ß2-Adrenergic receptor (ß2-AR) agonist formoterol suppresses differentiation of L6 myogenic cells by blocking PI3K-AKT pathway.

ß2-Adrenergic receptor (ß2-AR) is implicated in muscle metabolic activities such as glycogen metabolism, glucose uptake, lipolysis and muscle growth. However, the functional role of ß2-AR in the differentiation of skeletal muscle is largely unknown. Here, we examined the functional role of ß2-AR in L6 myoblast differentiation using the long-term-acting ß2-AR-specific agonist formoterol. We observed that formoterol treatment strongly suppressed L6 myoblast differentiation and the expression of myosin heavy chain (MHC) in a dose- and time-dependent manner. Showing that both long-acting agonist (formoterol) and short-acting agonist (terbutaline) inhibited the induction of MHC protein, whereas ß2-AR antagonist (ICI-118,551) upregulated MHC expression, we clearly demonstrated that ß2-AR is involved in L6 myoblast differentiation. Furthermore, our pharmacological inhibition study revealed that the PI3K-AKT pathway is the main signaling pathway for myotube formation. Formoterol inhibited the activation of PI3K-AKT signaling, but not that of ERK signaling. Moreover, formoterol selectively inhibited AKT activation by IGF-I, but not by insulin. Collectively, our findings reveal a previously undocumented role of ß2-AR activation in modulating the differentiation of L6 myoblasts.

Carboxy-terminal domain of Cas differentially modulates c-Jun expression, DNA synthesis, and membrane ruffling induced by insulin, EGF, and IGF-1.

p130 Crk-associated substrate (Cas) is an adaptor protein associating with many other signaling proteins and regulates a various biological processes including cell adhesion, migration, and growth factor stimulation. However, the exact functional role of Cas in growth factor signaling pathway was poorly understood. Here we investigated the role of Cas and its domains in the effects of insulin, EGF, and IGF-1 on c-Jun gene expression, DNA synthesis, cytoskeletal reorganization. We found that microinjection of anti-Cas antibody and C-terminal domain of Cas (Cas-CT) specifically inhibited EGF-induced, but not insulin- or IGF-1-induced, c-Jun expression. Cell cycle progression and cytoskeleton reorganization induced by insulin and EGF, but not by IGF-1, were inhibited by microinjected anti-Cas and Cas-CT. In contrast, microinjection of the substate domain (Cas-SD) of Cas did not have any inhibitory effects. These results revealed that the Cas-CT is differentially implicated in insulin and EGF-mediated, but not IGF-1-mediated, c-Jun expression, DNA synthesis and membrane ruffling.

Oncogenic N-Ras Stimulates SRF-Mediated Transactivation via H3 Acetylation at Lysine 9.

Signal transduction pathways regulate the gene expression by altering chromatin dynamics in response to mitogens. Ras proteins are key regulators linking extracellular stimuli to a diverse range of biological responses associated with gene regulation. In mammals, the three ras genes encode four Ras protein isoforms: H-Ras, K-Ras4A, K-Ras4B, and N-Ras. Although emerging evidence suggests that Ras isoforms differentially regulate gene expressions and are functionally nonredundant, the mechanisms underlying Ras specificity and Ras signaling effects on gene expression remain unclear. Here, we show that oncogenic N-Ras acts as the most potent regulator of SRF-, NF-κB-, and AP-1-dependent transcription. N-Ras-RGL2 axis is a distinct signaling pathway for SRF target gene expression such as Egr1 and JunB, as RGL2 Ras binding domain (RBD) significantly impaired oncogenic N-Ras-induced SRE activation. By monitoring the effect of Ras isoforms upon the change of global histone modifications in oncogenic Ras-overexpressed cells, we discovered that oncogenic N-Ras elevates H3K9ac/H3K23ac levels globally in the chromatin context. Importantly, chromatin immunoprecipitation (ChIP) assays revealed that H3K9ac is significantly enriched at the promoter and coding regions of Egr1 and JunB. Collectively, our findings define an undocumented role of N-Ras in modulating of H3 acetylation and in gene regulation.

Functional roles of BCAR3 in the signaling pathways of insulin leading to DNA synthesis, membrane ruffling and GLUT4 translocation.

Breast cancer anti-estrogen resistance 3 (BCAR3) is an SH2-containing signal transducer and is implicated in tumorigenesis of breast cancer cells. In this study, we found that BCAR3 mediates the induction of ERK activation and DNA synthesis by insulin, but not by IGF-1. Specifically, the SH2 domain of BCAR3 is involved in insulin-stimulated DNA synthesis. Differential tyrosine-phosphorylated patterns of the BCAR3 immune complex were detected in insulin and IGF-1 signaling, suggesting that BCAR3 is a distinct target molecule of insulin and IGF-1 signaling. Moreover, microinjection of BCAR3 inhibitory materials inhibited membrane ruffling induced by insulin, while this did not affect insulin-mediated GLUT4 translocation. Taken together, these results demonstrated that BCAR3 plays an important role in the signaling pathways of insulin leading to cell cycle progression and cytoskeleton reorganization, but not GLUT4 translocation.

BCAR3 regulates EGF-induced DNA synthesis in normal human breast MCF-12A cells.

BCAR3 (breast cancer anti-estrogen resistance 3) is a signal transducer containing an SH2 domain, a proline/serine-rich domain and a GDP-exchange factor homologous domain, whose role in signaling pathways is currently unclear. Furthermore, BCAR3 is implicated in anti-estrogen resistance of breast cancer cells. In the present study, we investigated the functional role of BCAR3 in a mitogenic signaling pathway of EGF in non-tumorigenic human breast epithelial MCF-12A cells. Microinjection of an anti-BCAR3 antibody, siRNAs targeting BCAR3 and an SH2 domain of BCAR3 inhibited EGF-induced DNA synthesis. Direct association of BCAR3 with activated EGF receptor and Cas was observed. Lastly, microinjection of a BCAR3 expression plasmid induced DNA synthesis. These findings suggest that the BCAR3 protein, through its SH2 domain, is involved in the signaling pathways of EGF leading to cell cycle progression, and that BCAR3 itself is part of a mitogenic signaling pathway.

Ethanol impairs insulin's actions through phosphatidylinositol 3-kinase.

Insulin plays an important role in cell metabolism and proliferation. In the present study, we examined the effect of ethanol on insulin actions such as glucose uptake, DNA synthesis, and c-Jun gene expression. Acute treatment with ethanol (200 mM) for 60 minutes inhibited insulin-stimulated 2-deoxyglucose uptake by 50% in 3T3-L1 adipocytes. Insulin-induced DNA synthesis and c-Jun protein expression were also reduced by ethanol treatment in Rat-1 fibroblasts overexpressing normal human insulin receptor. Ethanol has no effect on tyrosine phosphorylation of the insulin receptor and insulin receptor substrate (IRS)-1. However, association of the insulin receptor and IRS-1 with the Src homology 2 domain of the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) was reduced by ethanol. Pretreatment with the antidiabetic drug troglitazone, an insulin-sensitizer, reversed ethanol's inhibition. These results suggest that ethanol specifically inhibits the association of the insulin receptor and IRS-1 with the p85 subunit of PI3-kinase, which is required for increased glucose uptake, DNA synthesis, and c-Jun expression by insulin.

Kimchi and an active component, beta-sitosterol, reduce oncogenic H-Ras(v12)-induced DNA synthesis.

The Korean fermented vegetable food, kimchi, has been demonstrated to have anticancer functional properties. This study examined the effect of kimchi samples, methanol extracts of commercially grown baechu cabbage kimchi (CK) and organically grown baechu cabbage kimchi (OK), as well as the dichloromethane fraction (DCM fr.) from CK, and the active compound (AC), which has been identified as largely beta-sitosterol, from DCM fr., on the Ras-dependent signaling pathway. CK, OK, and DCM fr. exhibited a greater inhibition against the proliferation of Rat2 fibroblasts transformed with Ras(v12) (HO6) than parental Rat2 fibroblasts. In addition, OK and DCM fr. showed a higher inhibitory effect than CK. Furthermore, we employed the single-cell microinjection technique, combined with 3-bromo-5'-deoxyuridine incorporation, to examine the effects of kimchi samples on DNA synthesis induced by microinjected oncogenic Ras(v12). When the DCM fr. and AC were used to treat Rat1 fibroblasts overexpressing human insulin receptors (HIRc-B) and microinjected with oncogenic H-Ras(v12), the DNA synthesis of injected cells was decreased, suggesting that kimchi might block the signaling pathway of oncogenic Ras(v12), thus preventing the proliferation of transformed cells. This study provides additional evidence that kimchi and its active components, including beta-sitosterol, have potential in both the prevention and treatment of cancer, and presents convincing evidence that the anticancer effects may be a result of an inhibition of Ras oncogene signaling.

Leucine zipper domain of HIV-1 gp41 interacted specifically with alpha-catenin.

Interactions between viral and cellular proteins could explain the molecular mechanisms behind the viral life cycle of HIV-1. The envelope protein gp41 of HIV-1 specifically interacted with alpha-catenin, not with beta-catenin. This interaction was shown by in vitro protein assay and in vivo transfected cell systems. Microinjection of the DNA expressing HIV-1 gp160 and alpha-catenin, into the HeLa cell, resulted in the colocalization of gp41 and alpha-catenin. Interestingly the noncleavable mutant of gp160 and alpha-catenin were found to be colocalized in the cell membrane. Mapping of the interaction sites between these two proteins revealed that the leucine zipper-like structure, located between the first and second alpha-helix domains from the carboxy terminus of HIV-1 gp41, interacted strongly with the carboxy terminus of alpha-catenin.