CaMKII activates ASK1 and NF-kappaB to induce cardiomyocyte hypertrophy

Ca2+/calmodulin-dependent protein kinase (CaMK) is an important downstream target of Ca2+ in the hypertrophic signaling pathways. We previously showed that the activation of apoptosis signal-regulating kinase 1 (ASK1) or NF-kappaB is sufficient for cardiomyocyte hypertrophy. Infection of isolated neonatal cardiomyocytes with an adenoviral vector expressing CaMKIIdelta3 (AdCaMKIIdelta3) induced the activation of ASK1, while KN93, an inhibitor of CaMKII, inhibited phenylephrine-induced ASK1 activation. Overexpression of CaMKIIdelta3 induced characteristic features of in vitro cardiomyocyte hypertrophy. Infection of cardiomyocytes with an adenoviral vector expressing a dominant negative mutant of ASK1 (AdASK(KM)) inhibited the CaMKIIdelta3-induced hypertrophic responses. Overexpression of CaMKIIdelta3 increased the kappaB-dependent promoter/luciferase activity and induced IkappaBalpha degradation. Coinfection with AdCaMKIIdelta3 and AdASK(KM), and pre-incubation with KN93 attenuated CaMKIIdelta3- and phenylephrine-induced NF-kappaB activation, respectively. Expression of a degradation resistant mutant of IkappaBalpha inhibited CaMKIIdelta3-induced hypertrophic responses. These results indicate that CaMKIIdelta3 induces cardiomyocyte hypertrophy mediated through ASK1-NF-kappaB signal transduction pathway.     

A role for focal adhesion kinase in phenylephrine-induced hypertrophy of rat ventricular cardiomyocytes

A variety of agonists including phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Here we report that signals provided by extracellular matrix proteins (ECM) augment the PE-induced hypertrophic response of cardiomyocytes and provide evidence that ECM-dependent signaling is mediated in part by the protein tyrosine kinase, focal adhesion kinase (FAK). Addition of PE to cultured neonatal cardiomyocytes stimulated sarcomeric organization, increased cell size, and induced atrial natriuretic factor in cardiomyocytes plated on the ECM protein laminin or fibronectin. In contrast, cardiomyocytes plated on the non-adhesive substrate gelatin exhibited a reduced capacity to undergo these PE-stimulated hypertrophic changes. In cardiomyocytes cultured on ECM, PE stimulated a rapid increase in tyrosine phosphorylation of focal adhesion proteins including FAK, paxillin, and p130 Crk-associated substrate and subsequent formation of peripheral focal complexes. Inhibition of the PE-induced hypertrophic response by genistein and herbimycin-A indicated a requirement for protein tyrosine kinases in PE signaling. To determine whether activation of FAK is required for PE-induced hypertrophy, a dominant-interfering mutant form of FAK, termed FRNK (FAK-related non-kinase), was ectopically expressed in cardiomyocytes using a replication-defective adenovirus expression system. FRNK expression attenuated PE-stimulated hypertrophy as assessed by cell size, sarcomeric organization, and induction of atrial natriuretic factor. These data indicate that the signal transduction pathways leading to cardiomyocyte hypertrophy are strongly influenced by and/or dependent upon an integrin-mediated signaling process requiring FAK.     

Novel peroxisome proliferator-activated receptor (PPAR) gamma and PPARdelta ligands produce distinct biological effects

The peroxisome proliferator-activated receptors (PPARs) include three receptor subtypes encoded by separate genes: PPARalpha, PPARdelta, and PPARgamma. PPARgamma has been implicated as a mediator of adipocyte differentiation and the mechanism by which thiazolidinedione drugs exert in vivo insulin sensitization. Here we characterized novel, non-thiazolidinedione agonists for PPARgamma and PPARdelta that were identified by radioligand binding assays. In transient transactivation assays these ligands were agonists of the receptors to which they bind. Protease protection studies showed that ligand binding produced specific alterations in receptor conformation. Both PPARgamma and PPARdelta directly interacted with a nuclear receptor co-activator (CREB-binding protein) in an agonist-dependent manner. Only the PPARgamma agonists were able to promote differentiation of 3T3-L1 preadipocytes. In diabetic db/db mice all PPARgamma agonists were orally active insulin-sensitizing agents producing reductions of elevated plasma glucose and triglyceride concentrations. In contrast, selective in vivo activation of PPARdelta did not significantly affect these parameters. In vivo PPARalpha activation with WY-14653 resulted in reductions in elevated triglyceride levels with minimal effect on hyperglycemia. We conclude that: 1) synthetic non-thiazolidinediones can serve as ligands of PPARgamma and PPARdelta; 2) ligand-dependent activation of PPARdelta involves an apparent conformational change and association of the receptor ligand binding domain with CREB-binding protein; 3) PPARgamma activation (but not PPARdelta or PPARalpha activation) is sufficient to potentiate preadipocyte differentiation; 4) non-thiazolidinedione PPARgamma agonists improve hyperglycemia and hypertriglyceridemia in vivo; 5) although PPARalpha activation is sufficient to affect triglyceride metabolism, PPARdelta activation does not appear to modulate glucose or triglyceride levels.     

Viral induction of inflammatory cytokines in human epithelial cells follows a p38 mitogen-activated protein kinase-dependent but NF-kappa B-independent pathway

The initial step in an immune response toward a viral infection is the induction of inflammatory cytokines. This innate immune response is mediated by expression of a variety of cytokines exemplified by TNF-alpha and IL-1beta. A key signal for the recognition of intracellular viral infections is the presence of dsRNA. Viral infections and dsRNA treatment can activate several signaling pathways including the protein kinase R pathway, mitogen-activated protein kinase (MAPK) pathways, and NF-kappaB, which are important in the expression of inflammatory cytokines. We previously reported that activation of protein kinase R was required for dsRNA induction of TNF-alpha, but not for IL-1beta. In this study, we report that activation of the p38 MAPK pathway by respiratory viral infections is necessary for induction of inflammatory cytokines in human bronchial epithelial cells. Inhibition of p38 MAPK by two different pharmacological inhibitors showed that expression of both TNF-alpha and IL-1beta required activation of this signaling pathway. Interestingly, inhibition of NF-kappaB did not significantly reduce viral induction of either cytokine. Our data show that, during the initial infections of epithelial cells with respiratory viruses, activation of the p38 MAPK pathway is associated with induction of inflammation, and NF-kappaB activation may be less important than previously suggested.     

Allicin protects against cardiac hypertrophy and fibrosis via attenuating reactive oxygen species-dependent signaling pathways

Increased oxidative stress has been associated with the pathogenesis of chronic cardiac hypertrophy and heart failure. Since allicin suppresses oxidative stress in vitro and in vivo, we hypothesized that allicin would inhibit cardiac hypertrophy through blocking oxidative stress-dependent signaling. We examined this hypothesis using primary cultured cardiac myocytes and fibroblasts and one well-established animal model of cardiac hypertrophy. Our results showed that allicin markedly inhibited hypertrophic responses induced by Ang II or pressure overload. The increased reactive oxygen species (ROS) generation and NADPH oxidase activity were significantly suppressed by allicin. Our further investigation revealed this inhibitory effect on cardiac hypertrophy was mediated by blocking the activation of ROS-dependent ERK1/2, JNK1/2 and AKT signaling pathways. Additional experiments demonstrated allicin abrogated inflammation and fibrosis by blocking the activation of nuclear factor-κB and Smad 2/3 signaling, respectively. The combination of these effects resulted in preserved cardiac function in response to cardiac stimuli. Consequently, these findings indicated that allicin protected cardiac function and prevented the development of cardiac hypertrophy through ROS-dependent mechanism involving multiple intracellular signaling.     

Propofol depresses angiotensin II-induced cardiomyocyte hypertrophy in vitro

Cardiomyocyte hypertrophy is formed in response to pressure or volume overload, injury, or neurohormonal activation. The most important vascular hormone that contributes to the development of hypertrophy is angiotensin II (Ang II). Accumulating studies have suggested that reactive oxygen species (ROS) may play an important role in cardiac hypertrophy. Propofol is a general anesthetic that possesses antioxidant action. We therefore examined whether propofol inhibited Ang II-induced cardiomyocyte hypertrophy. Our results showed that both ROS formation and hypertrophic responses induced by Ang II in cardiomyocytes were partially blocked by propofol. Further studies showed that propofol inhibited the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen-activated protein kinase/ERK kinase 1/2 (MEK1/2) induced by Ang II via a decrease in ROS production. In addition, propofol also markedly attenuated Ang II-stimulated nuclear factor-kappaB (NF-kappaB) activation via a decrease in ROS production. In conclusion, propofol prevents cardiomyocyte hypertrophy by interfering with the generation of ROS and involves the inhibition of the MEK/ERK signaling transduction pathway and NF-kappaB activation.     

Human cytomegalovirus attenuates interleukin-1beta and tumor necrosis factor alpha proinflammatory signaling by inhibition of NF-kappaB activation

Viral infection is associated with a vigorous inflammatory response characterized by cellular infiltration and release of the proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). In the present study, we identified a novel function of human cytomegalovirus (HCMV) that results in inhibition of IL-1 and TNF-alpha signaling pathways. The effect on these pathways was limited to cells infected with the virus, occurred at late times of infection, and was independent of cell type or virus strain. IL-1 and TNF-alpha signaling pathways converge at a point upstream of NF-kappaB activation and involve phosphorylation and degradation of the NF-kappaB inhibitory molecule IkappaBalpha. The HCMV inhibition of IL-1 and TNF-alpha pathways corresponded to a suppression of NF-kappaB activation. Analysis of IkappaBalpha phosphorylation and degradation suggested that HCMV induced two independent blocks in NF-kappaB activation, which occurred upstream from the point of convergence of the IL-1 and TNF-alpha pathways. We believe that the ability of HCMV to inhibit these two major proinflammatory pathways reveals a critical aspect of HCMV biology, with possible importance for immune evasion, as well as establishment of infection in cell types persistently infected by this virus.     

Eicosapentaenoic acid prevents endothelin-1-induced cardiomyocyte hypertrophy in vitro through the suppression of TGF-beta 1 and phosphorylated JNK

The cardiovascular benefit of fish oil in humans and experimental animals has been reported. Endothelin (ET)-1 is a well-known cardiac hypertrophic factor. However, although many studies link a fish oil extract, eicosapentaenoic acid (EPA), to cardiac protection, the effects of EPA on cardiac hypertrophy and underlying mechanism(s) are unclear. The present study investigated whether EPA prevents ET-1-induced cardiomyocyte hypertrophy; the potential pathways likely to underlie such an effect were also investigated. Cardiomyocytes were isolated from neonatal rat heart, cultured for 3 days, and then treated for 24 h with vehicle only (control), treated with 0.1 nM ET-1 only, or pretreated with 10 microM EPA and then treated with 0.1 nM ET-1. The cells were harvested, and changes in cell surface area, protein synthesis, expression of a cytoskeletal (alpha-actinin) protein, and cell signaling were analyzed. ET-1 induced a 97% increase in cardiomyocyte surface area, a 72% increase in protein synthesis rate, and an increase in expression of alpha-actinin and signaling molecule [transforming growth factor-beta 1 (TGF-beta 1), c-Jun NH2-terminal kinase (JNK), and c-Jun]. Development of these ET-1-induced cellular changes was attenuated by EPA. Moreover, the hypertrophied cardiomyocytes showed a 1.5- and a 1.7-fold increase in mRNA expression of atrial and brain natriuretic peptides, the classical molecular markers of cardiac hypertrophy, respectively; these changes were also suppressed by EPA. Here we show that ET-1 induces cardiomyocyte hypertrophy and expression of hypertrophic markers, possibly mediated by JNK and TGF-beta 1 signaling pathways. These ET-1-induced effects were blocked by EPA, a major fish oil ingredient, suggesting that fish oil may have beneficial protective effects on cardiac hypertrophy.     

Silibinin attenuates cardiac hypertrophy and fibrosis through blocking EGFR‐dependent signaling

Cardiac hypertrophy is a major determinant of heart failure. The epidermal growth factor receptor (EGFR) plays an important role in cardiac hypertrophy. Since silibinin suppresses EGFR in vitro and in vivo, we hypothesized that silibinin would attenuate cardiac hypertrophy through disrupting EGFR signaling. In this study, we examined this hypothesis using neonatal cardiac myocytes and fibroblasts induced by angiotensin II (Ang II) and animal model by aortic banding (AB) mice. Our data revealed that silibinin obviously blocked cardiac hypertrophic responses induced by pressure overload. Meanwhile, silibinin markedly reduced the increased generation of EGFR. Moreover, these beneficial effects were associated with attenuation of the EGFR‐dependent ERK1/2, PI3K/Akt signaling cascade. We further demonstrated silibinin decreased inflammation and fibrosis by blocking the activation of NF‐κB and TGF‐β1/Smad signaling pathways in vitro and in vivo. Our results indicate that silibinin has the potential to protect against cardiac hypertrophy, inflammation, and fibrosis through blocking EGFR activity and EGFR‐dependent different intracellular signaling pathways. J. Cell. Biochem. 110: 1111–1122, 2010. Published 2010 Wiley‐Liss, Inc.     

Peroxisome proliferator-activated receptors and skin development

PPARs are nuclear hormone receptors. PPAR subtypes (alpha, gamma, delta, the latter a xPPARbeta homologue) were initially investigated in skin because of their known role in regulating lipid metabolism. Studies adding specific PPAR ligand activators to cultured skin or skin cells are compatible with the concepts that PPARalpha activation mediates early lipogenic steps common to the function of both skin epidermal cells (keratinocytes) and sebaceous cells (sebocytes), PPARgamma activation plays a unique role in stimulating sebocyte lipogenesis, and PPARdelta activation may contribute to lipid biosynthesis in both sebocytes and keratinocytes under certain circumstances. Epidermal keratinocytes appear to express small amounts of PPARalpha and PPARdelta mRNA and a trace of PPARgamma mRNA which is up-regulated with differentiation. Sebocytes express all subtypes; PPARgamma gene expression excedes that in epidermis. The emerging data on PPAR protein expression suggests that epidermis normally expresses predominantly PPARalpha, while sebocytes express more PPARgamma than PPARalpha. These expression patterns may change during hyperplasia, differentiation and inflammation. Gene disruption studies in mice are compatible with a contribution of PPARalpha to skin barrier function, suggest that PPARgamma is necessary for sebocyte differentiation, and indicate that PPARdelta can ameliorate inflammatory responses in skin. PPARs appear to play a role in keratinocyte synthesis of the lipids that they export to the intercellular space to form the skin permeability barrier. They also appear to be important for sebocyte formation of the intracellular fused lipid droplets that constitute the holocrine secretion of the sebaceous gland. In addition, they may play roles in keratinocyte growth and differentiation and the inhibition of skin inflammation by diverse mechanisms not necessarily related to fat metabolism.     

MLP参与心肌肥大发生过程与Calcineurin/NFAT信号通路有关

心肌肥大是心肌细胞面对多种病理刺激时的共同反应,以心肌细胞体积增大和胚胎期基因的重新表达为标志.心肌发育调控基因肌肉LIM蛋白(muscle LIM protein,MLP)的表达异常与心肌肥大有关.为研究MLP参与心肌肥大发生的分子机制,采用去氧肾上腺素（phenylephrine, PE）刺激大鼠原代培养心肌细胞,建立心肌细胞肥大模型,采用RNAi技术敲减MLP的表达,分析MLP与肥大信号通路钙调神经磷酸酶（calcineurin）/活化T细胞核因子（nuclear factor of activated T-cells, NFAT）的关系.结果显示, 原代培养的心肌细胞经一定浓度的PE刺激后细胞表面积增加,肥大标志蛋白ANP、BNP表达增高,并伴有MLP表达上调. RNAi方法敲减MLP的表达则明显抑制PE诱导的心肌细胞表面积增加和BNP表达增高,并且直接 影响NFAT的转录激活活性,提示MLP与心肌肥大的发生密切相关,并且可能是通过calcineurin/NFAT信号通路而参与心肌肥大的发生.     

Free cholesterol-loaded macrophages are an abundant source of tumor necrosis factor-alpha and interleukin-6: model of NF-kappaB- and map kinase-dependent inflammation in advanced atherosclerosis

Two key features of atherosclerotic plaques that precipitate acute atherothrombotic vascular occlusion ("vulnerable plaques") are abundant inflammatory mediators and macrophages with excess unesterified, or "free," cholesterol (FC). Herein we show that FC accumulation in macrophages leads to the induction and secretion of two inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). The increases in TNF-alpha and IL-6 mRNA and protein were mediated by FC-induced activation of the IkappaB kinase/NF-kappaB pathway as well as activation of MKK3/p38, Erk1/2, and JNK1/2 mitogen-activated protein kinases (MAPK). Activation of IkappaB kinase and JNK1/2 was needed for the induction of both cytokines. However, MKK3/p38 signaling was specifically involved in TNF-alpha induction, and Erk1/2 signaling was required for IL-6. Most interestingly, activation of all of the signaling pathways and induction of both cytokines required cholesterol trafficking to the endoplasmic reticulum (ER). The CHOP branch of the unfolded protein response, an ER stress pathway, was required for Erk1/2 activation and IL-6 induction. In contrast, one or more other ER-related pathways were responsible for activation of p38, JNK1/2, and IkappaB kinase/NF-kappaB and for the induction of TNF-alpha. These data suggest a novel scenario in which cytokines are induced in macrophages by endogenous cellular events triggered by excess ER cholesterol rather than by exogenous immune cell mediators. Moreover, this model may help explain the relationship between FC accumulation and inflammation in vulnerable plaques.