Acute oxidative stress is associated with cell proliferation in the mouse liver

Oxidative stress is known to produce tissue injury and to activate various signaling pathways. To investigate the molecular events linked to acute oxidative stress in mouse liver, we injected a toxic dose of paraquat. Liver necrosis was first observed, followed by histological marks of cell proliferation. Concomitantly, activation of the MAP kinase pathway and increased levels of the anti-apoptotic protein Bcl-XL were observed. Gene expression profiles revealed that the differentially expressed genes were potentially involved in cell proliferation. These data suggest that paraquat-induced acute oxidative stress triggers the activation of regeneration-related events in the liver.     

Carvedilol has stronger anti-inflammation and anti-virus effects than metoprolol in murine model with coxsackievirus B3-induced viral myocarditis

Aims

This study aims to compare the effects of carvedilol and metoprolol in alleviating viral myocarditis (VMC) induced by coxsackievirus B3 (CVB3) in mice.

Methods

A total of 116 Balb/c mice were included in this study. Ninety-six mice were inoculated intraperitoneally with CVB3 to induce VMC. The CVB3 inoculated mice were evenly divided into myocarditis group (n = 32), carvedilol group (n = 32) and metoprolol group (n = 32). Twenty mice (control group) were inoculated intraperitoneally with normal saline. Hematoxylin and eosin staining and histopathologic scoring were used to investigate the effects of carvedilol and metoprolol on myocardial histopathologic changes on days 3 and 5. In addition, serum cTn-I levels, cytokine levels and virus titers were determined using chemiluminescence immunoassay, enzyme-linked immunosorbent assay and plaque assay, respectively, on days 3 and 5. Finally, the levels of phosphorylated p38MAPK were studied using immunohistochemical staining and Western blotting on day 5.

Results

Carvedilol had a stronger effect than metoprolol in reducing the pathological scores of VMC induced by CVB3. Both carvedilol and metoprolol reduced the levels of cTn-I, but the effect of carvedilol was stronger. Carvedilol and metoprolol decreased the levels of myocardial pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokine, with the effects of carvedilol being stronger than those of metoprolol. Carvedilol had a stronger effect in reducing myocardial virus concentration compared with metoprolol. Carvedilol was stronger than metoprolol in decreasing the levels of myocardial phosphorylated p38MAPK.

Conclusions

In conclusion, carvedilol was more potent than metoprolol in ameliorating myocardial lesions in VMC, probably due to its stronger modulation of the balance between pro- and anti-inflammatory cytokines by inhibiting the activation of p38MAPK pathway through β1- and β2-adrenoreceptors.     

Examining the mechanism of Erk nuclear translocation using green fluorescent protein

In neuronal cells, the mitogen-activated protein kinase (MAP kinase) cascade is an important mediator of neurotrophin signaling from cell surface receptors to the nucleus, resulting in changes in gene expression. Nuclear localization of Erk is thought to be required for these effects. To examine the mechanism and regulation of Erk nuclear translocation, we have created a green fluorescent protein (GFP)-labeled Erk2 construct, which provides a sensitive means to follow the movement of Erk from the cytoplasm to the nucleus following receptor-mediated MAP kinase activation. Using this system in PC12 cells, we have examined a number of mechanisms that have been implicated in regulating the translocation of Erk. In PC12 cells, NGF and EGF induce a rapid translocation of GFP-Erk that requires Ras and Mek. We have found that prolonged phosphorylation of Erk is not required for the rapid and early influx of Erk into the nucleus following growth factor stimulation. Furthermore, following influx, GFP-Erk rapidly returned to the cytoplasm regardless of its phosphorylation state. The release of Erk from its cytoplasmic activator, Mek, followed by the dimerization of Erk, was sufficient to stimulate nuclear uptake, whereas Erk kinase activity was dispensable. PKA activity has been reported to be required for Erk translocation in PC12 cells. However, PKA activity was also not necessary for the early translocation of Erk into the nucleus by NGF or Ras, but it was able to induce a small influx of Erk that could be measured with GFP-Erk2.     

Oxysterols and calcium signal transduction

Ionised calcium (Ca²⁺) is a key second messenger, regulating almost every cellular process from cell death to muscle contraction. Cytosolic levels of this ion can be increased via gating of channel proteins located in the plasma membrane, endoplasmic reticulum and other membrane-delimited organelles. Ca²⁺ can be removed from cells by extrusion across the plasma membrane, uptake into organelles and buffering by anionic components. Ca²⁺ channels and extrusion mechanisms work in concert to generate diverse spatiotemporal patterns of this second messenger, the distinct profiles of which determine different cellular outcomes. Increases in cytoplasmic Ca²⁺ concentration are one of the most rapid cellular responses upon exposure to certain oxysterol congeners or to oxidised low-density lipoprotein, occurring within seconds of addition and preceding increases in levels of reactive oxygen species, or changes in gene expression. Furthermore, exposure of cells to oxysterols for periods of hours to days modulates Ca²⁺ signal transduction, with these longer-term alterations in cellular Ca²⁺ homeostasis potentially underlying pathological events within atherosclerotic lesions, such as hyporeactivity to vasoconstrictors observed in vascular smooth muscle, or ER stress-induced cell death in macrophages. Despite their candidate roles in physiology and disease, little is known about the molecular mechanisms that couple changes in oxysterol concentrations to alterations in Ca²⁺ signalling. This review examines the ways in which oxysterols could influence Ca²⁺ signal transduction and the potential roles of this in health and disease.     

Prosaposin: a new player in cell death prevention of U937 monocytic cells

We report that prosaposin binds to U937 and is active as a protective factor on tumor necrosis factor alpha (TNFalpha)-induced cell death. The prosaposin-derived saposin C binds to U937 cells in a concentration-dependent manner, suggesting that prosaposin behaves similarly. Prosaposin binding induces U937 cell death prevention, reducing both necrosis and apoptosis. This effect was inhibited by mitogen-activated protein ERK kinase (MEK) and sphingosine kinase (SK) inhibitors, indicating that prosaposin prevents cell apoptosis by activation of extracellular signal-regulated kinases (ERKs) and sphingosine kinase. Prosaposin led to rapid ERK phosphorylation in U937 cells as detected by anti-phospho-p44/42 mitogen-activated protein (MAP) kinase and anti-phosphotyrosine reactivity on ERK immunoprecipitates. It was partially prevented by apo B-100 and pertussis toxin (PT), suggesting that both lipoprotein receptor-related protein (LRP) receptor and Go-coupled receptor may play a role in the prosaposin-triggered pathway. Moreover, sphingosine kinase activity was increased by prosaposin treatment as demonstrated by the enhanced intracellular formation of sphingosine-1-phosphate (S-1-P). The observation that the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin prevented the prosaposin effect on cell apoptosis suggests that sphingosine kinase exerts its anti-apoptotic activity by the PI3K-Akt pathway. Thus, cell apoptosis prevention by prosaposin occurs through ERK phosphorylation and sphingosine kinase. The biological effect triggered by prosaposin might be extended to primary cells because it triggers Erk phosphorylation in peripheral blood mononuclear cells (PBMCs). This is the first evidence of a biological effect consequent to a signal transduction pathway triggered by prosaposin in cells of non-neurological origin.     

Muscular cell proliferative and protective effects of N-acetylcysteine by modulating activity of extracellular signal-regulated protein kinase

N-acetylcysteine (NAC), an antioxidant and a precursor of glutathione, is currently in clinical use for various pathological conditions. No data is available as to the relationship between NAC and muscular cell proliferation or muscular degenerative disease. In this study, we assessed the effect of NAC on growth of L6 myoblasts, a rat skeletal muscle cell line, under normal or bupivacaine-treated condition. Of interest, under normal growth conditions, NAC treatment concentration-dependently increased viability, cell number, and DNA incorporation of L6 cells. Remarkably, NAC treatment for 12 to 24 h led to increased phosphorylation of ERKs, a family of mitogen-activated protein kinase known to involve in cell proliferation, in L6 cells, and specific inhibition of ERKs by PD98059, a selective inhibitor of ERKs, greatly abolished the ability of NAC to increase the number of L6 cells. More importantly, pretreatment with NAC effectively blocked decrease in the number and ERKs phosphorylation in L6 cells induced by the exposure of bupivacaine, a local anesthetic with myotoxicity. These results collectively suggest that NAC has muscular cell proliferative and protective effects and the effects by NAC appear to be, in part, mediated via increase in ERKs activation.     

Regulation of MAPK pathways in response to purinergic stimulation of adult rat cardiac myocytes

We investigated the activation of mitogen-activated protein kinases (MAPKs) pathways by purinergic stimulation in cardiac myocytes from adult rat hearts. ATPS increased the phosphorylation (activation) of the extracellular signal regulated kinase 1 and 2 (ERK1/2) and p38 MAPK. ERK1/2 and p38 MAPK activation was differential, ERK1/2 being rapid and transient while that of p38 MAPK slow and sustained. Using selective inhibitors, activation of ERK1/2 was shown to involve protein kinase C and MEK1/2 while that of p38 MAPK was regulated by both protein kinase C and protein kinase A. Furthermore, we show that purinergic stimulation induces the phosphorylation of the MAPK downstream target, mitogen- and stress-activated protein kinase 1 (MSK1), in cardiac myocytes. The time course of MSK1 phosphorylation closely follows that of ERK activation. Inhibitors of the ERK and p38 MAPK pathways were tested on the phosphorylation of MSK1 at two different time points. The results suggest that ERKs initiate the response but both ERKs and p38 MAPK are required for the maintenance of the complete phosphorylation of MSK1. The temporal relationship of MSK1 phosphorylation and cPLA₂ translocation induced by purinergic stimulation, taken together with previous findings, is an indication that cPLA₂ may be a downstream target of MSK1.