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Pyroptosis is characterized by pore formation in the plasma membrane, cell swelling, and membrane disruption.1,2 These cellular events are also noted in necrosis, but not apoptosis, which is defined as a mode of regulated or programmed necrosis.2 Jorgensen et al3 have defined pyroptosis by the following 4 criteria: (1) programmed by an inflammatory caspase activation, (2) pore formation in the plasma membrane, (3) DNA damage with terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity at a lower intensity than apoptosis, and (4) ADP-ribose polymerase activation after the pyroptosis-mediated DNA damage. The inflammasome is a large complex containing NOD (nucleotide oligomerization domain)-like receptors (NLRs), procaspase-1, and apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD), which regulates both pyroptosis and the maturation and secretion of proinflammatory interleukin (IL)-1β and IL-18.4 A study by Xi et al5 from Wang’s laboratory in this issue of Circulation Research has now indicated a critical role for the caspase-1–inflammasome in regulating hyperhomocysteinemia-induced pyroptosis and apoptosis. In particular, they show that hyperhomocysteinemia-mediated aortic endothelial dysfunction induced by the depletion of cystathionine β-synthase was rescued by the depletion of caspase-1 and NLRP3. This suggests that the inflammasome plays a crucial role in regulating hyperhomocysteinemia-induced endothelial dysfunction. In this editorial, we will briefly review the regulatory mechanism of caspase-1– and 4/5/11–mediated pyroptosis in modulating endothelial cell pyroptosis/apoptosis and consequent endothelial dysfunction. Article, see p 1525 The caspase protease family has central roles in cell death and inflammation. Mammalian caspases can be divided into 3 groups based on their functions.6 Caspases 3, 6, and 7 are executioner caspases with a prodomain in the NH2-terminus that cleaves substrates essential for cellular homeostasis. Caspases 2, 8, 9, and 10 are initiator caspases with 2 DED (death-effector domain; caspases 8 and 10) or 1 …

Rationale: Disturbed flow induces proinflammatory and apoptotic responses in endothelial cells, causing them to become dysfunctional and subsequently proatherogenic. Objective: Although a possible link between SUMOylation of p53 and ERK5 detected during endothelial apoptosis and inflammation has been suggested, the mechanistic insights, especially under the proatherogenic flow condition, remain largely unknown. Methods and Results: SUMOylation of p53 and ERK5 was induced by disturbed flow but not by steady laminar flow. To examine the role of the disturbed flow–induced p53 and ERK5 SUMOylation, we used de-SUMOylation enzyme of sentrin/Small Ubiquitin-like MOdifier (SUMO)-specific protease 2 deficiency ( Senp2 +/− ) mice and observed a significant increase in endothelial apoptosis and adhesion molecule expression both in vitro and in vivo. These increases, however, were significantly inhibited in endothelial cells overexpressing p53 and ERK5 SUMOylation site mutants. Senp2 +/− mice exhibited increased leukocyte rolling along the endothelium, and accelerated formation of atherosclerotic lesions was observed in Senp2 +/− / Ldlr −/− , but not in Senp2 +/+ /Ldlr −/− , mice fed a high-cholesterol diet. Notably, the extent of lesion size in the aortic arch of Senp2 +/− / Ldlr −/− mice was much larger than that in the descending aorta, also suggesting a crucial role of the disturbed flow–induced SUMOylation of proteins, including p53 and ERK5 in atherosclerosis formation. Conclusions: These data show the unique role of sentrin/SUMO-specific protease 2 on endothelial function under disturbed flow and suggest that SUMOylation of p53 and ERK5 by disturbed flow contributes to the atherosclerotic plaque formation. Molecules involved in this newly discovered signaling will be useful targets for controlling endothelial cells dysfunction and consequently atherosclerosis formation.

Cancer treatments in general share various detrimental effects in common, especially upregulation of cardiovascular risk factors. Therefore, the science of onco-cardiology should not be restricted in scope to the side effects of each specific cancer drug. In particular, premature aging induced by cancer treatment may contribute to the chronic health problems of cancer survivors. About 1 660 000 people, including more than 12 000 children below the age of 18 years, are newly diagnosed with a malignancy in the United States every year.1 The American Cancer Society reported that in 2016 there are 15.5 million cancer survivors in the United States (http://www.cancer.org/cancer/news/news/report-number-of-cancer-survivors-continues-to-grow). At present, the 5-year survival rate of patients treated for cancer is 67%. Seventy-five percent of children in whom cancer is diagnosed today will live for at least 10 years; 20% will survive for longer than 35 years.1 Although these numbers are impressive compared with those from decades ago, further improvement of cancer survivors’ life span as well as quality of life and functional status is still necessary. Approximately 75% of cancer survivors have some form of chronic health problem. Cardiovascular diseases (CVDs) are the leading cause of morbidity and mortality in this population, particularly after recurrent or second malignancy. The risk of CVD in cancer survivors is 8× higher than that of the general population. The relative risks of coronary artery disease and heart failure in cancer survivors are 10× and 15× higher, respectively, than their siblings without cancer.1 Cancer treatments, including chemotherapy and radiation, can lead to both short- and long-term cardiovascular complications. Evidence of subclinical cardiac and vascular damage was observed in more than 50% of survivors 5 to 10 years after chemotherapy.1 Onco-cardiology is a medical subspecialty concerned with the diagnosis and treatment of CVDs and organ failure mediated by microcirculatory or macrocirculatory …

Rationale: Mitogen-activated protein kinase (MAPK) pathways provide a critical connection between extrinsic and intrinsic signals to cardiac hypertrophy. Extracellular signal-regulated protein kinase (ERK)5, an atypical MAPK is activated in the heart by pressure overload. However, the role of ERK5 plays in regulating hypertrophic growth and hypertrophy-induced apoptosis is not completely understood. Objective: Herein, we investigate the in vivo role and signaling mechanism whereby ERK5 regulates cardiac hypertrophy and hypertrophy-induced apoptosis. Methods and Results: We generated and examined the phenotypes of mice with cardiomyocyte-specific deletion of the erk5 gene (ERK5 cko ). In response to hypertrophic stress, ERK5 cko mice developed less hypertrophic growth and fibrosis than controls. However, increased apoptosis together with upregulated expression levels of p53 and Bad were observed in the mutant hearts. Consistently, we found that silencing ERK5 expression or specific inhibition of its kinase activity using BIX02189 in neonatal rat cardiomyocytes (NRCMs) reduced myocyte enhancer factor (MEF)2 transcriptional activity and blunted hypertrophic responses. Furthermore, the inhibition of MEF2 activity in NRCMs using a non-DNA binding mutant form of MEF2 was found to attenuate the ERK5-regulated hypertrophic response. Conclusions: These results reveal an important function of ERK5 in cardiac hypertrophic remodeling and cardiomyocyte survival. The role of ERK5 in hypertrophic remodeling is likely to be mediated via the regulation of MEF2 activity.

Unidirectional laminar flow is atheroprotective, in part by inhibiting cytokine-mediated endothelial cell (EC) inflammation and apoptosis. Previously, we showed that flow inhibited TNF-alpha signaling by preventing activation of JNK. Recently, PKCzeta was identified as the PKC isoform most strongly regulated by flow pattern, with increased PKCzeta activity in regions of disturbed flow versus unidirectional flow. Interestingly, PKCzeta is cleaved by caspases after TNF-alpha stimulation to generate a 50-kDa truncated form (CATzeta, catalytic domain of PKCzeta) with a higher kinase activity than the full-length protein. We hypothesized that flow would inhibit TNF-alpha-mediated PKCzeta cleavage and thereby CATzeta formation. We found that PKCzeta activity was required for TNF-alpha-mediated JNK and caspase-3 activation in ECs. PKCzeta was rapidly cleaved to generate CATzeta in cultured bovine and human aortic ECs and in intact rabbit vessels stimulated with TNF-alpha. This truncated form of PKCzeta enhanced JNK and caspase-3 activation. Interestingly, PKCzeta cleavage was prevented by inhibitors of PKCzeta, JNK, and caspase activities, suggesting that these enzymes, via regulating CATzeta formation, modulate caspase-3 activity in ECs. Finally, we found that flow reduced caspase-dependent processing of PKCzeta and caspase-3 activation. These results define a novel role for PKCzeta as a shared signaling mediator for flow and TNF-alpha, and important for flow-mediated inhibition of proinflammatory and apoptotic events in ECs.

Substantial evidence suggests that the progressive loss of cardiomyocytes caused by apoptosis significantly contributes to the development of heart failure. beta-Adrenergic receptor activation and subsequent persistent phosphodiesterase 3A (PDE3A) downregulation and concomitant inducible cAMP early repressor (ICER) upregulation (PDE3A/ICER feedback loop) has been proposed to play a key role in the pathogenesis of cardiomyocyte apoptosis. In contrast, insulin-like growth factor-1 can activate cell survival pathways, providing protection against cell death and restoring muscle function. In this study, we found that insulin-like growth factor-1 activates extracellular signal-regulated kinase 5 (ERK5) and inhibits PDE3A/ICER feedback loop. Insulin-like growth factor-1 normalized isoproterenol-mediated PDE3A downregulation and ICER upregulation via ERK5/MEF2 activation, and also inhibited isoproterenol-induced myocyte apoptosis. To determine the physiological relevance of ERK5 activation in regulating PDE3A/ICER feedback loop, we investigated the PDE3A/ICER expression and cardiomyocyte apoptosis in transgenic mice with cardiac specific expression of a constitutively active form of mitogen-activated protein (MAP)/extracellular signal-regulated protein kinase (ERK) kinase 5alpha (MEK5alpha) (CA-MEK5alpha-Tg). In wild-type mice, pressure overload- or doxorubicin-induced significant reduction of PDE3A expression and subsequent ICER induction. Cardiac specific expression of CA-MEK5alpha rescued pressure overload- or doxorubicin-mediated PDE3A downregulation and ICER upregulation and inhibited myocyte apoptosis as well as subsequent cardiac dysfunction in vivo. These data suggest that preventing the feedback loop of PDE3A/ICER by ERK5 activation could inhibit progression of myocyte apoptosis as well as cardiac dysfunction. These data suggest a new therapeutic paradigm for end stage of heart failure by inhibiting the PDE3A/ICER feedback loop via activating ERK5.

Growing evidence suggests that multiple spatially, temporally, and functionally distinct pools of cyclic nucleotides exist and regulate cardiac performance, from acute myocardial contractility to chronic gene expression and cardiac structural remodeling. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP and cyclic GMP, regulate the amplitude, duration, and compartmentation of cyclic nucleotide-mediated signaling. In particular, PDE3 enzymes play a major role in regulating cAMP metabolism in the cardiovascular system. PDE3 inhibitors, by raising cAMP content, have acute inotropic and vasodilatory effects in treating congestive heart failure but have increased mortality in long-term therapy. PDE3A expression is downregulated in human and animal failing hearts. In vitro, inhibition of PDE3A function is associated with myocyte apoptosis through sustained induction of a transcriptional repressor ICER (inducible cAMP early repressor) and thereby inhibition of antiapoptotic molecule Bcl-2 expression. Sustained induction of ICER may also cause the change of other protein expression implicated in human and animal failing hearts. These data suggest that the downregulation of PDE3A observed in failing hearts may play a causative role in the progression of heart failure, in part, by inducing ICER and promoting cardiac myocyte dysfunction. Hence, strategies that maintain PDE3A function may represent an attractive approach to circumvent myocyte apoptosis and cardiac dysfunction.

Hypoxia–reoxygenation can induce inflammation by activating nuclear factor (NF)-κB. In endothelial cells, this process is critical for the pathogenesis of many chronic inflammatory conditions, such as atherosclerosis and autoimmune disease. Recent publication from Evans’s laboratory shows the critical role of deubiquitinating enzyme Cezanne, regulating its extent of NF-κB activation and expression of inflammatory genes.1 In particular, they showed that the inhibition of polyubiquitination of TNF receptor associated factor (TRAF) 6 is a specific anti-inflammatory mechanism by Cezanne. In this editorial, we briefly review the TRAF6-mediated NF-κB signaling and other posttranslational modifications that play a key role in modulating endothelial cell inflammation. Article, see p 1583 NF-κB transcription factor complexes consist of a heterodimer of p65 (RelA) and p50 or p52.2 In most nonstimulated cells, p65-containing NF-κB complexes are kept in an inactive cytoplasmic form, bound to one family of inhibitor proteins, the inhibitory κBs (IκBs). Two IκB kinases, IKKα and IKKβ, target phosphorylation of IκB after hypoxia–reoxygenation, cytokine, or ultraviolet stress stimulation. Phosphorylation of IκBs promotes their ubiquitination and degradation by the proteasome, which releases the p65 complex, allowing it to translocate to the nucleus.3 An ubiquitin E2 conjugating enzyme of the ubiquitin-conjugating enzyme (Ubc)4/5 family and the SCF-βTrCP E3 ligase (Skp1-Cul1-F-box ligase containing the F-box beta protein βTrCP) execute ubiquitination of IκB. Once IκB is phosphorylated, p-transducin repeat containing protein 1 (βTrCP1) and βTrCP2 associate with phosphorylated IκB.4,5 The polyubiquitinated IκB is selectively degraded by the 26S proteasome, and then mature p52 and p65 subunits translocate into the nucleus to increase NF-κB activity.4 It is now well established that NF-κB activation is a key event in inflammation-based cardiovascular pathogenesis, including atherosclerotic plaque formation, diabetes mellitus–mediated endothelial inflammation, and ischemia/reperfusion injury.6 The regulatory subunit NF-κB essential modulator (NEMO/IKKγ) of the cytoplasmic IκB …

Abstract —Activation of the Na + /H + exchanger isoform-1 (NHE-1) by angiotensin II is an early signal transduction event that may regulate vascular smooth muscle cell (VSMC) growth and migration. Many signal transduction events stimulated by angiotensin II are mediated by the mitogen-activated protein (MAP) kinases. To define their roles in angiotensin II–mediated NHE-1 activity, VSMCs were treated with angiotensin II and the activities of p38, c-Jun N -terminal kinase (JNK), and extracellular signal–regulated kinases 1 and 2 (ERK1/2) were measured. Angiotensin II rapidly (peak, 5 minutes) activated p38 and ERK1/2, whereas JNK was activated more slowly (peak, 30 minutes). Because angiotensin II stimulated Na + /H + exchange within 5 minutes, the effects of p38 and ERK1/2 antagonists on Na + /H + exchange were studied. The MEK-1 inhibitor PD98059 decreased ERK1/2 activity and Na + /H + exchange stimulated by angiotensin II. In contrast, the specific p38 antagonist SKF-86002 increased Na + /H + exchange. Two mechanisms were identified that may mediate the effects of p38 and SKF-86002 on angiotensin II–stimulated Na + /H + exchange. First, angiotensin II activation of ERK1/2 was increased 1.5- to 2.5-fold (depending on assay technique) in the presence of SKF-86002, demonstrating that p38 negatively regulates ERK1/2. Second, the ability of angiotensin II–stimulated MAP kinases to phosphorylate a glutathione S -transferase fusion protein containing amino acids 625 to 747 of NHE-1 in vitro was analyzed. The relative activities of endogenous immunoprecipitated p38, ERK1/2, and JNK were 1.0, 2.0, and 0.05 versus control, respectively suggesting that p38 and ERK1/2, but not JNK, may phosphorylate NHE-1 in VSMC. These data indicate important roles for p38 and ERK1/2 in angiotensin II–mediated regulation of the Na + /H + exchanger in VSMC.

Abstract Angiotensin II is a multifunctional agonist for vascular smooth muscle cells (VSMCs), stimulating increases in signal events, cell growth, and ion flux. We previously defined protein kinase C (PKC)–dependent and –independent mechanisms by which angiotensin II stimulated activity of the Na + -H + exchanger isoform-1 (NHE-1) and identified a 90-kD kinase that exhibited increased activity in VSMCs isolated from genetically hypertensive rats. To determine whether this 90-kD kinase was p90 rsk (RSK), VSMCs were stimulated with 100 nmol/L angiotensin II, and NHE-1 kinase activity was measured by phosphorylation of recombinant NHE-1 (a glutathione S-transferase fusion protein containing amino acids 516 to 815 of the cytoplasmic carboxyl tail) in vitro. NHE-1 kinase (90 kD) activity was markedly decreased by immunodepletion of RSK. Characterization of RSK activation by angiotensin II revealed many similarities to the 90-kD NHE-1 kinase, including time course and NHE-1 domain phosphorylation, as well as regulation by extracellular signal–regulated kinases (ERK1/2), intracellular Ca 2+ , and PKC. Specifically, angiotensin II stimulated a rapid and transient (peak, 5 minutes) increase in RSK activity. Analysis of several NHE-1 fusion proteins revealed that only proteins containing amino acids 670 to 714 were phosphorylated by RSK. Inhibiting ERK1/2 (30 μmol/L PD098059 for 30 minutes) or chelating intracellular Ca 2+ prevented RSK activation. In contrast, downregulating PKC (1 μmol/L phorbol dibutyrate for 24 hours) had little effect. These findings establish RSK as a putative NHE-1 kinase and potential mediator of increased Na + -H + exchange in hypertension.

Rationale: The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, are principal therapeutic agents for the treatment of hypercholesteremia. Statins, however, appear to also exert cholesterol-independent pleiotropic effects, such as improvement of endothelial (EC) function, stabilization of fibrous plaques, and decrease vascular inflammation. It is now well established that statins are beneficial in the prevention of cardiovascular disease and also widely used for suppressing cardiac allograft rejection. Previously, we have reported anti-inflammatory effect of ERK5 kinase in ECs. Methods and Results: In this study, we screened small molecules that activate ERK5 using high throughput screening, and identified statins as strong activators of the transcriptional activity of ERK5. In particular, we have found that pitavastatin increases ERK5 transcriptional activity, KLF2 promoter activity, and eNOS mRNA expression in ECs. These effects are abolished by the depletion of ERK5, but not its direct upstream kinase, MEK5. In addition, pitavastatin directly and dose-dependently activates ERK5 kinase activity in an in vitro kinase reaction assay, suggesting that ERK5 is a direct target of this statin. To examine the functional role of EC ERK5 activation by the statin in vivo, we utilized inducible endothelial ERK5 knock out (EC-ERK5-KO) mice and evaluated the effect of pitavastatin on EC function and acute allograft rejection. Depletion of ERK5 in ECs resulted in significant EC inflammation and dysfunction in vivo. Although pitavastatin reduced leukocyte rolling and vascular reactivity in mesebteruc microvessels of diabetic mice and prolonged allograft survival in a full allomismatch combination model, these protective effects were lost in EC-ERK5-KO mice. Conclusion: These data suggest the crucial role of ERK5 in pleiotropic effects of statins on EC dysfunction and allograft rejection in vivo.

The formation of new blood vessels after ischemia and vascular injury is a critical process for tissue repair. A complex interplay of stress-activated signal transduction pathways is activated in response to ischemia and injury to initiate the repair process. Among these pathways, the hypoxia-inducible transcription factor-1 (HIF-1α) plays an essential role by transactivating the VEGF gene and activating a pattern of gene expression associated with mobilization, migration, and recruitment of endothelial cells and smooth muscle cells to form new blood vessels.1–3⇓⇓ The report by Jung et al4 in this issue of Circulation Research investigates the signaling pathway responsible for hypoxia-induced HIF-1α protein stabilization and concludes that a mechanism involving Src, Shc, Ras, and Raf-1 is critical in endothelial cells. In this editorial, we discuss 5 key issues raised by the work of Jung et al.4 (1) Signal transduction pathways that regulate protein stabilization are important pathophysiological mechanisms that may provide novel therapeutic approaches. (2) Shc isoforms represent a family of stress-regulated proteins that act as sensors for redox, oxygen species (ROS), and other genotoxic stresses. (3) Shc proteins mediate signal transduction in localized subcellular compartments, which are likely essential for hypoxia signaling pathways. (4) Cell migration in response to hypoxia likely involves multiple mechanisms including Shc. (5) Dominant-negative (DN) proteins represent an important technical advance to define signal transduction pathways, but appropriate controls must be used to avoid nonspecific effects. Shc is an adaptor protein that possesses no intrinsic enzymatic activity, yet alterations in its structure have profound functional consequences as shown by the fact that Shc was first identified as a protooncogene.5 The mammalian SHC locus encodes three different isoforms with molecular masses of 52 kDa (p52Shc), 46 kDa (p46Shc), and 66 kDa (p66Shc). These isoforms share a Src-homology2 (SH2) domain, a collagen-homology …

Rationale: Cardiac dysfunction is accelerated in DM patients after MI. Previously, we reported the critical role of ERK5 and CHIP association on CHIP Ub E3 ligase activity, which inhibits inducible cAMP early repressor (ICER)-mediated apoptosis and left ventricle (LV) dysfunction after MI in DM (DM + MI). Yet the regulatory mechanism of ERK5-CHIP has not been established. Objective: Since we found that p90RSK activation was increased in DM heart, we investigated whether p90RSK activation may inhibit ERK5-mediated CHIP activation, and subsequent ICER induction and apoptosis. Methods and Results: The inhibition of p90RSK activation prevented the reduction of ERK5-CHIP binding, CHIP activity, as well as ICER induction and cardiac apoptosis both in vitro after angiotensin II (ang II) stimulation and in vivo after DM + MI. p90RSK and CHIP share a same binding site with ERK5 C-terminal domain (aa571–807), and overexpression of both p90RSK and ERK5 (aa571–807) fragment, but not kinase dead mutant of p90RSK, inhibited ERK5-CHIP association, suggesting the critical role of p90RSK activation on ERK5-CHIP interaction, and competitive nature of p90RSK and CHIP against ERK5 association. Furthermore. LC-MS/MS analysis identified ERK5-S496 as being directly phosphorylated by p90RSK, and ERK5 S496A mutant significantly impaired ang II-mediated inhibition of CHIP Ub ligase activity, suggesting the critical role of Ser-496 phoaphorylation of ERK5 on CHIP activity. Therefore, p90RSK activation is critical for both p90RSK-ERK5 association as well as ERK5-Ser496 phosphorylation, and following disruption of ERK5-CHIP interaction and subsequent inhibition of CHIP Ub ligase activity. The reduction of CHIP Ub ligase activity and LV dysfunction were accelerated both in cardio-specific ERK5 knock out and wild type p90RSK transgenic mice (WT-p90RSK-Tg). Furthermore, double transgenic mice of WT-p90RSK and constitutively active form of MEK5α (specific ERK5 activator) inhibited single WT-p90RSK-Tg-medaited reduction of CHIP Ub ligase activity, LV dysfunction, and improved mortality after MI. Conclusions: These data strongly suggested that p90RSK activation accelerated cardiac dysfunction and apoptosis after DM + MI via inhibiting ERK5-CHIP module.

There is growing evidence that multiple mitogen-activated protein (MAP) kinases are activated during ischemia and/or reperfusion and may contribute to the structural and functional changes after myocardial ischemia. A new study by Yue et al1 in this issue of Circulation Research attempts to address the role of MAP kinases in ischemic myocardium and suggests that ERK1/ERK2 is part of a “survival” pathway whereas p38 and JNK mediate a “death” pathway in the ischemic myocardium. Myocyte loss during the acute stage of myocardial infarction involves both apoptotic and necrotic cell death.2 3 4 Therefore, it is reasonable to think that the balance of cell survival and death is critical during the pathological evolution of postischemic cardiac dysfunction. Recently, many scientists seeking to elucidate the intricate relationship between signal transduction and this balance of survival and death in ischemic myocardium have focused on MAP kinases. MAP kinases are highly conserved serine/threonine kinases that are activated in response to a wide variety of stimuli including growth factors, G protein–coupled receptors, and environmental stresses.5 Consequently, they play a role in numerous cell functions including growth and proliferation. The MAP kinases themselves require dual phosphorylation on a Thr-X-Tyr motif to become active. Three major MAP kinase cascades have been extensively studied in the heart: extracellular signal–regulated kinases (ERK1 and ERK2), c-Jun N-terminal kinases (JNK1 and JNK2), and p38 kinases, of which the p38α and p38β isoforms are found in the heart.5 Recently, a fourth MAP …

Bcr is a serine/threonine kinase activated by platelet-derived growth factor that is highly expressed in the neointima after vascular injury. Here, we demonstrate that Bcr is an important mediator of angiotensin (Ang) II and platelet-derived growth factor-mediated inflammatory responses in vascular smooth muscle cells (VSMCs). Among transcription factors that might regulate Ang II-mediated inflammatory responses we found that ligand-mediated peroxisome proliferator-activated receptor (PPAR)gamma transcriptional activity was significantly decreased by Ang II. Ang II increased Bcr expression and kinase activity. Overexpression of Bcr significantly inhibited PPARgamma activity. In contrast, knockdown of Bcr using Bcr small interfering RNA and a dominant-negative form of Bcr (DN-Bcr) reversed Ang II-mediated inhibition of PPARgamma activity significantly, suggesting the critical role of Bcr in Ang II-mediated inhibition of PPARgamma activity. Point-mutation and in vitro kinase analyses showed that PPARgamma was phosphorylated by Bcr at serine 82. Overexpression of wild-type Bcr kinase did not inhibit ligand-mediated PPARgamma1 S82A mutant transcriptional activity, indicating that Bcr regulates PPARgamma activity via S82 phosphorylation. DN-Bcr and Bcr small interfering RNA inhibited Ang II-mediated nuclear factor kappaB activation in VSMCs. DN-PPARgamma reversed DN-Bcr-mediated inhibition of nuclear factor kappaB activation, suggesting that PPARgamma is downstream from Bcr. Intimal proliferation in low-flow carotid arteries was decreased in Bcr knockout mice compared with wild-type mice, suggesting the critical role of Bcr kinase in VSMC proliferation in vivo, at least in part, via regulating PPARgamma/nuclear factor kappaB transcriptional activity.

Shear stress–induced extracellular signal-regulated kinase (ERK)5 activation and the consequent regulation of Kruppel-like factor 2 and endothelial nitric oxide synthase expression represents one of the antiinflammatory and vascular tone regulatory mechanisms maintaining normal endothelial function. Endothelial dysfunction is a major initiator of atherosclerosis, a vascular pathology often associated with diabetes. Small ubiquitin-like modifier (SUMO) covalently attaches to certain residues of specific target transcription factors and could inhibit its activity. We investigated whether H 2 O 2 and AGE (advanced glycation end products), 2 well-known mediators of diabetes, negatively regulated ERK5 transcriptional activity and laminar flow–induced endothelial nitric oxide synthase expression through ERK5 SUMOylation. H 2 O 2 and AGE induced endogenous ERK5 SUMOylation. In addition, ERK5 SUMOylation was increased in the aortas from diabetic mice. ERK5 transcriptional activity, but not kinase activity, was inhibited by expression of Ubc9 (SUMO E2 conjugase) or PIAS1 (E3 ligase), suggesting the involvement of ERK5 SUMOylation on its transcriptional activity. Point-mutation analyses showed that ERK5 is covalently modified by SUMO at 2 conserved sites, Lys6 and Lys22, and that the SUMOylation defective mutant of ERK5, dominant negative form of Ubc9 (DN-Ubc9), and small interfering RNA PIAS1 reversed H 2 O 2 and AGE–mediated reduction of shear stress–mediated ERK5/myocyte enhancer factor 2 transcriptional activity, as well as promoter activity of Kruppel-like factor 2. Finally, PIAS1 knockdown reversed the inhibitory effect of H 2 O 2 in shear stress–induced Kruppel-like factor 2 and endothelial nitric oxide synthase expression. These data clearly defined SUMOylation-dependent ERK5 transcriptional repression independent of kinase activity and suggested this process as among the molecular mechanisms of diabetes-mediated endothelial dysfunction.

Big MAP kinase 1 (BMK1 or ERK5) is a key mediator of endothelial cell (EC) function as shown by impaired embryonic angiogenesis and vascular collapse in BMK1 knockout mice. Hypoxia inducible factor 1α (HIF1α), a potent mediator of angiogenesis, is positively regulated by the MAP kinases, ERK1/2. Because BMK1 deficiency is associated with impaired angiogenesis we hypothesized that BMK1 might regulate HIF1α. To test this hypothesis, bovine lung microvascular ECs (BLMECs) were transfected with HIF1α and BMK1 cDNAs, and stimulated by hypoxia. HIF1α activity was measured by a reporter gene assay in which luciferase expression was driven by HIF1α activation. Hypoxia (1% O 2 , 24 hours) stimulated HIF1α activity by 5.1±0.6 fold. In the presence of dominant negative (DN)-BMK1, which inhibited BMK1 activity, hypoxia induced HIF1α activity was enhanced significantly to 6.4±0.4 fold. BMK1 activation by constitutively active (CA)-MEK5 inhibited HIF1α activity by 46±4%, suggesting BMK1 functions as a negative regulator of HIF1α activation. Activation of BMK1 reduced HIF1α protein levels. Ubiquitination inhibitors (30 μmol/L ALLN, 2 μmol/L lactacystin, or 100 nmol/L MG132) reduced the BMK1-mediated effect on HIF1α expression by >80%, suggesting that BMK1 stimulated HIF1α proteolysis. The negative effect of BMK1 on HIF1α was functionally important because transfection with CA-MEK5 significantly decreased EC migration by 68±10%, and inhibited angiogenesis (in vitro Matrigel assay) by 76±7%. In summary, BMK1 is a novel negative regulator of HIF1α and angiogenesis by increasing HIF1α ubiquitination and inhibiting HIF1α activity in endothelial cells.

Atherosclerosis involves cellular immune responses and altered vascular smooth muscle cell (VSMC) function. Nitric oxide (NO)/cGMP is uniquely capable of inhibiting key processes in atherosclerosis. In this study, we determined the effects of NO/cGMP and their molecular mechanisms in the regulation of NF-κB–dependent gene expression in VSMCs. We found that cGMP-elevating agents such as the NO donor S -nitroso- N -acetylpenicillamine (SNAP) and C-type natriuretic peptide (CNP), reduced TNF-α–induced NF-κB–dependent reporter gene expression in rat aortic VSMCs in a cGMP-dependent manner. The effects of SNAP and CNP on NF-κB are mediated by cAMP-dependent protein kinase (PKA) but not cGMP-dependent protein kinase (PKG) based on the findings that the selective PKA inhibitor, PKI, abolished the effects of SNAP and CNP on NF-κB, whereas the PKG inhibitor Rp-8-Br-PET-cGMP had no effect. Inhibition of cGMP-inhibited cAMP-hydrolyzing phosphodiesterase 3 (PDE3) blocked SNAP- and CNP-elicited effects on NF-κB–dependent transcription. Furthermore, cGMP analogues such as 8-pCPT-cGMP, which selectively activates PKG but does not inhibit PDE3, had no effect on NF-κB–mediated transcription. Activation of PKA by SNAP or cAMP-elevating agents not only inhibited TNF-α–induced NF-κB–dependent reporter gene expression but also reduced endogenous NF-κB–dependent adhesion molecule and chemokine expression. These results suggest that SNAP and CNP exert inhibitory effects on NF-κB–dependent transcription by activation of PKA via cGMP-dependent inhibition of PDE3 activity. Therefore, PDE3 is a novel mediator of inflammation in VSMCs.

Insulin-like growth factor (IGF)-1 and the type I IGF-1 receptor are important regulators of vascular function that may contribute to cardiovascular disease. We hypothesized that IGF-1 causes endothelial cell dysfunction and expression of neutrophil and monocyte adhesion molecules by enhancing pro-inflammatory cytokine signal transduction. Long-term IGF-1 treatment of endothelial cells potentiated c-Jun and nuclear factor NF-kappaB activation by tumor necrosis factor (TNF)-alpha and enhanced TNF-alpha-mediated adhesion molecule expression. In response to IGF-1 treatment, the expression of kinases in the c-Jun/c-Jun NH(2)-terminal kinase signaling pathway (MEKK1, MEK4, and JNK1/2) was unchanged, but expressions of insulin receptor substrate-1 and Grb2-associated binder-1 (Gab1) were significantly decreased. Because Gab1 is involved in both c-Jun and NF-kappaB activation by TNF-alpha, we focused on Gab1-dependent signaling. Gab1 inhibited c-Jun and NF-kappaB transcriptional activation by TNF-alpha. Interestingly, Gab1 inhibited c-Jun transcriptional activity induced by MEKK3 but not MEKK1 and MEK4. Gab1 associated with MEKK3, and a catalytically inactive form of MEKK3 inhibited TNF-alpha-induced c-Jun and NF-kappaB transcriptional activation, suggesting a critical role for Gab1 and MEKK3 in TNF-alpha signaling. These data demonstrate that Gab1 and MEKK3 play important roles in endothelial cell inflammation via regulating the activation of c-Jun and NF-kappaB. Furthermore, the IGF-1-mediated downregulation of Gab1 expression represents a novel mechanism to promote vascular inflammation and atherosclerosis.

Abstract —Reactive oxygen species (ROS) activate members of the Src kinase and mitogen-activated protein kinase superfamily, including big mitogen-activated protein kinase 1 (BMK1) and extracellular signal-regulated kinases (ERK1/2). A potentially important downstream effector of ERK1/2 is p90 ribosomal S6 kinase (p90RSK), which plays an important role in cell growth through the activation of several transcription factors, as well as the Na + /H + exchanger. Previously, we showed that Src regulates BMK1 via a redox-sensitive signaling pathway. Because ROS are generated during ischemia and reperfusion after ischemia, we assessed the effects of these stimuli (H 2 O 2 , ischemia, and reperfusion) in the activation of ERK1/2, p90RSK, Src, and BMK1 in perfused guinea pig hearts. H 2 O 2 (100 μmol/L) significantly activated all kinases. Ischemia alone stimulated p90RSK, Src, and BMK1 but not ERK1/2. These results suggest that p90RSK activation through ischemia occurs via a pathway other than ERK1/2. A role of Src in ischemia-mediated BMK1 activation was demonstrated through inhibition with the Src inhibitor 4-amino-5-(4-chlorophenyl)-7-( t -butyl)pyrazolo[3,4- d ]pyrimidine. Reperfusion after ischemia stimulated both p90RSK and ERK1/2. In contrast, although ROS increase during reperfusion after ischemia, the activities of both BMK1 and its upstream regulator, Src, were markedly attenuated by reperfusion after ischemia. The activation of C-terminal Src kinase during ischemia but not during reperfusion suggests that the attenuation of Src and BMK1 activity by reperfusion was not regulated by C-terminal Src kinase activity. The antioxidant N -2-mercaptopropionylglycine completely inhibited ERK1/2 and p90RSK activation by reperfusion but only partially inhibited ischemia-induced Src and BMK1 activation. The present study is the first to show the coregulation of Src and BMK1 by reperfusion after ischemia, which we propose to occur via a novel, ROS-independent pathway.