The closely related mitogen-activated protein kinase isoforms extracellular signal-regulated kinase 1 (ERK1) and ERK2 have been implicated in the control of cell proliferation, differentiation and survival. However, the specific in vivo functions of the two ERK isoforms remain to be analysed. Here, we show that disruption of the Erk2 locus leads to embryonic lethality early in mouse development after the implantation stage. Erk2 mutant embryos fail to form the ectoplacental cone and extra-embryonic ectoderm, which give rise to mature trophoblast derivatives in the fetus. Analysis of chimeric embryos showed that Erk2 functions in a cell-autonomous manner during the development of extra-embryonic cell lineages. We also found that both Erk2 and Erk1 are widely expressed throughout early-stage embryos. The inability of Erk1 to compensate for Erk2 function suggests a specific function for Erk2 in normal trophoblast development in the mouse, probably in regulating the proliferation of polar trophectoderm cells.

We studied the alterations in skeletal muscle protein breakdown in long lasting sepsis using a rat model that reproduces a sustained and reversible catabolic state, as observed in humans. Rats were injected intravenously with live Escherichia coli ; control rats were pair-fed to the intake of infected rats. Rats were studied in an acute septic phase (day 2 postinfection), in a chronic septic phase (day 6), and in a late septic phase (day 10). The importance of the lysosomal, Ca 2 ϩ -dependent, and ubiquitin-proteasome proteolytic processes was investigated using proteolytic inhibitors in incubated epitrochlearis muscles and by measuring mRNA levels for critical components of these pathways. Protein breakdown was elevated during the acute and chronic septic phases (when significant muscle wasting occurred) and returned to control values in the late septic phase (when wasting was stopped). A nonlysosomal and Ca 2 ϩ -independent process accounted for the enhanced proteolysis, and only mRNA levels for ubiquitin and subunits of the 20 S proteasome, the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates, paralleled the increased and decreased rates of proteolysis throughout. However, increased mRNA levels for the 14-kD ubiquitin conjugating enzyme E2, involved in substrate ubiquitylation, and for cathepsin B and m-calpain were observed in chronic sepsis. These data clearly support a major role for the ubiquitinproteasome dependent proteolytic process during sepsis but also suggest that the activation of lysosomal and Ca 2 ϩ -dependent proteolysis may be important in the chronic phase. (

Background-Myocardial infarction causes a rapid and largely irreversible loss of cardiac myocytes that can lead to sudden death, ventricular dilation, and heart failure. Members of the mitogen-activated protein kinase (MAPK) signaling cascade have been implicated as important effectors of cardiac myocyte cell death in response to diverse stimuli, including ischemia-reperfusion injury. Specifically, activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) has been associated with cardioprotection, likely through antagonism of apoptotic regulatory pathways. Methods and Results-To establish a causal relationship between ERK1/2 signaling and cardioprotection, we analyzed Erk1 nullizygous gene-targeted mice, Erk2 heterozygous gene-targeted mice, and transgenic mice with activated MEK1-ERK1/2 signaling in the heart. Although MEK1 transgenic mice were largely resistant to ischemia-reperfusion injury, Erk2 ϩ/Ϫ gene-targeted mice showed enhanced infarction areas, DNA laddering, and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick-end labeling (TUNEL) compared with littermate controls. In contrast, enhanced MEK1-ERK1/2 signaling protected hearts from DNA laddering, TUNEL, and preserved hemodynamic function assessed by pressure-volume loop recordings after ischemia-reperfusion injury. Conclusions-These data are the first to demonstrate that ERK2 signaling is required to protect the myocardium from ischemia-reperfusion injury in vivo. Key Words: ischemia Ⅲ cardiac output Ⅲ mitogen-activated protein kinases Ⅲ infarction Ⅲ signal transduction T he mitogen-activated protein kinases (MAPK) constitute an essential signal transduction cascade that occupies a central position in cell growth, differentiation, apoptosis, and transformation. 1 Although it is somewhat oversimplified, the MAPK signaling pathway consists of a sequence of successively acting kinases that ultimately result in the dual phosphorylation and activation of the terminal kinases p38, c-Jun N-terminal kinase (JNKs), and extracellular signal-regulated kinase (ERKs). 2 The MAPK signaling cascade is initiated in cardiac myocytes by G-protein-coupled receptors, receptor tyrosine kinases, cardiotrophin-1 (gp130 receptor), and stress stimuli. 3 The major upstream activators of ERK1/2 are 2 MAP kinase kinases (MAPKK), MEK1 and MEK2, which directly phosphorylate the dual site on ERK kinases (Thr-Glu-Tyr). 2 Within the heart, members of the MAPK cascade have been implicated in regulating myocyte survival after ischemia-reperfusion injury, oxidative stress, and anthracycline exposure. Indeed, a number of studies support the hypothesis that the MEK1-ERK1/2 branch of the MAPK pathway is cardioprotective by directly antagonizing myocyte apoptosis. 4 Although MEK1-ERK1/2 signaling is thought to protect the myocardium from apoptotic insults, definitive genetic data demonstrating a necessary function for this pathway in vivo have not been reported. Methods Murine In Vivo Ischemia-Reperfusion and Pressure-Volume AcquisitionAdult mice (aged 8 to 10 week...

The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinase signaling pathway plays an important role in the proliferative response of mammalian cells to mitogens. However, the individual contribution of the isoforms ERK1 and ERK2 to cell proliferation control is unclear. The two ERK isoforms have similar biochemical properties and recognize the same primary sequence determinants on substrates. On the other hand, analysis of mice lacking individual ERK genes suggests that ERK1 and ERK2 may have evolved unique functions. In this study, we used a robust genetic approach to analyze the individual functions of ERK1 and ERK2 in cell proliferation using genetically matched primary embryonic fibroblasts. We show that individual loss of either ERK1 or ERK2 slows down the proliferation rate of fibroblasts to an extent reflecting the expression level of the kinase. Moreover, RNA interference-mediated silencing of ERK1 or ERK2 expression in cells genetically disrupted for the other isoform similarly reduces cell proliferation. We generated fibroblasts genetically deficient in both Erk1 and Erk2. Combined loss of ERK1 and ERK2 resulted in a complete arrest of cell proliferation associated with G 1 arrest and premature replicative senescence. Together, our findings provide compelling genetic evidence for a redundant role of ERK1 and ERK2 in promoting cell proliferation.

Quantitative phosphoproteomics was used to measure the dynamic changes in phosphorylation of ERK1/2 MAP kinases consensus sequences in epithelial cells and to identify 128 novel candidate substrates involved in a broad spectrum of biological functions.

Background: The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established.

The development of new pharmacological approaches for preventing muscle wasting in cancer is an important goal because cachectic patients display a reduced response to chemotherapy and radiotherapy. Xanthine derivatives such as pentoxifylline inhibit tumour necrosis factor-alpha (TNF) production, which has been implicated in the signalling of muscle wasting. However, the effect of pentoxifylline has been inconclusive in clinical trials. We report here the first direct evidence that daily injections of torbafylline (also known as HWA 448), another xanthine derivative, had no effect by itself on muscle proteolysis in control healthy rats. In cancer rats, the drug blocked the lipopolysaccharide-induced hyperproduction of TNF and prevented muscle wasting. In these animals HWA 448 suppressed the enhanced proteasome-dependent proteolysis, which is sensitive to the proteasome inhibitor MG132, and the accumulation of high-molecular-mass ubiquitin (Ub) conjugates in the myofibrillar fraction. The drug also normalized the enhanced muscle expression of Ub, which prevails in the atrophying muscles from cancer rats. In contrast, HWA 448 did not reduce the increased expression of either the 14 kDa Ub conjugating enzyme E2 or the ATPase and non-ATPase subunits of the 19 S regulatory complex of the 26 S proteasome, including the non-ATPase subunit S5a, which recognizes polyUb degradation signals. Finally, the drug also prevented muscle wasting in septic rats (which exhibit increased TNF production), and was much more potent than pentoxifylline or other xanthine derivatives. Taken together, the data indicate that HWA 448 is a powerful inhibitor of muscle wasting that blocks enhanced Ub-proteasome-dependent proteolysis in situations where TNF production rises, including cancer and sepsis.

The epidermal growth factor receptor (EGFR) was recently identified as a signal transducer of G protein-coupled receptors (GPCRs). In this study, we have examined the contribution of EGFR transactivation to the growth-promoting effect of GPCRs on vascular smooth muscle cells. Activation of the Gq-coupled ANG II receptor or Gi-coupled lysophosphatidic acid receptor resulted in increased tyrosine phosphorylation and activation of EGFR. Specific inhibition of EGFR kinase activity by tyrphostin AG-1478 or expression of a dominant-negative EGFR mutant abolished this response. Importantly, inhibition of EGFR function strongly attenuated the global stimulation of protein synthesis by GPCR agonists in vitro in cultured aortic smooth muscle cells and in vivo in the rat aorta and in small resistance arteries. The growth inhibition was associated with a marked reduction of extracellular signal-regulated kinase and phosphoinositide 3-kinase pathway activity and the resulting suppression of eukaryotic translation initiation factor 4E and 4E binding protein 1 phosphorylation. Our results demonstrate that EGFR transactivation is a physiologically relevant action of GPCRs linked to translational control and protein synthesis.