and failing hearts, markedly attenuated the level of expression of the endogenous IL-18 antagonist IL-18BP. Cyclical mechanical stretch (10% cyclic equibiaxial stretch, 1Hz) induced hypertrophy of primary rabbit cardiomyocytes in vitro and enhanced ANP, IL-18, and IL-18Rα expression. Further, treatment with rhIL-18 induced its own expression and that of IL-18Rα via AP-1 activation, and induced cardiomyocyte hypertrophy in part via PI3K/Akt/GATA4 signaling. In contrast, IL-18 potentiated TNF-α-induced cardiomyocyte death, and by itself induced cardiac endothelial cell death. These results demonstrate that pressure overload is associated with enhanced IL-18 and its receptor expression in hypertrophied and failingrabbit hearts. Since IL-18BP expression is markedly inhibited, our results indicate a positive amplification in IL-18 proinflammatory signaling during pressure overload, and suggest IL-18 as a potential therapeutic target in pathological hypertrophy and cardiac failure.

OxLDL induces endothelial dysfunction and death via TRAF3IP2: inhibition by HDL3 and AMPK activators.

Oxidized low-density lipoprotein (oxLDL) induces endothelial cell death through the activation of NF-κB and AP-1 pathways. TRAF3IP2 is a redox-sensitive cytoplasmic adapter protein and an upstream regulator of IKK/NF-κB and JNK/AP-1. Here we show that oxLDL-induced death in human primary coronary artery endothelial cells (ECs) was markedly attenuated by the knockdown of TRAF3IP2 or the lectin-like oxLDL receptor 1 (LOX-1). Further, oxLDL induced Nox2/superoxide-dependent TRAF3IP2 expression, IKK/p65 and JNK/c-Jun activation, and LOX-1 upregulation, suggesting a reinforcing mechanism. Similarly, the lysolipids present in oxLDL (16:0-LPC and 18:0-LPC) and minimally modified LDL also upregulated TRAF3IP2 expression. Notably, whereas native HDL3 reversed oxLDL-induced TRAF3IP2 expression and cell death, 15-lipoxygenase-modified HDL3 potentiated its proapoptotic effects. The activators of the AMPK/Akt pathway, adiponectin, AICAR, and metformin, attenuated superoxide generation, TRAF3IP2 expression, and oxLDL/TRAF3IP2-mediated EC death. Further, both HDL3 and adiponectin reversed oxLDL/TRAF3IP2-dependent monocyte adhesion to endothelial cells in vitro. Importantly, TRAF3IP2 gene deletion and the AMPK activators reversed oxLDL-induced impaired vasorelaxation ex vivo. These results indicate that oxLDL-induced endothelial cell death and dysfunction are mediated via TRAF3IP2 and that native HDL3 and the AMPK activators inhibit this response. Targeting TRAF3IP2 could potentially inhibit progression of atherosclerotic vascular diseases.

Docosahexaenoic acid reverses angiotensin II-induced RECK suppression and cardiac fibroblast migration.

The omega-3 polyunsaturated fatty acids (ω-3 fatty acids) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been reported to inhibit or delay the progression of cardiovascular diseases, including myocardial fibrosis. Recently we reported that angiotensin II (Ang II) promotes cardiac fibroblast (CF) migration by suppressing the MMP regulator reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), through a mechanism dependent on AT1, ERK, and Sp1. Here we investigated the role of miR-21 in Ang II-mediated RECK suppression, and determined whether the ω-3 fatty acids reverse these effects. Ang II induced miR-21 expression in primary mouse cardiac fibroblasts (CFs) via ERK-dependent AP-1 and STAT3 activation, and while a miR-21 inhibitor reversed Ang II-induced RECK suppression, a miR-21 mimic inhibited both RECK expression and Ang II-induced CF migration. Moreover, Ang II suppressed the pro-apoptotic PTEN, and the ERK negative regulator Sprouty homologue 1 (SPRY1), but induced the metalloendopeptidase MMP2, all in a manner that was miR-21-dependent. Further, forced expression of PTEN inhibited Akt phosphorylation, Sp1 activation, and MMP2 induction. Notably, while both EPA and DHA reversed Ang II-mediated RECK suppression, DHA appeared to be more effective, and reversed Ang II-induced miR-21 expression, RECK suppression, MMP2 induction, and CF migration. These results indicate that Ang II-induced CF migration is differentially regulated by miR-21-mediated MMP induction and RECK suppression, and that DHA has the potential to upregulate RECK, and therefore may exert potential beneficial effects in cardiac fibrosis.

Acetylsalicylic acid inhibits IL-18-induced cardiac fibroblast migration through the induction of RECK.

The pathogenesis of cardiac fibrosis and adverse remodeling is thought to involve the ROS-dependent induction of inflammatory cytokines and matrix metalloproteinases (MMPs), and the activation and migration of cardiac fibroblasts (CF). Here we investigated the role of RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), a unique membrane-anchored MMP regulator, on IL-18-induced CF migration, and the effect of acetylsalicylic acid (ASA) on this response. In a Matrigel invasion assay, IL-18-induced migration of primary mouse CF was dependent on both IKK/NF-κB- and JNK/AP-1-mediated MMP9 induction and Sp1-mediated RECK suppression, mechanisms that required Nox4-dependent H(2)O(2) generation. Notably, forced expression of RECK attenuated IL-18-induced MMP9 activation and CF migration. Further, therapeutic concentrations of ASA inhibited IL-18-induced H(2)O(2) generation, MMP9 activation, RECK suppression, and CF migration. The salicylic acid moiety of ASA similarly attenuated IL-18-induced CF migration. Thus, ASA may exert potential beneficial effect in cardiac fibrosis through multiple protective mechanisms.

Angiotensin II stimulates cardiac fibroblast migration via the differential regulation of matrixins and RECK.

Sustained induction and activation of matrixins (matrix metalloproteinases or MMPs), and the destruction and deposition of extracellular matrix (ECM), are the hallmarks of cardiac fibrosis. The reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a unique membrane-anchored endogenous MMP regulator. We hypothesized that elevated angiotensin II (Ang II), which is associated with fibrosis in the heart, differentially regulates MMPs and RECK both in vivo and in vitro. Continuous infusion of Ang II into male C57Bl/6 mice for 2weeks resulted in cardiac fibrosis, with increased expressions of MMPs 2, 7, 9 and 14, and of collagens Ia1 and IIIa1. The expression of RECK, however, was markedly suppressed. These effects were inhibited by co-treatment with the Ang II type 1 receptor (AT1) antagonist losartan. In vitro, Ang II suppressed RECK expression in adult mouse cardiac fibroblasts (CF) via AT1/Nox4-dependent ERK/Sp1 activation, but induced MMPs 2, 14 and 9 via NF-κB, AP-1 and/or Sp1 activation. Further, while forced expression of RECK inhibits, its knockdown potentiates Ang II-induced CF migration. Notably, RECK overexpression reduced Ang II-induced MMPs 2, 9 and 14 activation, but enhanced collagens Ia1 and IIIa1 expression and soluble collagen release. These results demonstrate for the first time that Ang II suppresses RECK, but induces MMPs both in vivo and in vitro, and RECK overexpression blunts Ang II-induced MMP activation and CF migration in vitro. Strategies that upregulate RECK expression in vivo have the potential to attenuate sustained MMP expression, and blunt fibrosis and adverse remodeling in hypertensive heart diseases.

Interleukin-18 enhances IL-18R/Nox1 binding, and mediates TRAF3IP2-dependent smooth muscle cell migration. Inhibition by simvastatin.

We investigated the role of TRAF3 interacting protein 2 (TRAF3IP2), a redox-sensitive adapter protein and an upstream regulator of IKK and JNK in interleukin (IL)-18 induced smooth muscle cell migration, and the mechanism of its inhibition by simvastatin. The pleiotropic cytokine IL-18 induced human coronary artery SMC migration through the induction of TRAF3IP2. IL-18 induced Nox1-dependent ROS generation, TRAF3IP2 expression, and IKK/NF-κB and JNK/AP-1 activation. IL-18 induced its own expression and that of its receptor subunit IL-18Rα. Using co-IP/IB and GST pull-down assays, we show for the first time that the subunits of the IL-18R heterodimer physically associate with Nox1 under basal conditions, and IL-18 appears to enhance their binding. Importantly, the HMG-coA reductase inhibitor simvastatin attenuated IL-18-induced TRAF3IP2 induction. These inhibitory effects were reversed by mevalonate and geranylgeranylpyrophosphate (GGPP), but not by farnesylpyrophosphate (FPP). Interestingly, simvastatin, GGPP, FPP, or Rac1 inhibition did not modulate ectopically expressed TRAF3IP2. These results demonstrate that the promigratory effects of IL-18 are mediated through TRAF3IP2 in a redox-sensitive manner, and this may involve IL-18R/Nox1 physical association. Further, Simvastatin inhibits inducible, but not ectopically-xpressed TRAF3IP2. Targeting TRAF3IP2 may blunt progression of hyperplastic vascular diseases in vivo.

Advanced oxidation protein products induce cardiomyocyte death via Nox2/Rac1/superoxide-dependent TRAF3IP2/JNK signaling.

Advanced oxidation protein products (AOPPs) are formed during chronic oxidative stress as a result of reactions between plasma proteins and chlorinated oxidants. Their levels are elevated during various cardiovascular diseases. Because elevated AOPPs serve as independent risk factors for ischemic heart disease, and cardiomyocyte death is a hallmark of ischemic heart disease, we hypothesized that AOPPs will induce cardiomyocyte death. AOPP-modified mouse serum albumin (AOPP-MSA) induced significant death of neonatal mouse cardiomyocytes that was attenuated by knockdown of the receptor for advanced glycation end products, but not CD36. Notably, TRAF3-interacting protein 2 (TRAF3IP2; also known as CIKS or Act1) knockdown blunted AOPP-induced apoptosis. AOPP-MSA stimulated Nox2/Rac1-dependent superoxide generation, TRAF3IP2 expression, and TRAF3IP2-dependent JNK activation. The superoxide anion generating xanthine/xanthine oxidase system and hydrogen peroxide both induced TRAF3IP2 expression. Further, AOPP-MSA induced mitochondrial Bax translocation and release of cytochrome c into cytoplasm. Moreover, AOPP-MSA suppressed antiapoptotic Bcl-2 and Bcl-xL expression. These effects were reversed by TRAF3IP2 knockdown or forced expression of mutant JNK. Similar to its effects in neonatal cardiomyocytes, AOPP-MSA induced adult cardiomyocyte death in part via TRAF3IP2. These results demonstrate for the first time that AOPPs induce cardiomyocyte death via Nox2/Rac1/superoxide-dependent TRAF3IP2/JNK activation in vitro and suggest that AOPPs may contribute to myocardial injury in vivo. Thus TRAF3IP2 may represent a potential therapeutic target in ischemic heart disease.

CIKS (Act1 or TRAF3IP2) mediates high glucose-induced endothelial dysfunction.

Hyperglycemia-induced endothelial dysfunction is characterized by enhanced inflammatory cytokine and adhesion molecule expression, and endothelial-monocyte adhesion. The adapter molecule CIKS (connection to IKK and SAPK/JNK; also known as Act1 or TRAF3IP2) is an upstream regulator of NF-κB and AP-1, and plays a role in inflammation and injury. Here we show that high glucose (HG; 25mM vs. 5mM d-glucose)-induced endothelial-monocyte adhesion and inhibition of endothelial cell (EC) migration were both reversed by CIKS knockdown. In EC, HG induced CIKS mRNA and protein expression via DPI-inhibitable Nox4-dependent ROS generation. Further, HG induced CIKS transcription and enhanced CIKS promoter-dependent reporter gene activation via Nox4, ROS, AP-1 and C/EBP. Coimmunoprecipitation and immunoblotting revealed CIKS/IKKß/JNK physical association under basal conditions that was enhanced by HG treatment. Importantly, CIKS knockdown inhibited HG-induced (i) IKKß and JNK phosphorylation, (ii) p65 and c-Jun nuclear translocation, and (iii) NF-κB- and AP-1-dependent proinflammatory cytokine, chemokine, and adhesion molecule expression. Similar to HG, the deleterious metabolic products of chronic hyperglycemia, AGE-HSA, AOPPs-HSA and oxLDL, also induced CIKS-dependent endothelial dysfunction. Notably, aortas from streptozotocin-induced and the autoimmune type 1 diabetic NOD and Akita mice showed enhanced DPI-inhibitable ROS generation and CIKS expression. Since CIKS mediates high glucose-induced NF-κB and AP-1-dependent inflammatory signaling and endothelial dysfunction, targeting CIKS may delay progression of vascular diseases during diabetes mellitus and atherosclerosis.

Tuberin inhibits production of the matrix protein fibronectin in diabetes.

Exposure of proximal tubular epithelial cells to high glucose contributes to the accumulation of tubulointerstitial and matrix proteins in diabetic nephropathy, but how this occurs is not well understood. We investigated the effect of the signaling molecule tuberin, which modulates the mammalian target of rapamycin pathway, on renal hypertrophy and fibronectin expression. We found that the kidney mass was significantly greater in partially tuberin-deficient (TSC2(+/-) ) diabetic rats than wild-type diabetic rats. Furthermore, TSC2(+/-) rats exhibited significant increases in the basal levels of phospho-tuberin and fibronectin expression in the kidney cortex. Increased levels of phosphorylated tuberin associated with an increase in fibronectin expression in both wild-type and TSC2(+/-) diabetic rats. Treatment with insulin abrogated the diabetes-induced increase in fibronectin expression. In vitro, high glucose enhanced fibronectin expression in TSC2(+/-) primary proximal tubular epithelial cells; both inhibition of Akt and inhibition of the mammalian target of rapamycin could prevent this effect of glucose. In addition, forced expression of tuberin in tuberin-null cells abolished the expression of fibronectin protein. Taken together, these data suggest that tuberin plays a central role in the development of renal hypertrophy and in modulating the production of the matrix protein fibronectin in diabetes.

CIKS (Act1 or TRAF3IP2) mediates Angiotensin-II-induced Interleukin-18 expression, and Nox2-dependent cardiomyocyte hypertrophy.

Chronic elevation of angiotensin (Ang)-II can lead to myocardial inflammation, hypertrophy and cardiac failure. The adaptor molecule CIKS (connection to IKK and SAPK/JNK) activates the IκB kinase/nuclear factor (NF)-κB and JNK/activator protein (AP)-1 pathways in autoimmune and inflammatory diseases. Since Ang-II is a potent activator of NF-κB and AP-1, we investigated whether CIKS is critical in Ang-II-mediated cardiac hypertrophy. Here we report that Ang-II induced CIKS mRNA and protein expression, CIKS binding to IKK and JNK perhaps functioning as a scaffold protein, CIKS-dependent IKK/NF-κB and JNK/AP-1 activation, p65 and c-Jun phosphorylation and nuclear translocation, NF-κB- and AP-1-dependent IL-18 and MMP-9 induction, and hypertrophy of adult cardiomyocytes isolated from WT, but not CIKS-null mice. These results were recapitulated in WT-cardiomyocytes following CIKS knockdown. Infusion of Ang-II for 7days induced cardiac hypertrophy, increased collagen content, and upregulated CIKS mRNA and protein expression in WT mice, whereas cardiac hypertrophy and collagen deposition were markedly attenuated in the CIKS-null mice, despite a similar increase in systolic blood pressure and DPI-inhibitable superoxide generation in both types of animals. Further, Ang-II-induced IKK/p65 and JNK/c-Jun phosphorylation, NF-κB and AP-1 activation, and IL-18 and MMP-9 expression were also markedly attenuated in CIKS-null mice. These results demonstrate that CIKS is critical in Ang-II-induced cardiomyocyte hypertrophy and fibrosis, and that CIKS is an important intermediate in Ang-II-induced redox signaling. CIKS is a potential therapeutic target in cardiac hypertrophy, fibrosis, and congestive heart failure.

Angiotensin II enhances AT1-Nox1 binding and stimulates arterial smooth muscle cell migration and proliferation through AT1, Nox1, and interleukin-18.

The redox-sensitive transcription factors NF-κB and activator protein-1 (AP-1) are critical mediators of ANG II signaling. The promitogenic and promigratory factor interleukin (IL)-18 is an NF-κB- and AP-1-responsive gene. Therefore, we investigated whether ANG II-mediated smooth muscle cell (SMC) migration and proliferation involve IL-18. ANG II induced rat carotid artery SMC migration and proliferation and IL-18 and metalloproteinase (MMP)-9 expression via ANG II type 1 (AT(1)) receptor. ANG II-induced superoxide generation, NF-κB and AP-1 activation, and IL-18 and MMP-9 induction were all markedly attenuated by losartan, diphenyleneiodonium chloride (DPI), and Nox1 knockdown. Similar to ANG II, addition of IL-18 also induced superoxide generation, activated NF-κB and AP-1, and stimulated SMC migration and proliferation, in part via Nox1, and both ANG II and IL-18 induced NOX1 transcription in an AP-1-dependent manner. AT(1) physically associates with Nox1 in SMC, and ANG II enhanced this binding. Interestingly, exogenous IL-18 neither induced AT(1) binding to Nox1 nor enhanced the ANG II-induced increase in AT(1)/Nox1 binding. Importantly, IL-18 knockdown, or pretreatment with IL-18 neutralizing antibodies, or IL-18 binding protein, all attenuated the migratory and mitogenic effects of ANG II. Continuous infusion of ANG II for 7 days induced carotid artery hyperplasia in rats via AT(1) and was associated with increased AT(1)/Nox1 binding (despite lower AT(1) levels); increased DPI-inhibitable superoxide production; increased phospho-IKKß, JNK, p65, and c-Jun; and induction of IL-18 and MMP-9 in endothelium-denuded carotid arteries. These results indicate that IL-18 amplifies the ANG II-induced, redox-dependent inflammatory cascades by activating similar promitogenic and promigratory signal transduction pathways. The ANG II/Nox1/IL-18 pathway may be critical in hyperplastic vascular diseases, including atherosclerosis and restenosis.

Interleukin-17A stimulates cardiac fibroblast proliferation and migration via negative regulation of the dual-specificity phosphatase MKP-1/DUSP-1.

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.

Angiotensin-II type 1 receptor and NOX2 mediate TCF/LEF and CREB dependent WISP1 induction and cardiomyocyte hypertrophy.

Angiotensin-II (Ang-II) plays a key role in myocardial hypertrophy, remodeling and failure. We investigated whether Ang-II-induced cardiomyocyte hypertrophy is dependent on WNT1 inducible signaling pathway protein 1 (WISP1), a pro growth factor. Ang-II induced hypertrophy and WISP1 expression in neonatal rat cardiomyocytes (NRCM), effects that were significantly inhibited by pre-treatment with the AT1 antagonist losartan and by WISP1 knockdown. Further, Ang-II induced WISP1 was superoxide-dependent, and inhibited by DPI, an inhibitor of NADPH oxidases, and by knockdown of NOX2. AT1 was physically associated with NOX2 both in vitro and in vivo, and Ang-II increased this interaction in vivo. Ang-II induced WISP1 expression via superoxide/Akt/GSK3ß/ß-catenin/TCF/LEF and by Akt-dependent CREB activation. Further, Ang-II also activated CREB via superoxide-mediated p38 MAPK and ERK activation. Continuous infusion of Ang-II for 7days induced myocardial hypertrophy in rats, and was associated with increased Akt, p-Akt, p-p38 MAPK, p-ERK1/2, and WISP1 expression. These results demonstrate that Ang-II induced cardiomyocyte hypertrophy is mediated through AT1, NOX2 and the induction of WISP1, and may involve the direct interaction of AT1 with NOX2. Thus targeting both WISP1 and NOX2 may have a therapeutic potential in improving cardiomyocyte survival and growth following myocardial injury and remodeling. This article is part of a Special Issue entitled 'Possible Editorial'.

Interleukin-18/WNT1-inducible signaling pathway protein-1 signaling mediates human saphenous vein smooth muscle cell proliferation.

We demonstrate for the first time that the pro-inflammatory cytokine interleukin (IL)-18 stimulates rapid and significant proliferation of SMC derived from human saphenous vein (VSMC), but not coronary artery. IL-18 also stimulates VSMC growth. Further investigations revealed that IL-18-induced VSMC proliferation was Wnt inducible secreted protein-1 (WISP1) dependent. In addition to inducing its own expression via phosphatidylinositol 3-kinase/Akt-dependent IKK/NF-κB activation, IL-18 stimulated glycogen synthase kinase 3ß phosphorylation and degradation, ß-catenin nuclear translocation and stabilization, T-cell factor-lymphoid enhancer binding factor (TCF-LEF) activation, and WISP1 induction. Moreover, WISP1 induced its own expression, and that of survivin and multiple matrix metalloproteinases via ß-catenin/TCF-LEF interaction. WISP1 also activated AP-1, but not NF-κB, and induced matrix metalloproteinase (MMP)9 transcription in part via AP-1. Interestingly, WISP1 failed to regulate tissue inhibitors of matrix metalloproteinases (TIMP) expression. These novel findings indicate that IL-18 induces a series of signaling events that result in WISP1-mediated VSMC proliferation, survival and MMP induction that are key components of vein graft stenosis and this may be amplified by IL-18 and WISP1 autoregulation and cross-regulation.

Novel regulation of parkin function through c-Abl-mediated tyrosine phosphorylation: implications for Parkinson's disease.

Mutations in parkin, an E3 ubiquitin ligase, are the most common cause of autosomal-recessive Parkinson's disease (PD). Here, we show that the stress-signaling non-receptor tyrosine kinase c-Abl links parkin to sporadic forms of PD via tyrosine phosphorylation. Under oxidative and dopaminergic stress, c-Abl was activated in cultured neuronal cells and in striatum of adult C57BL/6 mice. Activated c-Abl was found in the striatum of PD patients. Concomitantly, parkin was tyrosine-phosphorylated, causing loss of its ubiquitin ligase and cytoprotective activities, and the accumulation of parkin substrates, AIMP2 (aminoacyl tRNA synthetase complex-interacting multifunctional protein 2) (p38/JTV-1) and FBP-1.STI-571, a selective c-Abl inhibitor, prevented tyrosine phosphorylation of parkin and restored its E3 ligase activity and cytoprotective function both in vitro and in vivo. Our results suggest that tyrosine phosphorylation of parkin by c-Abl is a major post-translational modification that leads to loss of parkin function and disease progression in sporadic PD. Moreover, inhibition of c-Abl offers new therapeutic opportunities for blocking PD progression.

Regulation of PI 3-K, PTEN, p53, and mTOR in Malignant and Benign Tumors Deficient in Tuberin.

The tuberous sclerosis complex (TSC) is caused by mutation in either of 2 tumor suppressor genes, TSC-1 (encodes hamartin) and TSC-2 (encodes tuberin). In humans, deficiency in TSC1/2 is associated with benign tumors in many organs, including renal angiomyolipoma (AML) but rarely renal cell carcinoma (RCC). In contrast, deficiency of TSC function in the Eker rat is associated with RCC. Here, we have investigated the activity of PI 3-K and the expression of PTEN, p53, tuberin, p-mTOR, and p-p70S6K in both Eker rat RCC and human renal AML. Compared to normal tissue, increased PI 3-K activity was detected in RCC of Eker rats but not in human AML tissue. In contrast, PTEN was highly expressed in AML but significantly reduced in the renal tumors of Eker rats. Phosphorylation on Ser(2448) of mTOR and Thr(389) of p70S6K were significantly increased in both RCC and AML compared to matching control tissue. Total tuberin was significantly decreased in AML while completely lost in RCC of Eker rats. Our data also show that while p53 protein expression is lost in rat RCC, it was highly elevated in AML. These novel data provide evidence that loss of TSC-2, PTEN, and p53 as well as activation of PI 3-K and mTOR is associated with kidney cancer in the Eker rat, while sustained expression of TSC-2, PTEN, and p53 may prevent progression of kidney cancer in TSC patients.

EMMPRIN activates multiple transcription factors in cardiomyocytes, and induces interleukin-18 expression via Rac1-dependent PI3K/Akt/IKK/NF-kappaB andMKK7/JNK/AP-1 signaling.

The transmembrane glycoprotein extracellular matrix metalloproteinase inducer (EMMPRIN), and the pleiotropic proinflammatory cytokine interleukin (IL)-18, play critical roles in myocardial remodeling, by inducing matrix degrading metalloproteinases (MMPs). Previously we showed that IL-18 induces EMMPRIN expression in cardiomyocytes via MyD88/IRAK4/TRAF6/JNK-dependent Sp1 activation. Here in reciprocal studies we demonstrate that EMMPRIN is a potent inducer of IL-18 transcription, protein expression and protein secretion in primary mouse cardiomyocytes. We show for the first time that EMMPRIN stimulates the activation of NF-kappaB, AP-1, CREB, and ATF-2 in cardiomyocytes, and induces IL-18 expression via Rac1-dependent PI3K/Akt/IKK/NF-kappaB and MKK7/JNK/AP-1 signaling. Moreover, EMMPRIN induces robust time-dependent induction of various MMP mRNAs. EMMPRIN also induces the mRNA of TIMPs 1 and 3, but in a delayed fashion. These results suggest that IL-18-induced EMMPRIN expression may favor net MMP expression and ECM destruction, and thus identify both as potential therapeutic targets in countering adverse myocardial remodeling.

Regulation of phagocyte NADPH oxidase by hydrogen peroxide through a Ca(2+)/c-Abl signaling pathway.

The importance of H(2)O(2) as a cellular signaling molecule has been demonstrated in a number of cell types and pathways. Here we explore a positive feedback mechanism of H(2)O(2)-mediated regulation of the phagocyte respiratory burst NADPH oxidase (NOX2). H(2)O(2) induced a dose-dependent stimulation of superoxide production in human neutrophils, as well as in K562 leukemia cells overexpressing NOX2 system components. Stimulation was abrogated by the addition of catalase, the extracellular Ca(2+) chelator BAPTA, the T-type Ca(2+) channel inhibitor mibefradil, the PKCdelta inhibitor rottlerin, or the c-Abl nonreceptor tyrosine kinase inhibitor imatinib mesylate or by overexpression of a dominant-negative form of c-Abl. H(2)O(2) induced phosphorylation of tyrosine 311 on PKCdelta and this activating phosphorylation was blocked by treatment with rottlerin, imatinib mesylate, or BAPTA. Rac GTPase activation in response to H(2)O(2) was abrogated by BAPTA, imatinib mesylate, or rottlerin. In conclusion, H(2)O(2) stimulates NOX2-mediated superoxide generation in neutrophils and K562/NOX2 cells via a signaling pathway involving Ca(2+) influx and c-Abl tyrosine kinase acting upstream of PKCdelta. This positive feedback regulatory pathway has important implications for amplifying the innate immune response and contributing to oxidative stress in inflammatory disorders.

WNT1-inducible signaling pathway protein-1 activates diverse cell survival pathways and blocks doxorubicin-induced cardiomyocyte death.

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.