Takafumi Senokuchi

Statins Activate Peroxisome Proliferator-Activated Receptor γ Through Extracellular Signal-Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase–Dependent Cyclooxygenase-2 Expression in Macrophages

Both statins and peroxisome proliferator-activated receptor (PPAR)γ ligands have been reported to protect against the progression of atherosclerosis. In the present study, we investigated the effects of statins on PPARγ activation in macrophages. Statins increased PPARγ activity, which was inhibited by mevalonate, farnesylpyrophosphate, or geranylgeranylpyrophosphate. Furthermore, a farnesyl transferase inhibitor and a geranylgeranyl transferase inhibitor mimicked the effects of statins. Statins inhibited the membrane translocations of Ras, RhoA, Rac, and Cdc42, and overexpression of dominant-negative mutants of RhoA (DN-RhoA) and Cdc42 (DN-Cdc42), but not of Ras or Rac, increased PPARγ activity. Statins induced extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) activation. However, DN-RhoA and DN-Cdc42 activated p38 MAPK, but not ERK1/2. ERK1/2- or p38 MAPK–specific inhibitors abrogated statin-induced PPARγ activation. Statins induced cyclooxygenase (COX)-2 expression and increased intracellular 15-deoxy-&Dgr;12,14-prostaglandin J2 (15d-PGJ2) levels through ERK1/2- and p38 MAPK–dependent pathways, and inhibitors or small interfering RNA of COX-2 inhibited statin-induced PPARγ activation. Statins also activate PPARα via COX-2–dependent increases in 15d-PGJ2 levels. We further demonstrated that statins inhibited lipopolysaccharide-induced tumor necrosis factor α or monocyte chemoattractant protein-1 mRNA expression, and these effects by statins were abrogated by the PPARγ antagonist T0070907 or by small interfering RNA of PPARγ or PPARα. Statins also induced ATP-binding cassette protein A1 or CD36 mRNA expression, and these effects were suppressed by small interfering RNAs of PPARγ or PPARα. In conclusion, statins induce COX-2–dependent increase in 15d-PGJ2 level through a RhoA- and Cdc42-dependent p38 MAPK pathway and a RhoA- and Cdc42-independent ERK1/2 pathway, thereby activating PPARγ. Statins also activate PPARα via COX-2–dependent pathway. These effects of statins may explain their antiatherogenic actions.

Macrophage deficiency of p38α MAPK promotes apoptosis and plaque necrosis in advanced atherosclerotic lesions in mice

ER stress occurs in macrophage-rich areas of advanced atherosclerotic lesions and contributes to macrophage apoptosis and subsequent plaque necrosis. Therefore, signaling pathways that alter ER stress-induced apoptosis may affect advanced atherosclerosis. Here we placed Apoe-/- mice deficient in macrophage p38alpha MAPK on a Western diet and found that they had a marked increase in macrophage apoptosis and plaque necrosis. The macrophage p38alpha-deficient lesions also exhibited a significant reduction in collagen content and a marked thinning of the fibrous cap, which suggests that plaque progression was advanced in these mice. Consistent with our in vivo data, we found that ER stress-induced apoptosis in cultured primary mouse macrophages was markedly accelerated under conditions of p38 inhibition. Pharmacological inhibition or genetic ablation of p38 suppressed activation of Akt in cultured macrophages and in atherosclerotic lesions. In addition, inhibition of Akt enhanced ER stress-induced macrophage apoptosis, and expression of a constitutively active myristoylated Akt blocked the enhancement of ER stress-induced apoptosis that occurred with p38 inhibition in cultured cells. Our results demonstrate that p38alpha MAPK may play a critical role in suppressing ER stress-induced macrophage apoptosis in vitro and advanced lesional macrophage apoptosis in vivo.

Mitochondrial Dysfunction and Increased Reactive Oxygen Species Impair Insulin Secretion in Sphingomyelin Synthase 1-null Mice

Sphingomyelin synthase 1 (SMS1) catalyzes the conversion of ceramide to sphingomyelin. Here, we generated and analyzed SMS1-null mice. SMS1-null mice exhibited moderate neonatal lethality, reduced body weight, and loss of fat tissues mass, suggesting that they might have metabolic abnormality. Indeed, analysis on glucose metabolism revealed that they showed severe deficiencies in insulin secretion. Isolated mutant islets exhibited severely impaired ability to release insulin, dependent on glucose stimuli. Further analysis indicated that mitochondria in mutant islet cells cannot up-regulate ATP production in response to glucose. We also observed additional mitochondrial abnormalities, such as hyperpolarized membrane potential and increased levels of reactive oxygen species (ROS) in mutant islets. Finally, when SMS1-null mice were treated with the anti-oxidant N-acetyl cysteine, we observed partial recovery of insulin secretion, indicating that ROS overproduction underlies pancreatic β-cell dysfunction in SMS1-null mice. Altogether, our data suggest that SMS1 is important for controlling ROS generation, and that SMS1 is required for normal mitochondrial function and insulin secretion in pancreatic β-cells.

SIRT7 Controls Hepatic Lipid Metabolism by Regulating the Ubiquitin-Proteasome Pathway

Sirtuins (SIRT1-7) have attracted considerable attention as regulators of metabolism over the past decade. However, the physiological functions and molecular mechanisms of SIRT7 are poorly understood. Here we demonstrate that Sirt7 knockout mice were resistant to high-fat diet-induced fatty liver, obesity, and glucose intolerance, and that hepatic triglyceride accumulation was also attenuated in liver-specific Sirt7 knockout mice. Hepatic SIRT7 positively regulated the protein level of TR4/TAK1, a nuclear receptor involved in lipid metabolism, and as a consequence activated TR4 target genes to increase fatty acid uptake and triglyceride synthesis/storage. Biochemical studies revealed that the DDB1-CUL4-associated factor 1 (DCAF1)/damage-specific DNA binding protein 1 (DDB1)/cullin 4B (CUL4B) E3 ubiquitin ligase complex interacted with TR4, leading to its degradation, while binding of SIRT7 to the DCAF1/DDB1/CUL4B complex inhibited the degradation of TR4. In conclusion, we propose that hepatic SIRT7 controls lipid metabolism in liver by regulating the ubiquitin-proteasome pathway.

Oxidized Low Density Lipoprotein Activates Peroxisome Proliferator-activated Receptor-α (PPARα) and PPARγ through MAPK-dependent COX-2 Expression in Macrophages

It has been reported that oxidized low density lipoprotein (Ox-LDL) can activate both peroxisome proliferator-activated receptor-α (PPARα) and PPARγ. However, the detailed mechanisms of Ox-LDL-induced PPARα and PPARγ activation are not fully understood. In the present study, we investigated the effect of Ox-LDL on PPARα and PPARγ activation in macrophages. Ox-LDL, but not LDL, induced PPARα and PPARγ activation in a dose-dependent manner. Ox-LDL transiently induced cyclooxygenase-2 (COX-2) mRNA and protein expression, and COX-2 specific inhibition by NS-398 or meloxicam or small interference RNA of COX-2 suppressed Ox-LDL-induced PPARα and PPARγ activation. Ox-LDL induced phosphorylation of ERK1/2 and p38 MAPK, and ERK1/2 specific inhibition abrogated Ox-LDL-induced COX-2 expression and PPARα and PPARγ activation, whereas p38 MAPK-specific inhibition had no effect. Ox-LDL decreased the amounts of intracellular long chain fatty acids, such as arachidonic, linoleic, oleic, and docosahexaenoic acids. On the other hand, Ox-LDL increased intracellular 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) level through ERK1/2-dependent overexpression of COX-2. Moreover, 15d-PGJ2 induced both PPARα and PPARγ activation. Furthermore, COX-2 and 15d-PGJ2 expression and PPAR activity were increased in atherosclerotic lesions of apoE-deficient mice. Finally, we investigated the involvement of PPARα and PPARγ on Ox-LDL-induced mRNA expression of ATP-binding cassette transporter A1 and monocyte chemoattractant protein-1. Interestingly, specific inhibition of PPARα and PPARγ suppressed Ox-LDL-induced ATP-binding cassette transporter A1 mRNA expression and enhanced Ox-LDL-induced monocyte chemoattractant protein-1 mRNA expression. In conclusion, Ox-LDL-induced increase in 15d-PGJ2 level through ERK1/2-dependent COX-2 expression is one of the mechanisms of PPARα and PPARγ activation in macrophages. These effects of Ox-LDL may control excess atherosclerotic progression.

Telmisartan Exerts Antiatherosclerotic Effects by Activating Peroxisome Proliferator-Activated Receptor-γ in Macrophages

Objective—Telmisartan, an angiotensin type I receptor blocker (ARB), protects against the progression of atherosclerosis. Here, we investigated the molecular basis of the antiatherosclerotic effects of telmisartan in macrophages and apolipoprotein E–deficient mice. Methods and Results—In macrophages, telmisartan increased peroxisome proliferator-activated receptor-&ggr; (PPAR&ggr;) activity and PPAR ligand-binding activity. In contrast, 3 other ARBs, losartan, valsartan, and olmesartan, did not affect PPAR&ggr; activity. Interestingly, high doses of telmisartan activated PPAR&agr; in macrophages. Telmisartan induced the mRNA expression of CD36 and ATP-binding cassette transporters A1 and G1 (ABCA1/G1), and these effects were abrogated by PPAR&ggr; small interfering RNA. Telmisartan, but not other ARBs, inhibited lipopolysaccharide-induced mRNA expression of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-&agr;, and these effects were abrogated by PPAR&ggr; small interfering RNA. Moreover, telmisartan suppressed oxidized low-density lipoprotein-induced macrophage proliferation through PPAR&ggr; activation. In apolipoprotein E−/− mice, telmisartan increased the mRNA expression of ABCA1 and ABCG1, decreased atherosclerotic lesion size, decreased the number of proliferative macrophages in the lesion, and suppressed MCP-1 and tumor necrosis factor-&agr; mRNA expression in the aorta. Conclusion—Telmisartan induced ABCA1/ABCG1 expression and suppressed MCP-1 expression and macrophage proliferation by activating PPAR&ggr;. These effects may induce antiatherogenic effects in hypertensive patients.

Voltage-gated K+ channel KCNQ1 regulates insulin secretion in MIN6 β-cell line

KCNQ1, located on 11p15.5, encodes a voltage-gated K(+) channel with six transmembrane regions, and loss-of-function mutations in the KCNQ1 gene cause hereditary long QT syndrome. Recent genetic studies have identified that single nucleotide polymorphisms located in intron 15 of the KCNQ1 gene are strongly associated with type 2 diabetes and impaired insulin secretion. In order to understand the role of KCNQ1 in insulin secretion, we introduced KCNQ1 into the MIN6 mouse β-cell line using a retrovirus-mediated gene transfer system. In KCNQ1 transferred MIN6 cells, both the density of the KCNQ1 current and the density of the total K(+) current were significantly increased. In addition, insulin secretion by glucose, pyruvate, or tolbutamide was significantly impaired by KCNQ1-overexpressing MIN6 cells. These results suggest that increased KCNQ1 protein expression limits insulin secretion from pancreatic β-cells by regulating the potassium channel current.

Nifedipine Induces Peroxisome Proliferator-Activated Receptor-γ Activation in Macrophages and Suppresses the Progression of Atherosclerosis in Apolipoprotein E-Deficient Mice

Objective—Nifedipine, an L-type calcium channel blocker, protects against the progression of atherosclerosis. We investigated the molecular basis of the antiatherosclerotic effect of nifedipine in macrophages and apolipoprotein E-deficient mice. Methods and Results—In macrophages, nifedipine increased peroxisome proliferator-activated receptor-&ggr; (PPAR&ggr;) activity without increasing PPAR&ggr;-binding activity. Amlodipine, another L-type calcium channel blocker, and 1,2-bis-(o-aminophenoxy)-ethane-N,N,-N′,N′-tetraacetic acid tetraacetoxy-methyl ester (BAPTA-AM), a calcium chelator, decreased PPAR&ggr; activity, suggesting that nifedipine does not activate PPAR&ggr; via calcium channel blocker activity. Inactivation of extracellular signal-regulated kinase 1/2 suppressed PPAR&ggr;2-Ser112 phosphorylation and induced PPAR&ggr; activation. Nifedipine suppressed extracellular signal-regulated kinase 1/2 activation and PPAR&ggr;2-Ser112 phosphorylation, and mutating PPAR&ggr;2-Ser112 to Ala abrogated nifedipine-mediated PPAR&ggr; activation. These results suggested that nifedipine inhibited extracellular signal-regulated kinase 1/2 activity and PPAR&ggr;2-Ser112 phosphorylation, leading to PPAR&ggr; activation. Nifedipine inhibited lipopolysaccharide-induced monocyte chemoattractant protein-1 expression and induced ATP-binding cassette transporter A1 mRNA expression, and these effects were abrogated by small interfering RNA for PPAR&ggr;. Furthermore, in apolipoprotein E-deficient mice, nifedipine treatment decreased atherosclerotic lesion size, phosphorylation of PPAR&ggr;2-Ser112 and extracellular signal-regulated kinase 1/2, and monocyte chemoattractant protein-1 mRNA expression and increased ATP-binding cassette transporter A1 expression in the aorta. Conclusion—Nifedipine unlike amlodipine inhibits PPAR&ggr;-Ser phosphorylation and activates PPAR&ggr; to suppress monocyte chemoattractant protein-1 expression and induce ATP-binding cassette transporter A1 expression in macrophages. These effects may induce antiatherogenic effects in hypertensive patients.

Sirt7 Contributes to Myocardial Tissue Repair by Maintaining Transforming Growth Factor-β Signaling Pathway.

Background— Sirt7, 1 of the 7 members of the mammalian sirtuin family, promotes oncogenic transformation. Tumor growth and metastasis require fibrotic and angiogenic responses. Here, we investigated the role of Sirt7 in cardiovascular tissue repair process. Methods and Results— In wild-type mice, Sirt7 expression increased in response to acute cardiovascular injury, including myocardial infarction and hind-limb ischemia, particularly at the active wound healing site. Compared with wild-type mice, homozygous Sirt7-deficient (Sirt7−/−) mice showed susceptibility to cardiac rupture after myocardial infarction, delayed blood flow recovery after hind-limb ischemia, and impaired wound healing after skin injury. Histological analysis showed reduced fibrosis, fibroblast differentiation, and inflammatory cell infiltration in the border zone of infarction in Sirt7−/− mice. In vitro, Sirt7−/− mouse–derived or Sirt7 siRNA–treated cardiac fibroblasts showed reduced transforming growth factor-&bgr; signal activation and low expression levels of fibrosis-related genes compared with wild-type mice–derived or control siRNA–treated cells. These changes were accompanied by reduction in transforming growth factor receptor I protein. Loss of Sirt7 activated autophagy in cardiac fibroblasts. Transforming growth factor-&bgr; receptor I downregulation induced by loss of Sirt7 was blocked by autophagy inhibitor, and interaction of Sirt7 with protein interacting with protein kinase-C&agr; was involved in this process. Conclusion— Sirt7 maintains transforming growth factor receptor I by modulating autophagy and is involved in the tissue repair process.

Sirt7 Contributes to Myocardial Tissue Repair by Maintaining TGF-β Signaling Pathway

Background— Sirt7, 1 of the 7 members of the mammalian sirtuin family, promotes oncogenic transformation. Tumor growth and metastasis require fibrotic and angiogenic responses. Here, we investigated the role of Sirt7 in cardiovascular tissue repair process. Methods and Results— In wild-type mice, Sirt7 expression increased in response to acute cardiovascular injury, including myocardial infarction and hind-limb ischemia, particularly at the active wound healing site. Compared with wild-type mice, homozygous Sirt7-deficient (Sirt7−/−) mice showed susceptibility to cardiac rupture after myocardial infarction, delayed blood flow recovery after hind-limb ischemia, and impaired wound healing after skin injury. Histological analysis showed reduced fibrosis, fibroblast differentiation, and inflammatory cell infiltration in the border zone of infarction in Sirt7−/− mice. In vitro, Sirt7−/− mouse–derived or Sirt7 siRNA–treated cardiac fibroblasts showed reduced transforming growth factor-&bgr; signal activation and low expression levels of fibrosis-related genes compared with wild-type mice–derived or control siRNA–treated cells. These changes were accompanied by reduction in transforming growth factor receptor I protein. Loss of Sirt7 activated autophagy in cardiac fibroblasts. Transforming growth factor-&bgr; receptor I downregulation induced by loss of Sirt7 was blocked by autophagy inhibitor, and interaction of Sirt7 with protein interacting with protein kinase-C&agr; was involved in this process. Conclusion— Sirt7 maintains transforming growth factor receptor I by modulating autophagy and is involved in the tissue repair process.