Cycloheximide-induced cPLA(2) activation is via the MKP-1 down-regulation and ERK activation

Extracellular signal-regulated kinase (ERK)-dependent phosphorylation is an important regulator for cytosolic phospholipase A(2) (cPLA(2)). In this study, we found that the protein synthesis inhibitor cycloheximide can potentiate thapsigargin-induced arachidonic acid (AA) release concomitant with ERK phosphorylation from murine RAW 264.7 macrophages. The cycloheximide effect is not due to the activation of p38 mitogen-activated protein kinase (MAPK) nor c-Jun NH(2)-terminal kinase (JNK), because the activator of both MAPKs anisomycin does not elicit AA release. Cycloheximide effect is additive to the tyrosine phosphatase inhibitor orthovanadate since these two stimuli induced sustained ERK activation respectively through inhibition of the translation and activity of MAPK phosphatase-1 (MKP-1).     

Antarctic fish can survive prolonged exposure to elevated temperatures

The Antarctic notothenioid Pagothenia borchgrevinki was collected from the stenothermal waters of McMurdo Sound in the summers of 2004, 2005 and 2006. Acclimation ability at 4° C was tested in healthy P. borchgrevinki and in individuals infected with x‐cell gill disease. All healthy fish successfully acclimated to 4° C, establishing compensatory changes in resting oxygen consumption rate (R_rest) and critical swimming speed (U_crit) during a 1 month acclimation period, which were maintained during a longer, 6 month acclimation period. In contrast, individuals infected with x‐cell disease were unable to acclimate to 4° C, demonstrating significantly reduced survival rates compared with healthy individuals at 4° C. Measurements of R_rest suggest that limitations in the ability of x‐cell fish to uptake oxygen from the external milieu may have a negative effect on their survival at 4° C.     

Tissue transglutaminase activation modulates inflammation in cystic fibrosis via PPARgamma down-regulation

Cystic fibrosis (CF), the most common life-threatening inherited disease in Caucasians, is due to mutations in the CF transmembrane conductance regulator (CFTR) gene and is characterized by airways chronic inflammation and pulmonary infections. The inflammatory response is not secondary to the pulmonary infections. Indeed, several studies have shown an increased proinflammatory activity in the CF tissues, regardless of bacterial infections, because inflammation is similarly observed in CFTR-defective cell lines kept in sterile conditions. Despite recent studies that have indicated that CF airway epithelial cells can spontaneously initiate the inflammatory cascade, we still do not have a clear insight of the molecular mechanisms involved in this increased inflammatory response. In this study, to understand these mechanisms, we investigated ex vivo cultures of nasal polyp mucosal explants of CF patients and controls, CFTR-defective IB3-1 bronchial epithelial cells, C38 isogenic CFTR corrected, and 16HBE normal bronchial epithelial cell lines. We have shown that a defective CFTR induces a remarkable up-regulation of tissue transglutaminase (TG2) in both tissues and cell lines. The increased TG2 activity leads to functional sequestration of the anti-inflammatory peroxisome proliferator-activated receptor gamma and increase of the classic parameters of inflammation, such as TNF-alpha, tyrosine phosphorylation, and MAPKs. Specific inhibition of TG2 was able to reinstate normal levels of peroxisome proliferator-activated receptor-gamma and dampen down inflammation both in CF tissues and CFTR-defective cells. Our results highlight an unpredicted central role of TG2 in the mechanistic pathway of CF inflammation, also opening a possible new wave of therapies for sufferers of chronic inflammatory diseases.     

Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation.

Forty-one ionizing radiation-sensitive strains of Deinococcus radiodurans were evaluated for their ability to survive 6 weeks of desiccation. All exhibited a substantial loss of viability upon rehydration compared with wild-type D. radiodurans. Examination of chromosomal DNA from desiccated cultures revealed a time-dependent increase in DNA damage, as measured by an increase in DNA double-strand breaks. The evidence presented suggests that D. radiodurans' ionizing radiation resistance is incidental, a consequence of this organism's adaptation to a common physiological stress, dehydration.     

Changed genome heterochromatinization upon prolonged activation of the Raf/ERK signaling pathway

The Raf/ERK (Extracellular Signal Regulated Kinase) signal transduction pathway controls numerous cellular processes, including growth, differentiation, cellular transformation and senescence. ERK activation is thought to involve complex spatial and temporal regulation, to achieve a high degree of specificity, though precisely how this is achieved remains to be confirmed. We report here that prolonged activation of a conditional form of c-Raf-1 (BXB-ER) leads to profound changes in the level and distribution of a heterochromatic histone mark. In mouse fibroblasts, the heterochromatic trimethylation of lysine 9 in histone H3 (H3K9Me3) is normally confined to pericentromeric regions. However, following ERK activation a genome-wide redistribution of H3K9Me3 correlates with loss of the histone modification from chromocentres and the appearance of numerous punctuate sites throughout the interphase nucleus. These epigenetic changes during interphase correlate with altered chromosome structure during mitosis, where robust H3K9Me3 signals appear within telomeric heterochromatin. This pattern of heterochromatinization is distinct from previously described oncogene induced senescence associated heterochromatin foci (SAHF), which are excluded from telomeres. The H3K9Me3 histone mark is known to bind the major heterochromatin protein HP1 and we show that the alterations in the distribution of this histone epistate correlate with redistribution of HP1β throughout the nucleus. Interestingly while ERK activation is fully reversible, the observed chromatin changes induced by epigenetic modifications are not reversible once established. We describe for the first time a link from prolonged ERK activation to stable changes in genome organization through redistribution of heterochromatic domains involving the telomeres. These epigenetic changes provide a possible mechanism through which prolonged activation of Raf/ERK can lead to growth arrest or the induction of differentiation, senescence and cancer.     

BMP4 Signaling Acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells

Extrinsic BMP and LIF signaling collaboratively maintain mouse embryonic stem cell (ESC) pluripotency, whereas appropriate ERK activity is essential for ESC fate commitment. However, how the extrinsic signals restrain appropriate ERK activity remains elusive. Here, we show that, whereas LIF sustains relatively high ERK activity, BMP4 can steadily attenuate ERK activity by upregulating ERK-specific dual-specificity phosphatase 9 (DUSP9). This upregulation requires Smad1/5 and Smad4 and specifically occurs to DUSP9, but not other DUSPs, and only in ESCs. Through DUSP9-mediated inhibition of ERK activity, BMP signaling reinforces the self-renewal status of mouse ESCs together with LIF. Upon LIF withdrawal, ESCs spontaneously undergo neural differentiation, during which process DUSP9 can partially mediate BMP inhibition on neural commitment. Collectively, our findings identify DUSP9 as a critical mediator of BMP signaling to control appropriate ERK activity critical for ESC fate determination.     

M-CSF-mediated macrophage differentiation but not proliferation is correlated with increased and prolonged ERK activation

M-CSF is a cytokine essential for both the proliferation and differentiation of monocytes/macrophages. In this study, we established a new M-CSF-mediated differentiation-inducing system, and examined how the level and duration of the activation of ERK preceded M-CSF-mediated differentiation. TF-1-fms human leukemia cells rapidly proliferated in response to M-CSF. However, in the presence of a phorbol ester, TPA, TF-1-fms cells definitely switched their responsiveness to M-CSF from proliferation to differentiation, as evidenced by a more drastic morphological change and the appearance of cells with a higher level of phagocytic activity. In TF-1-fms cells expressing HIV-1 Nef protein in a conditionally active-manner, both M-CSF-mediated proliferation and M-CSF/TPA-mediated differentiation were inhibited by the activation of Nef. The Nef-active cells showed perturbed patterns of ERK activation. Under the proliferation-inducing conditions (TPA-free), parental or Nef-inactive cells showed modest ERK activation following M-CSF stimulation, whereas Nef-active cells showed an earlier and transient ERK activation, despite a decrease in their proliferation rate. Under the differentiation-inducing conditions, parental or Nef-inactive cells showed increased and prolonged ERK activation following M-CSF stimulation, whereas Nef-active cells showed transient ERK activation. These results supported the idea that the increased and prolonged ERK activation led to M-CSF-mediated macrophage differentiation but not to proliferation.     

Muscle and blood ammonia and lactate responses to prolonged exercise with hyperoxia

Investigations using nonsteady-state and fatiguing exercise protocols have demonstrated a strong relationship between ammonia and lactate metabolism and have suggested a cause and effect relationship between these two variables. We investigated the lactate-ammonia response using prolonged exercise and inspiration of hyperoxic gas (60% O2-40% N2). The exercise consisted of either 70-75% maximal O2 uptake (VO2 max) for 40 min (series 1, n = 6) or 75-80% VO2max for 30 min (series 2, n = 6) with the subjects inspiring room air on one occasion and hyperoxia in the other test. In both series blood ammonia rose continuously throughout the exercise regardless of the inspired gas treatment; in contrast blood lactate did not increase after 10 min with room air, and with hyperoxia blood lactate was reduced. Muscle lactate and ammonia (series 2; vastus lateralis) had responses similar to the blood data. The data demonstrated no apparent lactate-ammonia relationship with prolonged exercise or in response to hyperoxia, suggesting that ammonia production can be independent of lactate metabolism. The data also suggest that type I fibers can be a major source of ammonia in humans.     

Responses of baboons to prolonged hyperoxia: physiology and qualitative pathology.

Cardiopulmonary responses to prolonged hyperoxia and their relationships to the development of lung pathology have not been fully characterized in primates. In this study, circulatory hemodynamics and pulmonary function, vascular permeability, and leukocyte sequestration were measured in male baboons after 100% O2 exposure and related to ultrastructural changes of lung injury by electron microscopy. Three groups of animals were exposed to 100% O2 in an exposure cage for 40, 66, and 80 h, respectively. A fourth group of animals was exposed in a cage for 80 h and then anesthetized and ventilated with 100% O2 for additional time. These animals were exposed for a total duration of 110 h or until death from the injury. Physiological responses to hyperoxia were characterized by decreases in total lung capacity and inspiratory capacity at 80 and 110 h. A significant increase in pulmonary leukocyte accumulation was noted by 80 h. Extravascular lung water and permeability surface-area product increased at 80 and 110 h. Cardiac output and stroke volume also decreased, and systemic vascular resistance increased after 80 and 110 h of hyperoxia. Histopathological changes were present in the lungs of all but the 40-h exposure group. Animals exposed for 66 h showed endothelial injury and neutrophil accumulation. By 80 h, animals showed endothelial cell destruction, interstitial edema, and type I cell injury. At 110 h, animals showed substantial destruction of endothelial and type I epithelial cells, exposure of alveolar basement membrane, congestion of capillaries, and substantial interstitial edema. The data indicate that histological changes by electron microscopy precede physiological responses to hyperoxic pulmonary injury in baboons by as much as 14 h and that the physiological responses to early hyperoxic injury are relatively insensitive to the pathological injury.     

The protein tyrosine phosphatase SHP-2 regulates interleukin-1-induced ERK activation in fibroblasts

Focal adhesion complexes are actin-rich, cytoskeletal structures that mediate cell adhesion to the substratum and also selectively regulate signal transduction pathways required for interleukin (IL)-1beta signaling to the MAP kinase, ERK. IL-1-induced ERK activation is markedly diminished in fibroblasts deprived of focal adhesions whereas activation of p38 and JNK is unaffected. While IL-1 signaling is known to involve the activity of protein and lipid kinases including MAP kinases, FAK, and PI3K, little is known about the role of phosphatases in the regulation of IL-1 signal generation and attenuation. Here we demonstrate that SHP-2, a protein tyrosine phosphatase present in focal adhesions, modulates IL-1-induced ERK activation and the transient actin stress fiber disorganization that occurs following IL-1 treatment in human gingival fibroblasts. Using a combination of immunoblotting, immunoprecipitation, and immunostaining we show that SHP-2 is present in nascent focal adhesions and undergoes phosphorylation on tyrosine 542 in response to IL-1 stimulation. Blocking anti-SHP-2 antibodies, electoporated into the cytosol of fibroblasts, inhibited IL-1-induced ERK activation, actin filament assembly, and cell contraction, indicating a role for SHP-2 in these processes. In summary, our data indicate that SHP-2, a focal adhesion-associated protein, participates in IL-1-induced ERK activation likely via an adaptor function.     

Sphingosine-1-phosphate promotes mouse melanocyte survival via ERK and Akt activation

In this study, we investigated the signalling pathways induced by ultraviolet B (UVB) and the effects of sphingosine-1-phosphate on UVB-induced apoptosis of mouse melanocytes, Mel-Ab, and observed the cytoprotective effects of sphingosine-1-phosphate on UVB-induced apoptosis. Since sphingosine-1-phosphate is a well-known mitogenic agent, we thought it possible that the mitogenic effect of sphingosine-1-phosphate might contribute to cell survival. However, we found that sphingosine-1-phosphate significantly inhibits DNA synthesis. We next examined the regulation of the three major subfamilies of mitogen-activated protein (MAP) kinases and of the Akt pathway by sphingosine-1-phosphate against UVB-induced apoptosis. UVB irradiation resulted in the remarkable and sustained activation of c-Jun N-terminal kinase (JNK), while p38 MAP kinase was only transiently activated. The basal level of extracellular signal-regulated protein kinase (ERK) phosphorylation decreased 30 min after UVB irradiation, whereas the basal level of Akt phosphorylation was unaffected by UVB. We also found that sphingosine-1-phosphate potently stimulates the phosphorylation of both ERK and Akt, which are involved in the cell survival-signalling cascade. Furthermore, the specific inhibition of the ERK and Akt pathways by PD98059 and LY294002, respectively, restored the cytoprotective effect induced by sphingosine-1-phosphate. On the other hand, the p38 inhibitor SB203580 additively enhanced the cytoprotective effect on sphingosine-1-phosphate. Based on these results, we conclude that the activation of p38 MAP kinase plays an important role in UVB-induced apoptosis, and that sphingosine-1-phosphate probably exert its cytoprotective effect in Mel-Ab cells through ERK and Akt activation.     

Erythropoietin receptor Y479 couples to ERK1/2 activation via recruitment of phospholipase Cgamma

Red blood cell development is primarily controlled by erythropoietin (EPO). Several studies have revealed the importance of EPO-R Y343 and Y479 for erythroid cell growth, differentiation, and survival. In order to isolate critical signaling proteins that bind to EPO-R, we initiated a Cloning of Ligand Target (COLT) screen using a murine embryonic day 16 phage library and a biotinylated EPO-R Y343 phosphopeptide. One of the clones isolated encodes Phospholipase C (PLC)gamma1. PLCgamma1 is rapidly tyrosine phosphorylated upon EPO stimulation and associates with EPO-R in an SH2-domain-dependent manner. Although PLCgamma1 bound EPO-R Y343, Y401, Y429, Y431, and Y479 in the COLT screen, PLCgamma1 required Y479 for association with EPO-R in Ba/F3-EPO-R cells. Studies have identified EPO-R Y479 as important for ERK activation. Since PI3-kinase binds EPO-R Y479, one group has suggested that ERK activation downstream of PI3-kinase accounts for the importance of this residue in EPO signaling. However, we show that inhibition of PI3-kinase does not abolish ERK activation. Furthermore, we demonstrate interaction of PLCgamma1 with Grb2 and SOS2. Hence, we have identified a novel adapter function for PLCgamma1 in EPO signaling in which recruitment of PLCgamma1 to EPO-R may lead to activation of the ERK pathway.     

Cdc25A and ERK interaction: EGFR-independent ERK activation by a protein phosphatase Cdc25A inhibitor, compound 5

Extracellular signal-regulated kinase (ERK) plays a central role in regulating cell growth, differentiation, and apoptosis. We previously found that 2-(2-mercaptoethanol)-3-methyl-1,4-napthoquinone or Compound 5 (Cpd 5), is a Cdc25A protein phosphatase inhibitor and causes prolonged, strong ERK phosphorylation which is triggered by epidermal growth factor receptor (EGFR) activation. We now report that Cpd 5 can directly cause ERK phosphorylation by inhibiting Cdc25A activity independently of the EGFR pathway. We found that Cdc25A physically interacted with and de-phosphorylated phospho-ERK both in vitro and in cell culture. Inhibition of Cdc25A activity by Cpd 5 resulted in ERK hyper-phosphorylation. Transfection of Hep3B human hepatoma cells with inactive Cdc25A mutant enhanced Cpd 5 action on ERK phosphorylation, whereas over-expression of Cdc25A attenuated this Cpd 5 action. Furthermore, endogenous Cdc25A knock-down by Cdc25A siRNA resulted in a constitutive-like ERK phosphorylation and Cpd 5 treatment further enhanced it. In EGFR-devoid NR6 fibroblasts and MEK (ERK kinase) mutated MCF7 cells, Cpd 5 treatment also resulted in ERK phosphorylation, providing support for the idea that Cpd 5 can directly act on ERK phosphorylation by inhibiting Cdc25A activity. These data suggest that phospho-ERK is likely another Cdc25A substrate, and Cpd 5-caused ERK phosphorylation is probably regulated by both EGFR-dependent and EGFR-independent pathways.     

Solution structure of ERK2 binding domain of MAPK phosphatase MKP-3: structural insights into MKP-3 activation by ERK2

MAP kinases (MAPKs), which control mitogenic signal transduction in all eukaryotic organisms, are inactivated by dual specificity MAPK phosphatases (MKPs). MKP-3, a prototypical MKP, achieves substrate specificity through its N-terminal domain binding to the MAPK ERK2, resulting in the activation of its C-terminal phosphatase domain. The solution structure and biochemical analysis of the ERK2 binding (EB) domain of MKP-3 show that regions that are essential for ERK2 binding partly overlap with its sites that interact with the C-terminal catalytic domain, and that these interactions are functionally coupled to the active site residues of MKP-3. Our findings suggest a novel mechanism by which the EB domain binding to ERK2 is transduced to cause a conformational change of the C-terminal catalytic domain, resulting in the enzymatic activation of MKP-3.