Yoji Kyotani

S-nitrosylation regulates mitochondrial quality control via activation of parkin

Parkin, a ubiquitin E3 ligase of the ring between ring fingers family, has been implicated in mitochondrial quality control. A series of recent reports have suggested that the recruitment of parkin is regulated by phosphorylation. However, the molecular mechanism that activates parkin to induce mitochondrial degradation is not well understood. Here, and in contrast to previous reports that S-nitrosylation of parkin is exclusively inhibitory, we identify a previously unrecognized site of S-nitrosylation in parkin (Cys323) that induces mitochondrial degradation. We demonstrate that endogenous S-nitrosylation of parkin is in fact responsible for activation of its E3 ligase activity to induce aggregation and degradation. We further demonstrate that mitochondrial uncoupling agents result in denitrosylation of parkin, and that prevention of denitrosylation restores mitochondrial degradation. Our data indicates that NO both positive effects on mitochondrial quality control, and suggest that targeted S-nitrosylation could provide a novel therapeutic strategy against Parkinsons disease.

Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation.

Exendin-4 Prevents Vascular Smooth Muscle Cell Proliferation and Migration by Angiotensin II via the Inhibition of ERK1/2 and JNK Signaling Pathways.

Angiotensin II (Ang II) is a main pathophysiological culprit peptide for hypertension and atherosclerosis by causing vascular smooth muscle cell (VSMC) proliferation and migration. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, is currently used for the treatment of type-2 diabetes, and is believed to have beneficial effects for cardiovascular diseases. However, the vascular protective mechanisms of GLP-1 receptor agonists remain largely unexplained. In the present study, we examined the effect of exendin-4 on Ang II-induced proliferation and migration of cultured rat aortic smooth muscle cells (RASMC). The major findings of the present study are as follows: (1) Ang II caused a phenotypic switch of RASMC from contractile type to synthetic proliferative type cells; (2) Ang II caused concentration-dependent RASMC proliferation, which was significantly inhibited by the pretreatment with exendin-4; (3) Ang II caused concentration-dependent RASMC migration, which was effectively inhibited by the pretreatment with exendin-4; (4) exendin-4 inhibited Ang II-induced phosphorylation of ERK1/2 and JNK in a pre-incubation time-dependent manner; and (5) U0126 (an ERK1/2 kinase inhibitor) and SP600125 (a JNK inhibitor) also inhibited both RASMC proliferation and migration induced by Ang II stimulation. These results suggest that exendin-4 prevented Ang II-induced VSMC proliferation and migration through the inhibition of ERK1/2 and JNK phosphorylation caused by Ang II stimulation. This indicates that GLP-1 receptor agonists should be considered for use in the treatment of cardiovascular diseases in addition to their current use in the treatment of diabetes mellitus.

Neuroprotective effects of pramipexole against tunicamycin-induced cell death in PC12 cells.

1 Pramipexole (PPX), a dopamine D2 and D3 receptor agonist, exerts neuroprotective effects via both dopamine receptor‐mediated and non‐dopaminergic mechanisms. In the present study, we demonstrate that PPX reduces the toxicity of tunicamycin, a typical endoplasmic reticulum (ER) stressor, in PC12h cells, a subline of PC12 cells. 2 The PC12h cells were treated with 300 μmol / L PPX in the presence of 0.5 μmol / L tunicamycin for 24 h. The neuroprotective effects of PPX against tunicamycin‐induced cell death were evaluated using 3‐(4,5‐dimethyl‐2 thiazoyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays, Hoechst 33258 staining and western blot analysis. 3 Tunicamycin (0.2, 0.3 and 0.5 μg / mL) dose‐dependently decreased MTT activity and increased LDH release from PC12h cells. Treatment with 300 μmol / L PPX rescued the tunicamycin‐induced decrease in cell viability. 4 Spiperone (10 μmol / L), a dopamine D2 and D4 receptor antagonist, had no effect on PPX neuroprotection against tunicamycin in these cells. Marker proteins of ER stress and apoptosis are known to be upregulated by tunicamycin, but we detected no significant effects of PPX on these factors. 5 In conclusion, we speculate that a combination of several mechanisms may be involved in PPX‐induced neuroprotection.

Azelnidipine Inhibits Cultured Rat Aortic Smooth Muscle Cell Death Induced by Cyclic Mechanical Stretch

Acute aortic dissection is the most common life-threatening vascular disease, with sudden onset of severe pain and a high fatality rate. Clarifying the detailed mechanism for aortic dissection is of great significance for establishing effective pharmacotherapy for this high mortality disease. In the present study, we evaluated the influence of biomechanical stretch, which mimics an acute rise in blood pressure using an experimental apparatus of stretching loads in vitro, on rat aortic smooth muscle cell (RASMC) death. Then, we examined the effects of azelnidipine and mitogen-activated protein kinase inhibitors on mechanical stretch-induced RASMC death. The major findings of the present study are as follows: (1) cyclic mechanical stretch on RASMC caused cell death in a time-dependent manner up to 4 h; (2) cyclic mechanical stretch on RASMC induced c-Jun N-terminal kinase (JNK) and p38 activation with peaks at 10 min; (3) azelnidipine inhibited RASMC death in a concentration-dependent manner as well as inhibited JNK and p38 activation by mechanical stretch; and (4) SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) protected against stretch-induced RASMC death; (5) Antioxidants, diphenylene iodonium and tempol failed to inhibit stretch-induced RASMC death. On the basis of the above findings, we propose a possible mechanism where an acute rise in blood pressure increases biomechanical stress on the arterial walls, which induces RASMC death, and thus, may lead to aortic dissection. Azelnidipine may be used as a pharmacotherapeutic agent for prevention of aortic dissection independent of its blood pressure lowering effect.

Olmesartan inhibits cultured rat aortic smooth muscle cell death induced by cyclic mechanical stretch through the inhibition of the c-Jun N-terminal kinase and p38 signaling pathways.

Acute aortic dissection (AAD) is a life-threating disease; however, there is almost no effective pharmacotherapy for it. An increase in c-Jun N-terminal kinase (JNK) phosphorylation and smooth muscle cell (SMC) apoptosis is observed tissues in patients with AAD. Therefore, we hypothesized that an acute rise in blood pressure leads to SMC death through phosphorylation of JNK or p38, which may cause AAD. We investigated the influence of cyclic mechanical stretch, which mimics an acute increase in blood pressure, on cultured rat aortic SMCs (RASMCs) and examined the changes in JNK and p38 phosphorylation. Further, we investigated the effect of olmesartan, an angiotensin II receptor blocker, on stretch-induced RASMC death. We found that mechanical stretch-induced RASMC death in a time-dependent manner, which correlated with the phosphorylation of JNK and p38. Olmesartan inhibited RASMC death and the phosphorylation of JNK and p38. JNK and p38 inhibitors reversed stretch-induced RASMC death. These results suggest that acute mechanical stretch causes JNK and p38 phosphorylation, which may result in SMC death leading to aortic dissection. Olmesartan may be used for pharmacotherapy to prevent aortic dissection, independent of its blood pressure-lowering effect, through its inhibition of JNK and p38 phosphorylation.

Prevention of the wortmannin-induced inhibition of phosphoinositide 3-kinase by sulfhydryl reducing agents

The effects of the sulfhydryl reducing agents 2-mercaptoethanol and dithiothreitol on wortmannin-induced inhibition of phosphoinositide 3-kinase (PI3K) were studied in order to examine whether the sulfhydryl reducing agents directly affect the wortmannin inhibition of PI3K. These reducing agents are commonly used to stabilize enzyme structures by maintaining protein sulfhydryl groups in the reduced state. Preincubation of wortmannin with millimolar levels of 2-mercaptoethanol, a sulfhydryl derivative of ethanol, markedly prevented subsequent wortmannin-induced inhibition of PI3K. In contrast, ethanol, 2-mercaptoethanol lacking sulfhydryl group, and 2-(methylthio)ethanol, a methyl derivative of the sulfhydryl group of 2-mercaptoethanol, had little effect on the wortmannin-induced inhibition of PI3K, which suggests that the prevention of wortmannin-induced inhibition by 2-mercaptoethanol occurs through the sulfhydryl group of this agent. Moreover, dithiothreitol, a second sulfhydryl reducing agent, also markedly prevented wortmannin-induced inhibition of PI3K. These results indicate that the wortmannin-induced inhibition of PI3K is markedly prevented by millimolar concentrations of sulfhydryl reducing agents such as 2-mercaptoethanol and dithiothreitol in the medium, presumably by the binding of wortmannin to the agents.

Vascular smooth muscle cell response to cyclic mechanical stretch and aortic dissection

Acute aortic dissection is the most common life-threatening vascular disease, with sudden onset of severe pain and a high fatality rate. The pulsatile nature of blood flow exposes vascular smooth muscle cells (VSMCs) in the vessel wall to cyclic mechanical stretch (CMS), which evokes VSMC death, phenotypic switching, and migration, leading to aortic dissection. We have revealed that CMS of rat aortic smooth muscle cells (RASMCs) caused JNK- and p38-dependent cell death and that a calcium channel blocker, azelnidipine and an angiotensin II receptor antagonist, olmesartan decreased the phosphorylation of JNK and p38 and, subsequently, decreased cell death by CMS. JNK and p38 inhibitors also inhibited CMS-induced cell death. In addition, we showed that the expression of Cxcl1 and Cx3cl1 chemokines was induced by CMS in a JNK-dependent manner. Expression of Cxcl1 was also induced in VSMCs by hypertension produced by abdominal aortic constriction in mouse. In addition, antagonists against the receptors for CXCL1 and CX3CL1 increased cell death, indicating that CXCL1 and CX3CL1 protect RASMCs from CMS-induced cell death. We also revealed that STAT1 is activated in RASMCs subjected to CMS. Taken together, these results indicate that CMS of VSMCs induces inflammation-related gene expression, including that of CXCL1 and CX3CL1, and activates JNK and p38 MAP kinases, which may play important roles in the stress response against CMS caused by acute rise in blood pressure.

Intermittent hypoxia‐induced epiregulin expression by IL‐6 production in human coronary artery smooth muscle cells

Patients with obstructive sleep apnea (OSA) experience repetitive episodes of desaturation and resaturation of blood oxygen (known as intermittent hypoxia or IH), during sleep. We showed previously that IH induced excessive proliferation of rat vascular smooth muscle cells through upregulation of members of the epidermal growth factor family, especially epiregulin (EREG), and the erbB2 receptor. In this study, we exposed human coronary artery smooth muscle cells to IH and found that IH significantly increased the expression of EREG. IH increased the production of interleukin‐6 (IL‐6) in smooth muscle cells, and the addition of IL‐6 induced EREG expression. Small interfering RNA for IL‐6 or IL‐6 receptor attenuated the IH‐induced increase in EREG. IL‐6 may play a pivotal role in EREG upregulation by IH and consequently OSA‐related atherosclerosis.