p38␣ mitogen-activated protein (MAP) kinase is a broadly expressed signaling molecule that participates in the regulation of cellular responses to stress as well as in the control of proliferation and survival of many cell types. We have used cell lines derived from p38␣ knockout mice to study the role of this signaling pathway in the regulation of apoptosis. Here, we show that cardiomyocytes and fibroblasts lacking p38␣ are more resistant to apoptosis induced by different stimuli. The reduced apoptosis of p38␣-deficient cells correlates with decreased expression of the mitochondrial proapoptotic protein Bax and the apoptosis-inducing receptor Fas/CD-95. Cells lacking p38␣ also have increased extracellular signalregulated kinase (ERKs) MAP kinase activity, and the up-regulation of this survival pathway seems to be at least partially responsible for the reduced levels of apoptosis in the absence of p38␣. Phosphorylation of the transcription factor STAT3 on Ser-727, mediated by the extracellular signal-regulated kinase MAP kinase pathway, may contribute to the decrease in both Bax and Fas expression in p38␣؊/؊ cells. Thus, p38␣ seems to sensitize cells to apoptosis via both up-regulation of proapoptotic proteins and down-regulation of survival pathways. INTRODUCTIONThe family of p38 mitogen-activated protein kinases (MAPKs) are strongly activated by stress and inflammatory cytokines, but nonstressful stimuli can also activate p38 MAPKs, leading to the regulation of cellular functions such as proliferation, differentiation, and survival. Four different p38 MAPK family members have been identified, p38␣, ␤, ␥, and ␦, also known as stress-activated kinase (SAPK)2a, SAPK2b, SAPK3, and SAPK4, respectively, which may have both overlapping and specific functions (reviewed by Cohen, 1997;Nebreda and Porras, 2000;Ono and Han, 2000;Kyriakis and Avruch, 2001). p38␣ is broadly expressed and is also the most abundant p38 family member present in most cell types. Targeted inactivation of the mouse p38␣ gene results in embryonic death due to a placental defect (Adams et al., 2000;Mudgett et al., 2000;Tamura et al., 2000). Studies using cell lines have suggested an important role for p38 MAPKs in the regulation of cardiomyocyte differentiation, hypertrophy, and apoptosis (Wang et al., 1998;Zechner et al., 1998;Mackay and Mochly-Rosen, 1999;Saurin et al., 2000; Stephanou et al., 2000). However, p38␣ does not seem to play a critical role in the development of the embryonic heart in mice (Adams et al., 2000), although heterozygous p38␣ϩ/Ϫ mice have been reported to be less sensitive to myocardial cell death caused by ischemia-reperfusion (Otsu et al., 2003). In addition, cardiac-specific expression of dominant negative forms of p38␣ and their activators MKK3 and MKK6 enhances cardiac hypertrophy in transgenic mice and/or makes the mice resistant to cardiac fibrosis (Braz et al., 2003;Zhang et al., 2003b).The role of p38 MAPKs in apoptosis depends on the cell type and the stimuli (reviewed by Nebreda and Porras, 2000). Most of the d...

Class IA phosphoinositide 3-kinase (PI3K) are enzymes comprised of a p85 regulatory and a p110 catalytic subunit that induce formation of 3-polyphosphoinositides, which activate numerous downstream targets. PI3K controls cell division. Of the 2 ubiquitous PI3K isoforms, ␣ has selective action in cell growth and cell cycle entry, but no specific function in cell division has been described for ␤. We report here a unique function for PI3K␤ in the control of DNA replication. PI3K␤ regulated DNA replication through kinase-dependent and kinase-independent mechanisms. PI3K␤ was found in the nucleus, where it associated PKB. Modulation of PI3K␤ activity altered the DNA replication rate by controlling proliferating cell nuclear antigen (PCNA) binding to chromatin and to DNA polymerase ␦. PI3K␤ exerted this action by regulating the nuclear activation of PKB in S phase, and in turn phosphorylation of PCNA negative regulator p21 Cip . Also, p110␤ associated with PCNA and controlled PCNA loading onto chromatin in a kinase-independent manner. These results show a selective function of PI3K␤ in the control of DNA replication.

PIK3R2 encodes a ubiquitous regulatory subunit (p85β) of PI3K, an enzyme that generates 3-polyphosphoinositides at the plasma membrane. PI3K activation triggers cell survival and migration. We found that p85β expression is elevated in breast and colon carcinomas and that its increased expression correlates with PI3K pathway activation and tumor progression. p85β expression induced moderate PIP 3 generation at the cell membrane and enhanced cell invasion. In accordance, genetic alteration of pik3r2 expression levels modulated tumor progression in vivo. Increased p85β expression thus represents a cellular strategy in cancer progression.A ctivation of class I PI3K is involved in the pathogenesis of cancer. PI3Ks are lipid kinases that phosphorylate membrane phosphoinositides [i.e., phosphatidylinositol (PtdIns)] to generate PtdIns(3,4)P 2 (PIP 2 ) and PtdIns(3,4,5)P 3 (PIP 3 ). PI3K is composed of a regulatory and a p110 catalytic subunit. Four genes encode the highly conserved p110 catalytic subunit (PIK3CA, CB, CD, and CG). p110α, β, and δ associate with p85 regulatory subunits and are activated mainly by growth factor receptors; p110γ associates with distinct regulatory subunits and is activated preferentially by G protein-coupled receptors (1-3). Three genes encode p85-type regulatory subunits: PIK3R1 (p85α, p55α, p50α), PIK3R2 (p85β), and PIK3R3 (p55γ). R1 and R2 are ubiquitously expressed and R3 expression is tissue-restricted (4).p85β is expressed at lower levels than p85α in most tissues (5-7). Whereas mice deficient in Pik3r2 develop normally and exhibit only moderate metabolic and immunological defects (7) Pik3r1 −/− mice die perinatally (8). p85α controls p110 stability and blocks p110 activity during quiescence (9). The inhibitory role of p85α on p110 activity explains why WT PIK3R1 expression is normally reduced in tumors, and that p85α mutations that relieve p110 from p85 inhibition have been found in cancer (10). Despite extensive analysis of p85α mutations in tumors (10-12), p85β involvement in cancer is less well studied. Here we analyzed the potential contribution of p85β in cancer.Results p85β Expression Is Increased in Breast and Colon Carcinomas. By using microarray technology, we performed a preliminary survey of the expression of the genes that form part of the PI3K pathway in a collection of clinical breast (n = 14) and colon carcinomas (n = 12). Comparison of PI3K subunit expression showed that mRNA levels of PIK3R2 (which encodes p85β) were increased in nearly half the carcinoma samples examined, whereas PIK3R1 (which encodes p85α) was decreased (Fig. S1). To study this finding in more detail, we compared 20 colon adenocarcinomas (CCs) and 35 breast carcinomas (BCs) with normal surrounding tissue. Tumor and normal samples had comparable numbers of epithelial cells, and normal tissue had a low percentage of malignant cells (0-10%).To evaluate p85β expression levels, we prepared extracts from normal and tumor samples and analyzed p85 levels by Western blot (WB). We generated anti-p85β Abs an...

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Cardiac differentiation involves cross-regulation of several transcription factors, such as Mef2C, regulated by p38a MAP kinase. We analysed the role of p38a in cardiac differentiation. Either the absence or inhibition of p38a impairs MEF2C nuclear localization in cardiomyocytes, colocalising with vimentin at the perinuclear region. As a consequence, expression of the Mef2C targets, ANF and myocardin, is drastically downregulated. In contrast, Mlc2v and crt are mainly unaltered, probably by the strong Mef2B upregulation, conpensating for the impaired Mef2C transactivity. In addition, p38a deficiency leads to a decrease in the phosphorylated Mlc2v fraction and a-actinin accumulation causing sarcomere disorganisation. We propose a critical role for p38a in early stages of cardiac differentiation by modulation of Mef2C localisation and sarcomeric assembly.

Trying to define the precise role played by insulin regulating the survival of brown adipocytes, we have used rat fetal brown adipocytes maintained in primary culture. The effect of insulin on apoptosis and the mechanisms involved were assessed. Different from the known effects of insulin as a survival factor, we have found that long-term treatment (72 h) with insulin induces apoptosis in rat fetal brown adipocytes. This process is dependent on the phosphatidylinositol 3-kinase/mammalian target of rapamycin/p70 S6 kinase pathway. Short-term treatment with the conditioned medium from brown adipocytes treated with insulin for 72 h mimicked the apoptotic effect of insulin. During the process, caspase 8 activation, Bid cleavage, cytochrome c release, and activation of caspases 9 and 3 are sequentially produced. Treatment with the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (Z-VAD), prevents activation of this apoptotic cascade. The antioxidants, ascorbic acid and superoxide dismutase, also impair this process of apoptosis. Moreover, generation of reactive oxygen species (ROS), probably through reduced nicotinamide adenine dinucleotide phosphate oxidases, and a late decrease in reduced glutathione content are produced. According to this, antioxidants prevent caspase 8 activation and Bid cleavage, suggesting that ROS production is an important event mediating this process of apoptosis. However, the participation of uncoupling protein-1, -2, and -3 regulating ROS is unclear because their levels remain unchanged upon insulin treatment for 72 h. Our data suggest that the prolonged hyperinsulinemia might cause insulin resistance through the loss of brown adipose tissue.

The small GTP-ase Rac-1 can trigger p38 MAPK activation and, in turn, p38a can regulate signalling pathways that potentially impinge on Rac-1 activity. We have investigated the cross-talk between p38a and Rac-1 and found that p38a regulates the association between Rac-1 and caveolin-1 in serum-deprived cardiomyocytes. This interaction depends on cell attachment and correlates with higher levels of active Rac-1. Actin organization might regulate the formation of Rac-1-caveolin-1 complexes. In contrast, the Rac-1-caveolin-1 interaction is almost undetectable in the presence of serum, where Rac-1 activity is negatively regulated by p38a. Our results indicate that p38a can differentially contribute to Rac-1 activation depending on the presence of serum.

Mitosis, the final phase of cell division, includes the processes of nuclear division and cytosolic division (cytokinesis). Cytokinesis occurs when DNA separation terminates, and involves a number of proteins that induce furrowing at the region of cell separation, formation of new membrane, and abscission. This process is remarkably complex, and the list of proteins that regulate it is long. Our understanding is limited as to how these players are organized in space and time to ensure that the cytosol divides equally, and only after nuclear division. Class I(A) PI3K (phosphoinositide 3-kinase) is an enzyme activated by growth factor receptor stimulation, but it is re-activated in early mitosis and regulates mitosis entry. By the end of mitosis, PI3K activity is low; at this point, the class I(A) PI3K regulatory subunit p85 contributes to co-ordination of the cytoskeletal changes required for cytokinesis. The impact of these observations on current models of cytokinesis execution is discussed here.