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.

Alcohol consumption shows a J-shaped association with lower urinary tract symptoms in the general screening population.

PURPOSE: Controversial and contradictory data on the association between alcohol consumption and lower urinary tract symptoms are currently available in the literature. In this study we determined the association between alcohol consumption and lower urinary tract symptoms, including voiding and storage symptoms, in a large general screening population. MATERIALS AND METHODS: This cross-sectional study included 30,196 men 30 years old or older participating in a comprehensive health evaluation at the Seoul National University Hospital Healthcare System Gangnam Center. Men with a history of prostate related medical problems such as prostate cancer, prostate surgery or prostatitis were excluded from study. Using the International Prostate Symptom Score, lower urinary tract symptoms were defined as a score of 8 or greater, indicating moderate to severe symptoms. We used logistic regression analysis to determine the association between alcohol consumption and lower urinary tract symptoms. RESULTS: After adjustment for eligible covariates, graphing of the association between alcohol consumption and the risk of moderate to severe lower urinary tract symptoms showed a J-shaped curve. Compared with nondrinkers, the odds ratios of moderate to severe lower urinary tract symptoms were 0.91 (95% CI 0.84-0.98) in men who drank 0 to 10 gm daily and 1.19 (95% CI 1.07-1.33) in those who drank 40 or more gm daily. This is a cross-sectional study with data from self-reported alcohol consumption and, therefore, the reported amounts of alcohol consumption might be underestimated. CONCLUSIONS: To the best of our knowledge this is the largest population based study to evaluate the relationship between alcohol consumption and moderate to severe lower urinary tract symptoms, including voiding and storage symptoms. In men alcohol consumption shows a J-shaped curve relationship with the risk of moderate to severe lower urinary tract symptoms.

Metabolic syndrome and accompanying hyperinsulinemia have favorable effects on lower urinary tract symptoms in a generally healthy screened population.

PURPOSE: We assessed the association of metabolic syndrome, insulin resistance and lower urinary tract symptoms in a large, screened adult population. MATERIALS AND METHODS: We analyzed 33,841 Korean men 30 years old or older who underwent routine health assessments from October 2003 to February 2010. Metabolic syndrome was defined according to the modified Adult Treatment Panel III guidelines. Lower urinary tract symptoms were assessed using the International Prostate Symptom Score. Blood samples were drawn in the morning after patients had fasted at least 12 hours. RESULTS: Lower urinary tract symptoms had a marginally negative association with metabolic syndrome after adjusting for age (p = 0.045). This negative association became more significant as the number of metabolic syndrome components increased (p trend <0.01), especially voiding symptoms (p trend <0.01). Increasing the level of fasting insulin and the severity of insulin resistance were associated with a lower age adjusted OR for lower urinary tract symptoms (p <0.01 and 0.03, respectively). However, the diabetes group with high HbA1c (8.0% or greater) had a higher age adjusted OR for lower urinary tract symptoms, especially storage symptoms. The group with metabolic syndrome plus insulin resistance had lower total International Prostate Symptom Score, voiding symptoms, storage symptoms and quality of life scores than those without metabolic syndrome and/or insulin resistance (p <0.01, 0.01, 0.047 and 0.03, respectively). CONCLUSIONS: Metabolic syndrome, insulin resistance and the accompanying hyperinsulinemia may have favorable effects on lower urinary tract symptoms in the early compensatory stage, especially voiding symptoms. However, advanced diabetes may have unfavorable effects on lower urinary tract symptoms, especially storage symptoms. Hyperinsulinemia in patients with metabolic syndrome or insulin resistance may be a key factor in this phenomenon.

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.

Efficacy of protein A-HRP in an immunological study of black rockfish (Sebastes schlegeli Higendorf) humoral immune responses.

The efficacy of protein A-horse radish peroxidase (HRP), as compared to that of mouse polyclonal antibody raised against purified Ig, in detection of black rockfish (Sebastes schlegeli Higendorf) immunoglobulin (Ig) was examined. Protein A affinity chromatography successfully purified Ig from black rockfish serum; the purified-Ig could be visualised as two protein bands (MW 70 and 25kDa) following resolution with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. In SDS-PAGE immunoblot profiles of the purified-Ig, the mouse polyclonal antibody recognised both the light chain and heavy chains of rockfish Ig, whereas protein A-HRP immunostained only the heavy chain of rockfish Ig. These results suggest that protein A-HRP may be used to detect rockfish antibody-antigen complexes in immunoassays. In a 2-DE immunoblot assay for exploring antigenic profiles of Lactococcus garvieae KG9408, protein A-HRP successfully detected specific antibodies to antigenic proteins of L. garvieae in the rockfish Ig. In addition, enzyme linked immunosorbent assay (ELISA) showed a high correlation between the results obtained for positivity of L. garvieae when protein A-HRP and the mouse polyclonal antibody-was used to analyse samples from 25 diseased rockfish. These results collectively indicate that protein A-HRP has a high affinity for Ig, and may be useful for new investigations into the humoral immune responses of rockfish.

Experimental evaluation of pathogenicity of Lactococcus garvieae in black rockfish (Sebastes schlegeli)

Black rockfish (Sebastes schlegeli) is an important mariculture species in Korea. The production of this fish is drastically declined due to bacterial diseases, particularly streptococcosis caused by Lactococcus garvieae. The bacterial surface characteristics of SJ7 and TY6 were found to have capsule but not NB13 and YS18. The experiential evaluation of L. garvieae pathogenicity, the capsular isolates showed high cumulative mortality i.e. SJ7 (100%) and TY6 (60%) compared to non-capsular isolates. Based on this result the capsular isolates L. garvieae were highly suspected as the causative agent of streptococcosis in rockfish.