Roscovitine inhibits ERK1/2 activation induced by angiotensin II in vascular smooth muscle cells

Roscovitine is a potent CDK inhibitor often used as a biological tool in cell-cycle studies, but its working mechanism and real targets in vascular smooth muscle cells (VSMCs) remain unclear. In this study, we observed that ERK1/2 phosphorylation induced by Ang II was abrogated by pretreating VSMCs with roscovitine for 15h. Pretreating VSMCs with roscovitine also inhibited Ang II-induced c-Jun expression and phosphorylation. We further demonstrated that roscovitine could suppress the DNA binding activity of c-Jun and activation of angiotensinogen promoter by Ang II. These results suggest that roscovitine represses Ang II-induced angiotensinogen expression by inhibiting activation of ERK1/2 and c-Jun.     

Regulation of vascular smooth muscle cell bioenergetic function by protein glutathiolation

Protein thiolation by glutathione is a reversible and regulated post-translational modification that is increased in response to oxidants and nitric oxide. Because many mitochondrial enzymes contain critical thiol residues, it has been hypothesized that thiolation reactions regulate cell metabolism and survival. However, it has been difficult to differentiate the biological effects due to protein thiolation from other oxidative protein modifications. In this study, we used diamide to titrate protein glutathiolation and examined its impact on glycolysis, mitochondrial function, and cell death in rat aortic smooth muscle cells. Treatment of cells with diamide increased protein glutathiolation in a concentration-dependent manner and had comparably little effect on protein-protein disulfide formation. Diamide increased mitochondrial proton leak and decreased ATP-linked mitochondrial oxygen consumption and cellular bioenergetic reserve capacity. Concentrations of diamide above 200 μM promoted acute bioenergetic failure and caused cell death, whereas lower concentrations of diamide led to a prolonged increase in glycolytic flux and were not associated with loss of cell viability. Depletion of glutathione using buthionine sulfoximine had no effect on basal protein thiolation or cellular bioenergetics but decreased diamide-induced protein glutathiolation and sensitized the cells to bioenergetic dysfunction and death. The effects of diamide on cell metabolism and viability were fully reversible upon addition of dithiothreitol. These data suggest that protein thiolation modulates key metabolic processes in both the mitochondria and cytosol.     

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.     

Angiotensin II induces tyrosine nitration and activation of ERK1/2 in vascular smooth muscle cells

Angiotensin II (Ang II) induces a prominent and sustained nitration and activation of ERK1/2 in rat vascular smooth muscle cells, both mediated via AT1 receptor. Nitration and activation was also shown for recombinant non-activated extracellular signal-regulated kinase (ERK) and MEK. Nitration and phosphorylation of ERK1/2 by Ang II was significantly inhibited by NAD(P)H inhibitors and scavengers of oxygen and nitrogen reactive species and completely blocked by a selective inducible nitric-oxide synthase inhibitor. MEK inhibitor U0126 did not affect ERK nitration but completely blocked activation. These data indicate that Ang II nitrates and activates ERK1/2 via a reactive species-sensitive pathway.     

Alpha-lipoic acid regulates the autophagy of vascular smooth muscle cells in diabetes by elevating hydrogen sulfide level

Dysfunctional vascular smooth muscle (VSM) plays a vital role in the process of atherosclerosis in patients with type 2 diabetes mellitus (T2DM). Alpha-lipoic acid (ALA) can prevent the altered VSM induced by diabetes. However, the precise mechanism underlying the beneficial effect of ALA is not well understood. This study aimed to determine whether ALA ameliorates VSM function by elevating hydrogen sulfide (H₂S) level in diabetes and whether this effect is associated with regulation of autophagy of VSM cells (VSMCs). We found decreased serum H₂S levels in Chinese patients and rats with type 2 diabetes mellitus (T2DM). ALA treatment could increase H₂S level, which reduced the autophagy-related index and activation of the 5′-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway, thereby protecting vascular function in rats with T2DM. Propargylglycine (PPG), a cystathionine-γ-lyase inhibitor, could weaken the ALA effect. In cultured VSMCs, high glucose level also reduced H₂S level, upregulated the autophagy-related index and activated the AMPK/mTOR pathway, which were reversed by concomitant application of sodium hydrosulfide (NaHS, an H₂S donor) or ALA. The protective effect of NaHS or ALA was attenuated by rapamycin (an autophagy activator), 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (an AMPK activator) or PPG. In contrast, Compound C (an AMPK inhibitor) enhanced the effect of ALA or NaHS. ALA may have a protective effect on VSMCs in T2DM by elevating H₂S level and downregulating autophagy via the AMPK/mTOR pathway. This study provides a new target for addressing diabetic macroangiopathy.     

Sphingosine kinase 1 expression is regulated by signaling through PI3K, AKT2, and mTOR in human coronary artery smooth muscle cells

Sphingosine kinase 1 (SphK1) is a lipid kinase implicated in mitogenic signaling pathways in vascular smooth muscle cells. We demonstrate that human coronary artery smooth muscle (HCASM) cells require SphK1 for growth and that SphK1 mRNA and protein levels are elevated in PDGF stimulated HCASM cells. To determine the mechanism of PDGF-induced SphK1 expression, we used pharmacological inhibitors of the PI3K/AKT/mTOR signaling pathway. Wortmannin, SH-5, and rapamycin significantly blocked PDGF-stimulated induction of SphK1 mRNA and protein expression, indicating a regulatory role of the PI3K/AKT/mTOR pathway in SphK1 expression. To determine which isoform of AKT regulates SphK1 mRNA and protein levels, siRNAs specific for AKT1, AKT2, and AKT3 were used. We show that AKT2 siRNA significantly blocked PDGF-stimulated increases in SphK1 mRNA and protein expression levels as well as SphK1 enzymatic activity levels. In contrast, AKT1 or AKT3 siRNA did not have an effect. Together, these results demonstrate that the PI3K/AKT/mTOR signaling pathway is involved in regulation of SphK1, with AKT2 playing a key role in PDGF-induced SphK1 expression in HCASM cells.     

(S)-ZJM-289, a nitric oxide-releasing derivative of 3-n-butylphthalide, protects against ischemic neuronal injury by attenuating mitochondrial dysfunction and associated cell death

Pharmacological compounds that release nitric oxide (NO) have been recognized as the potential therapeutic agents for acute stroke. (S)-ZJM-289 is a novel NO-releasing derivative of 3-n-butylphthalide (NBP) with enhanced anti-platelet and anti-thrombotic actions. The present study was performed to investigate the neuroprotective effects and related mechanisms of (S)-ZJM-289 on ischemic neuronal injury in vitro and in vivo. Primary cortical neuronal cultures were exposured to oxygen-glucose deprivation followed by recovery (OGD/R), a model of ischemia-like injury, and treated with (S)-ZJM-289 before OGD. In vitro results showed that (S)-ZJM-289 attenuated OGD/R-induced neuronal injury, which was associated with the maintenance of mitochondrial integrity and function by alleviating intracellular calcium overload and reactive oxygen species (ROS) accumulation, preventing mitochondrial membrane depolarization and preserving respiratory chain complexes activities. Moreover, (S)-ZJM-289 treatment suppressed mitochondrial release of cytochrome c (cyt c) and nuclear translocation of apoptosis-inducing factor (AIF), thereby blocking mitochondria-mediated cell death, which may be partially mediated by up-regulation of Hsp70. The neuroprotection by (S)-ZJM-289 was also studied using a model of middle cerebral artery occlusion (MCAO). Oral administration of (S)-ZJM-289 at the onset of reperfusion for 3d significantly reduced the brain infarct size, improved neurological deficit and prevented neuronal loss and apoptosis. In current study, (S)-ZJM-289 appears to be more potent in ischemic neuroprotection than NBP, in particular at the lower doses, which may be due to the synergistic action of NBP and NO. These findings point to that (S)-ZJM-289 could be an attractive alternative to NBP in preventing the process of ischemia/reperfusion (I/R) injury.     

Deficiency of peroxisome proliferator-activated receptor α attenuates apoptosis and promotes migration of vascular smooth muscle cells

Peroxisome proliferator-activated receptor (PPAR) α is widely expressed in the vasculature and has pleiotropic and lipid-lowering independent effects, but its role in the growth and function of vascular smooth muscle cells (VSMCs) during vascular pathophysiology is still unclear. Herein, VSMC-specific PPARα-deficient mice (Ppara^ΔSMC) were generated by Cre-LoxP site-specific recombinase technology and VSMCs were isolated from mice aorta. PPARα deficiency attenuated VSMC apoptosis induced by angiotensin (Ang) II and hydrogen peroxide, and increased the migration of Ang II-challenged cells.     

Induction of glutathione synthesis and heme oxygenase 1 by the flavonoids butein and phloretin is mediated through the ERK/Nrf2 pathway and protects against oxidative stress

Butein and phloretin are chalcones that are members of the flavonoid family of polyphenols. Flavonoids have well-known antioxidant and anti-inflammatory activities. In rat primary hepatocytes, we examined whether butein and phloretin affect tert-butylhydroperoxide (tBHP)-induced oxidative damage and the possible mechanism(s) involved. Treatment with butein and phloretin markedly attenuated tBHP-induced peroxide formation, and this amelioration was reversed by l-buthionine-S-sulfoximine [a glutamate cysteine ligase (GCL) inhibitor] and zinc protoporphyrin [a heme oxygenase 1 (HO-1) inhibitor]. Butein and phloretin induced both HO-1 and GCL protein and mRNA expression and increased intracellular glutathione (GSH) and total GSH content. Butein treatment activated the ERK1/2 signaling pathway and increased Nrf2 nuclear translocation, Nrf2 nuclear protein-DNA binding activity, and ARE-luciferase reporter activity. The roles of the ERK signaling pathway and Nrf2 in butein-induced HO-1 and GCL catalytic subunit (GCLC) expression were determined by using RNA interference directed against ERK2 and Nrf2. Both siERK2 and siNrf2 abolished butein-induced HO-1 and GCLC protein expression. These results suggest the involvement of ERK2 and Nrf2 in the induction of HO-1 and GCLC by butein. In an animal study, phloretin was shown to increase GSH content and HO-1 expression in rat liver and decrease carbon tetrachloride-induced hepatotoxicity. In conclusion, we demonstrate that butein and phloretin up-regulate HO-1 and GCL expression through the ERK2/Nrf2 pathway and protect hepatocytes against oxidative stress.     

Vascular smooth muscle cell proliferation as a therapeutic target. Part 1: molecular targets and pathways

Cardiovascular diseases are a major cause of human death worldwide. Excessive proliferation of vascular smooth muscle cells contributes to the etiology of such diseases, including atherosclerosis, restenosis, and pulmonary hypertension. The control of vascular cell proliferation is complex and encompasses interactions of many regulatory molecules and signaling pathways. Herein, we recapitulated the importance of signaling cascades relevant for the regulation of vascular cell proliferation. Detailed understanding of the mechanism underlying this process is essential for the identification of new lead compounds (e.g., natural products) for vascular therapies.     

Depletion of cytosolic or mitochondrial thioredoxin increases CYP2E1-induced oxidative stress via an ASK-1-JNK1 pathway in HepG2 cells

Thioredoxin is an important reducing molecule in biological systems. Increasing CYP2E1 activity induces oxidative stress and cell toxicity. However, whether thioredoxin protects cells against CYP2E1-induced oxidative stress and toxicity is unknown. SiRNA were used to knockdown either cytosolic (TRX-1) or mitochondrial thioredoxin (TRX-2) in HepG2 cells expressing CYP2E1 (E47 cells) or without expressing CYP2E1 (C34 cells). Cell viability decreased 40-60% in E47 but not C34 cells with 80-90% knockdown of either TRX-1 or TRX-2. Depletion of either thioredoxin also potentiated the toxicity produced either by a glutathione synthesis inhibitor or by TNFα in E47 cells. Generation of reactive oxygen species and 4-HNE protein adducts increased in E47 but not C34 cells with either thioredoxin knockdown. GSH was decreased and adding GSH completely blocked E47 cell death induced by either thioredoxin knockdown. Lowering TRX-1 or TRX-2 in E47 cells caused an early activation of ASK-1, followed by phosphorylation of JNK1 after 48 h of siRNA treatment. A JNK inhibitor caused a partial recovery of E47 cell viability after thioredoxin knockdown. In conclusion, knockdown of TRX-1 or TRX-2 sensitizes cells to CYP2E1-induced oxidant stress partially via ASK-1 and JNK1 signaling pathways. Both TRX-1 and TRX-2 are important for defense against CYP2E1-induced oxidative stress.     

Alternative cell death of Apaf1-deficient neural progenitor cells induced by withdrawal of EGF or insulin

Background

Various forms of cell death, such as apoptotic, autophagic and non-lysosomal types, are implicated in normal physiological processes. Apoptotic protease activating factor 1 (Apaf1) is an important component of the intrinsic apoptotic pathway. Deficiency of Apaf1 results in an accumulation of neural progenitor cells (NPCs) in the developing central nervous system and thus, in perinatal lethality. A small percentage of the mutant mice, however, are viable and grow to maturity. The occurrence of such normal mutants implicates alternative cell death pathways during neurogenesis.

Methods

NPCs prepared from wild-type or Apaf1-deficient embryos were cultured in growth factor-deprived medium and examined for cell death, caspase activation and morphological alterations. Generation of reactive oxygen species (ROS) and the effects of antioxidants were examined.

Results

Wild-type NPCs underwent apoptosis within 24 hours of withdrawal of epidermal growth factor (EGF) or insulin, whereas Apaf1-deficient NPCs underwent cell death but showed no signs of apoptosis. Autophagy was not necessarily accompanied by cell death. Cell death of the Apaf1-deficient NPCs resembled necroptosis—necrosis-like programmed cell death. The necroptosis inhibitor necrostatin-1, however, failed to inhibit the cell death. ROS accumulation was detected in NPCs deprived of growth factors, and an antioxidant partially suppressed the non-apoptotic cell death of Apaf1-deficient NPCs.

Conclusions

These data indicate that after withdrawal EGF or insulin withdrawal, the Apaf1-deficient cells underwent non-apoptotic cell death. ROS generation may partially participate in the cell death.

General Significance

Non-apoptotic cell death in NPCs may be a compensatory mechanism in the developing CNS of Apaf1-deficient embryos.     

HSP90 is required for TAK1 stability but not for its activation in the pro-inflammatory signaling pathway

The protein kinase transforming-growth-factor-β-activated kinase-1 (TAK1) is a key regulator in the pro-inflammatory signaling pathway and is activated by tumor necrosis factor-α, interleukin-1 (IL-1) and lipopolysaccharide (LPS). We describe the identification of TAK1 as a client protein of the 90 kDa heat-shock protein (Hsp90)/cell division cycle protein 37 (Cdc37) chaperones. However, Hsp90 is not required for the activation of TAK1 as short exposure to the Hsp90 inhibitor, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) did not affect its activation by LPS or IL-1. Prolonged treatment of cells with 17-AAG inhibits Hsp90 and downregulates TAK1. Our results suggest that Hsp90 is required for the folding and stability of TAK1 but is displaced and no longer required when TAK1 is complexed to TAK1-binding protein-1 (TAB1).

Structured summary

MINT-6797182:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with CDC37 (uniprotkb:Q16543) and HSP90 (uniprotkb:P07900) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797194:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with TAB1 (uniprotkb:Q15750), HSP90 (uniprotkb:P07900) and CDC37 (uniprotkb:Q16543) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797248:

TAK1 (uniprotkb:Q62073) physically interacts (MI:0218) with HSP90 (uniprotkb:P07901), CDC37 (uniprotkb:Q61081), TAB2 (uniprotkb:Q99K90) and TAB1 (uniprotkb:Q8CF89) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797232:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with HSP90 (uniprotkb:P07900) and CDC37 (uniprotkb:Q16543) by pull down (MI:0096)

MINT-6797216:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with TAB2 (uniprotkb:Q9NYJ8), CDC37 (uniprotkb:Q16543), HSP90 (uniprotkb:P07900) and TAB1 (uniprotkb:Q15750) by anti bait coimmunoprecipitation (MI:0006)

     

Activation of cultured astrocytes by amphotericin B: Stimulation of NO and cytokines production and changes in neurotrophic factors production

Amphotericin B (AmB) is a polyene antibiotic and reported to be one of a few reagents having therapeutic effects on prion diseases, such as the delay in the appearing of the clinical signs and the prolongation of the survival time. In prion diseases, glial cells have been suggested to play important roles by proliferating and producing various factors such as nitric oxide, proinflammatory cytokines, and neurotrophic factors. However, the therapeutic mechanism of AmB on prion diseases remains elusive. We have previously reported that AmB changed the expression of neurotoxic and neurotrophic factors in microglia (Motoyoshi et al., 2008, Neurochem. Int. 52, 1290–1296). In the present study, we examined the effects of AmB on cellular functions of rat cultured astrocytes. We found that AmB could activate astrocytes to produce nitric oxide via inducible nitric oxide synthase induction. AmB also induced mRNA expression of interleukin-1β and tumor necrosis factor-α, and productions of their proteins in astrocytes. Moreover, AmB changed levels of neurotrophic factor mRNAs and proteins. Among three neurotrophic factors examined here, neurotrophin-3 mRNA expression and its protein production in the cells were down-regulated by AmB stimulation. On the other hand, AmB significantly enhanced the amounts of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor proteins in the cells and the medium. These results suggest that AmB might show therapeutic effects on prion diseases by controlling the expression and production of such mediators in astrocytes.     

The role of metallothionein in oncogenesis and cancer prognosis

The antiapoptotic, antioxidant, proliferative, and angiogenic effects of metallothionein (MT)-I+II has resulted in increased focus on their role in oncogenesis, tumor progression, therapy response, and patient prognosis. Studies have reported increased expression of MT-I+II mRNA and protein in various human cancers; such as breast, kidney, lung, nasopharynx, ovary, prostate, salivary gland, testes, urinary bladder, cervical, endometrial, skin carcinoma, melanoma, acute lymphoblastic leukemia (ALL), and pancreatic cancers, where MT-I+II expression is sometimes correlated to higher tumor grade/stage, chemotherapy/radiation resistance, and poor prognosis. However, MT-I+II are downregulated in other types of tumors (e.g. hepatocellular, gastric, colorectal, central nervous system (CNS), and thyroid cancers) where MT-I+II is either inversely correlated or unrelated to mortality. Large discrepancies exist between different tumor types, and no distinct and reliable association exists between MT-I+II expression in tumor tissues and prognosis and therapy resistance. Furthermore, a parallel has been drawn between MT-I+II expression as a potential marker for prognosis, and MT-I+II's role as oncogenic factors, without any direct evidence supporting such a parallel. This review aims at discussing the role of MT-I+II both as a prognostic marker for survival and therapy response, as well as for the hypothesized role of MT-I+II as causal oncogenes.