Almudena Porras

p38 MAP kinases: beyond the stress response.

We are grateful to Anne Ephrussi and Giulio Superti-Furga for comments on the manuscript. We apologize to the authors whose original work is not included in the references owing to space limitations.

Essential Role of p38α MAP Kinase in Placental but Not Embryonic Cardiovascular Development

p38alpha MAP kinase is activated in response to many cellular stresses and also regulates the differentiation and/or survival of various cell types in vitro, including skeletal muscle cells and cardiomyocytes. Here we show that targeted inactivation of the mouse p38alpha gene results in embryonic lethality at midgestation correlating with a massive reduction of the myocardium and malformation of blood vessels in the head region. However, this defect appears to be secondary to insufficient oxygen and nutrient transfer across the placenta. When the placental defect was rescued, p38alpha(-/-) embryos developed to term and were normal in appearance. Our results indicate that p38alpha is required for placental organogenesis but is not essential for other aspects of mammalian embryonic development.

p38 MAPK enhances STAT1-dependent transcription independently of Ser-727 phosphorylation

The transcription factor signal transducer and activator of transcription 1 (STAT1) requires phosphorylation at both Tyr-701 and Ser-727 for full activation. IFN-γ induces phosphorylation of both residues, whereas stress signals like UV or lipopolysaccharide stimulate phosphorylation of Ser-727 only. Using p38α mitogen-activated protein kinase (MAPK)-deficient cells, we show that the stress-induced phosphorylation of Ser-727 requires p38α MAPK activity, whereas IFN-γ-stimulated Ser-727 phosphorylation occurs independently of the p38α pathway. Consistently, IFN-γ stimulated expression of the STAT1 target gene IRF1 to a similar extent in both wild-type and p38α-deficient cells. However, stress-induced activation of the p38 MAPK pathway considerably enhanced the IFN-γ-induced expression of both the endogenous IRF1 gene and a reporter driven by the IFN-γ-activated sequence element of the IRF1 promoter. This enhancement occurred independently of increased phosphorylation of Ser-727 by the p38 pathway. Taken together, these results demonstrate an interaction between IFN-γ signaling and the p38 pathway that leads to increased transcriptional activation by STAT1 independently of phosphorylation at Ser-727.

p38α Mediates Cell Survival in Response to Oxidative Stress via Induction of Antioxidant Genes EFFECT ON THE p70S6K PATHWAY

Background: p38α MAPK is activated by stress stimuli, which can regulate cell death. Results: In response to H2O2, p38α MAPK increases SOD and catalase levels, impairs ROS accumulation, and leads to cell survival. Conclusion: p38α MAPK signals survival under moderate oxidative stress through up-regulation of antioxidant defenses. Significance: To know how p38α regulates ROS levels is important for cell homeostasis. We reveal a novel pro-survival role for mammalian p38α in response to H2O2, which involves an up-regulation of antioxidant defenses. The presence of p38α increases basal and H2O2-induced expression of the antioxidant enzymes: superoxide-dismutase 1 (SOD-1), SOD-2, and catalase through different mechanisms, which protects from reactive oxygen species (ROS) accumulation and prevents cell death. p38α was found to regulate (i) H2O2-induced SOD-2 expression through a direct regulation of transcription mediated by activating transcription factor 2 (ATF-2) and (ii) H2O2-induced catalase expression through regulation of protein stability and mRNA expression and/or stabilization. As a consequence, SOD and catalase activities are higher in WT MEFs. We also found that this p38α-dependent antioxidant response allows WT cells to maintain an efficient activation of the mTOR/p70S6K pathway. Accordingly, the loss of p38α leads to ROS accumulation in response to H2O2, which causes cell death and inactivation of mTOR/p70S6K signaling. This can be rescued by either p38α re-expression or treatment with the antioxidants, N-acetyl cysteine, or exogenously added catalase. Therefore, our results reveal a novel homeostatic role for p38α in response to oxidative stress, where ROS removal is favored by antioxidant enzymes up-regulation, allowing cell survival and mTOR/p70S6K activation.

Met acts on Mdm2 via mTOR to signal cell survival during development

Coordination of cell death and survival is crucial during embryogenesis and adulthood, and alteration of this balance can result in degeneration or cancer. Growth factor receptors such as Met can activate phosphatidyl-inositol-3′ kinase (PI3K), a major intracellular mediator of growth and survival. PI3K can then antagonize p53-triggered cell death, but the underlying mechanisms are not fully understood. We used genetic and pharmacological approaches to uncover Met-triggered signaling pathways that regulate hepatocyte survival during embryogenesis. Here, we show that PI3K acts via mTOR (Frap1) to regulate p53 activity both in vitro and in vivo. mTOR inhibits p53 by promoting the translation of Mdm2, a negative regulator of p53. We also demonstrate that the PI3K effector Akt is required for Met-triggered Mdm2 upregulation, in addition to being necessary for the nuclear translocation of Mdm2. Inhibition of either mTOR or Mdm2 is sufficient to block cell survival induced by Hgf-Met in vitro. Moreover, in vivo inhibition of mTOR downregulates Mdm2 protein levels and induces p53-dependent apoptosis. Our studies identify a novel mechanism for Met-triggered cell survival during embryogenesis, involving translational regulation of Mdm2 by mTOR. Moreover, they reinforce mTOR as a potential drug target in cancer.

TNF-α induces apoptosis in rat fetal brown adipocytes in primary culture

The effect of TNF‐α on cell death in rat fetal brown adipocytes maintained in primary culture was determined. TNF‐α inhibited proliferation and induced apoptosis in these cells. Most of the cells undergoing apoptosis after TNF‐α treatment did not express PCNA, suggesting an induction of apoptosis by TNF‐α in non‐proliferative cells. IGF‐I but not EGF prevented TNF‐α‐induced apoptosis.

p38 Mitogen-Activated Protein Kinase Mediates Tumor Necrosis Factor-α-Induced Apoptosis in Rat Fetal Brown Adipocytes1

Tumor necrosis factor-alpha (TNFalpha) induces apoptosis and cell growth inhibition in primary rat fetal brown adipocytes. Here, we examine the role played by some members of the mitogen-activated protein kinase (MAPK) superfamily. TNFalpha activates extracellular regulated kinase-1/2 (ERK1/2) and p38MAPK. Inhibition of p38MAPK by either SB203580 or SB202190 highly reduces apoptosis induced by TNFalpha, whereas ERK inhibition potentiates it. Moreover, cotransfection of an active MKK3 mutant and p38MAPK induces apoptosis. p38MAPK inhibition also prevents TNFalpha-induced cell cycle arrest, whereas MEK1 inhibition enhances this effect, which correlates with changes in proliferating cell nuclear antigen expression, but not in cyclin D1. c-Jun and activating transcription factor-1 are potential downstream effectors of p38MAPK and ERKs upon TNFalpha treatment. Thus, TNFalpha-induced c-Jun messenger RNA expression requires ERKs activation, whereas p38MAPK inhibition enhances its expression. In addition, TNFalpha-induced activating transcription factor-1 phosphorylation is extensively decreased by SB203580. However, TNFalpha-induced NF-kappaB DNA-binding activity is independent of p38MAPK and ERK activation. On the other hand, C/EBP homology protein does not appear to mediate the actions of TNFalpha, because its expression is almost undetectable and even reduced by TNFalpha. Finally, although TNFalpha induces c-Jun N-terminal kinase (JNK) activation, transfection of a dominant negative of either JNK1 or JNK2 had no effect on TNFalpha-induced apoptosis. These results suggest that p38MAPK mediates TNFalpha-induced apoptosis and cell cycle arrest, whereas ERKs do the opposite, and JNKs play no role in this process of apoptosis.

TNF‐α inhibits UCP‐1 expression in brown adipocytes via ERKs

Tumor necrosis factor‐α (TNF‐α) activates extracellular‐regulated kinases (ERKs) and p38 mitogen‐activated protein kinase (p38MAPK), and inhibits the expression of uncoupling protein‐1 (UCP‐1) and adipocyte‐specific genes in rat fetal brown adipocytes. MEK inhibition with PD98059 abolished the inhibitory effect of TNF‐α on UCP‐1, but not on adipogenic genes. In contrast, inhibition of p38MAPK with SB203580 potentiated the negative effect of TNF‐α on UCP‐1 and adipogenic genes. The inhibitory action of TNF‐α was partially correlated with changes in C/EBPα and β protein levels and in their DNA binding activity, suggesting a role for these transcription factors. However, other transcription factors might explain the different regulation of UCP‐1 and adipogenic genes by ERKs.

Activation of p38MAPK by TGF-β in fetal rat hepatocytes requires radical oxygen production, but is dispensable for cell death

We have previously found that transforming growth factor‐β (TGF‐β) induces an increase in radical oxygen species (ROS) production that mediates its apoptotic effects in fetal hepatocytes. In this paper we show that TGF‐β activates p38 mitogen‐activated protein kinase (p38MAPK) and ROS may be responsible for this activation. Activation of p38MAPK occurs late, coincident with the maximal production of ROS, it is inhibited by radical scavengers and it is accentuated by the presence of glutathione synthesis inhibitors. However, p38MAPK does not appear to be involved in any of the apoptotic events: loss of Bcl‐xL levels, cytochrome c release, cleavage of caspase substrates and loss of cell viability.

Adrenergic regulation of the uncoupling protein expression in foetal rat brown adipocytes in primary culture

The adrenergic and T3 modulation of UCP expression in non-proliferative foetal brown adipocyte primary cultures were studied. The UCP in the cultured cells was determined by immunological detection of the protein and by quantification of the mitochondrial GDP-binding. Our results showed a relative increase of 65-75% in UCP levels and 60-80% in the mitochondrial GDP-binding capacity under beta-adrenergic stimulatory conditions, while neither alpha 1-adrenergic agonists nor T3 showed an effect.