The MEK/ERK and PI3K/AKT pathways are often concurrently activated by separate genetic alterations in colorectal cancer (CRC), which is associated with CRC progression and poor survival. However, how activating both pathways is required for CRC metastatic progression remains unclear. Our recent study showed that both ERK and AKT signaling are required to activate eIF4E-initiated cap-dependent translation via convergent regulation of the translational repressor 4E-BP1 for maintaining CRC transformation. Here, we identified that the activation of cap-dependent translation by cooperative ERK and AKT signaling is critical for promotion of CRC motility and metastasis. In CRC cells with coexistent mutational activation of ERK and AKT pathways, inhibition of either MEK or AKT alone showed limited activity in inhibiting cell migration and invasion, but combined inhibition resulted in profound effects. Genetic blockade of the translation initiation complex by eIF4E knockdown or expression of a dominant active 4E-BP1 mutant effectively inhibited migration, invasion and metastasis of CRC cells, whereas overexpression of eIF4E or knockdown of 4E-BP1 had the opposite effect and markedly reduced their dependence on ERK and AKT signaling for cell motility. Mechanistically, we found that these effects were largely dependent on the increase in mTORC1-mediated survivin translation by ERK and AKT signaling. Despite the modest effect of survivin knockdown on tumor growth, reduction of the translationally-regulated survivin profoundly inhibited motility and metastasis of CRC. These findings reveal a critical mechanism underlying the translational regulation of CRC metastatic progression, and suggest that targeting cap-dependent translation may provide a promising treatment strategy for advanced CRC.

Mobilization of intracellular Ca2ϩ is a critical cellular response to lysophosphatidic acid (LPA) in many cell types. Recent identification of endothelial differentiation gene (Edg) 2 and Edg4 as subtypes of G protein-coupled receptors for LPA allowed examination of the Ca 2ϩ mobilization mediated specifically by each subtype. To reduce endogenous background levels while enhancing recombinant receptor-specific signals, the aequorin luminescence method was used to quantify cytoplasmic Ca 2ϩ

Sphingosine 1-phosphate (S1P) regulates cell proliferation, apoptosis, motility, and neurite retraction. Contradictory reports propose that S1P acts as either an intracellular second messenger or an extracellular ligand for cell-surface receptors. Hence, the precise signaling mechanisms mediating the diverse cellular effects of S1P remain to be determined. Here, we investigate whether S1P stimulation of cell proliferation, survival, and related signaling events can be mediated by the recently cloned Edg family members of G protein-coupled receptors. We observed that S1P treatment significantly increased proliferation of HTC4 hepatoma cells stably transfected with human S1P receptor Edg3 or Edg5, which was attributable to stimulation of cell growth and inhibition of apoptosis caused by serum starvation. Edg3 and Edg5 transduced S1P-evoked signaling events relevant to cell proliferation and survival, including activation of the ERK/MAP kinases, and immediate-early induction of c-Jun and c-Fos. Trancriptional activation of reporter genes for the c-fos promoter and the serum response element by Edg3 and Edg5 transfected in Jurkat cells was inhibited by pertussis toxin and C3 exoenzyme, implicating G i/o -and Rho-dependent pathways. Our data also indicated that Edg3 and Edg5 mediated the serum response element activation through transcriptional factors Elk-1 and serum response factor. Thus, specific G protein-coupled receptors Edg3 and Edg5 account for, at least in part, S1P-induced cell proliferation, survival, and related signaling events.Several types of sphingolipids, such as ceramide, sphingosine, and sphingosine 1-phosphate (S1P), 1 have attracted increasing attention as mediators of important cellular functions (1-3). S1P, in particular, is implicated in cell proliferation (4 -6), suppression of apoptosis (7,8), modulation of cell motility, tumor invasiveness (9, 10), platelet activation (11), and neurite retraction (12). Cellular signaling by S1P evokes activation of the mitogen-activated protein kinases (MAP kinases) (9, 13), inward rectifying K ϩ channels (I k(Ach) ) (14, 15), and intracellular Ca 2ϩ mobilization (4, 14, 16 -20). Despite extensive observations in various cell types, the precise signaling mechanisms by which S1P exerts its cellular effects remain undetermined. The major uncertainty is whether S1P acts intracellularly as a second messenger, or extracellularly as a receptor ligand, or both. The second messenger hypothesis is based mainly on the following observations: first, S1P stimulated Ca 2ϩ release directly from isolated endoplasmic reticulum preparations through an inositol trisphosphate-independent mechanism (17, 18); second, inhibition of S1P production by sphingosine kinase inhibitors, e.g. N,N-dimethylsphingosine or dihydrosphingosine, blocked cell proliferation induced by platelet-derived growth factor or serum, and Ca 2ϩ mobilization induced by IgE or muscarinic receptor stimulation (21,22). These observations, together with the finding that the intracellular level of S1P was eleva...

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Ischemic heart disease is a leading cause of death in human population and protection of myocardial infarction (MI) associated with ischemia-reperfusion (I/R) remains a challenge. MG53 is an essential component of the cell membrane repair machinery that protects injury to the myocardium. We investigated the therapeutic value for using the recombinant human MG53 (rhMG53) protein for treatment of MI. Using Langendorff perfusion of isolated mouse heart, we found that I/R caused injury to cardiomyocytes and release of endogenous MG53 into the extracellular solution. rhMG53 protein applied to the perfusion solution concentrated at injury sites on cardiomyocytes to facilitate cardioprotection. With rodent models of I/R-induced MI, we established the in vivo dosing range for rhMG53 in cardioprotection. Using a porcine model of angioplasty-induced MI, the cardioprotective effect of rhMG53 was evaluated. Intravenous administration of rhMG53, either prior to or post ischemia, reduced infarct size and troponin I release in the porcine model when examined at 24 hours post reperfusion. Echocardiogram and histological analyses revealed that the protective effects for rhMG53 observed following acute MI led to long-term improvement in cardiac structure and function in the porcine model when examined at 4 weeks post operation. Our study supports the concept that rhMG53 could have potential therapeutic value for treatment of MI in human patients with ischemic heart diseases.

Methionine adenosyltransferase 2B (MAT2B) encodes for two variant proteins V1 and V2 that promote cell growth. Using in-solution proteomics, GIT1 (G-protein-coupled receptor kinase-interacting protein 1) was identified as a potential interacting partner of MAT2B. Here we examined the functional significance of this interplay. Coimmunoprecipitation experiments examined protein interactions. Tissue expression levels of proteins were examined using immunohistochemistry and Western blotting. The expression levels of the proteins were varied using transient knockdown or overexpression to observe the effect of alterations in each protein on the entire complex. Direct interaction among the individual proteins was further verified using in vitro translated and recombinant proteins. We found both MAT2B variants interact with GIT1. Overexpression of V1, V2 or GIT1 activated MEK1, ERK, raised cyclin D1 protein level and increased growth, while the opposite occurred when V1, V2 or GIT1 was knocked down. MAT2B and GIT1 require each other to activate MEK1/ERK and increase growth. MAT2B directly interacts with MEK1, GIT1 and ERK2. Expression level of V1, V2 or GIT1 directly influenced recruitment of GIT1 or MAT2B and ERK2 to MEK1, respectively. In pull down assays, MAT2B directly promoted binding of GIT1 and ERK2 to MEK1. MAT2B and GIT1 interact and are overexpressed in most human liver and colon cancer specimens. Increased expression of V1, V2 or GIT1 promoted growth in an orthotopic liver cancer model; while increased expression of either V1 or V2 with GIT1 further enhanced growth and lung metastasis. Conclusion MAT2B and GIT1 form a scaffold, which recruits and activates MEK and ERK to promote growth and tumorigenesis. This novel MAT2B/GIT1 complex may provide a potential therapeutic gateway in human liver and colon cancer.

Lysophosphatidic acid (LPA) from platelets and mononuclear phagocytes regulates T cell functions through endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs). Human blood unactivated CD4+ T cells express predominant ly Edg-4 LPA R over marginal levels of Edg-2 LPA R, as assessed by semiquantitative PCR and Western blots. After mitogen activation, the CD4+ T cells express Ed g-2 Rs at approximately one half the level of Edg-4 Rs. Secretion of IL-2 by unactivated Edg-4 R-predominant CD4+ T cells incubated with anti-CD3 plus anti-CD28 antibodies was suppressed significantly and by up to 60% by 10-10 M to 10-6 M LPA, whereas secretion of IL-2 by mitogen-activated Edg-2 R and Edg-4 R codominant CD4+ T cells was enhanced by up to twofold by the same concentrations of LPA. The possibility that the two Edg Rs transduce different LPA signals to CD4+ T cells was supported by findings that IL-2 secretion was inhibited by mouse anti-Edg-4 R monoclonal antibody, but enhanced by mouse anti-Edg-2 R monoclonal antibody. The separate effects of each LPA R were studied in Jurkat T cell transfectants expressing principally human Edg-2 Rs (Jurkat-T-2) or Edg-4 Rs (Jurkat-T-4) and stimulated with anti-CD3 plus phorbol myristate acetate. LPA and anti-Edg-4 R antibody suppressed IL-2 secretion by stimulated Jurkat-T-4 cells, whereas LPA and anti-Edg-2 R antibody enhanced IL-2 secretion by stimulated Jurkat-T-2 cells. Activation-induced alterations in the relative levels of Edg-2 and -4 Rs on CD4+ T cells thus reverse the effects of LPA on T cell receptor-stimulated generation of IL-2.

PF suppresses rat AA at least partly by inhibiting abnormal proliferation of synoviocytes and the production of IL-1, PGE2, IL-6, VEGF and GM-CSF by synoviocytes and reducing Gi and COX-2 expression in synovium.