Role of the cytosolic phospholipase A2-linked cascade in signaling by an oncogenic, constitutively active Ha-Ras isoform

Activation of Ras signaling by growth factors has been associated with gene regulation and cell proliferation. Here we characterize the contributory role of cytosolic phospholipase A(2) in the oncogenic Ha-Ras(V12) signaling pathway leading to activation of c-fos serum response element (SRE) and transformation in Rat-2 fibroblasts. Using a c-fos SRE-luciferase reporter gene, we showed that the transactivation of SRE by Ha-Ras(V12) is mainly via a Rac-linked cascade, although the Raf-mitogen-activated protein kinase cascade is required for full activation. In addition, Ha-Ras(V12)-induced DNA synthesis was significantly attenuated by microinjection of recombinant Rac(N17), a dominant negative mutant of Rac1. To identify the mediators downstream of Rac in the Ha-Ras(V12) signaling, we investigated the involvement of cytosolic phospholipase A(2). Oncogenic Ha-Ras(V12)-induced SRE activation was significantly inhibited by either pretreatment with mepacrine, a phospholipase A(2) inhibitor, or cotransfection with the antisense oligonucleotide of cytosolic phospholipase A(2). We also found cytosolic phospholipase A(2) to be situated downstream of Ha-Ras(V12) in a signal pathway leading to transformation. Together, these results are indicative of mediatory roles of Rac and cytosolic phospholipase A(2) in the signaling pathway by which Ha-Ras(V12) transactivates c-fos SRE and transformation. Our findings point to cytosolic phospholipase A(2) as a novel potential target for suppressing oncogenic Ha-Ras(V12) signaling in the cell.     

Preoperative Flexible Bronchoscopy in Patients with Persistent Ground-Glass Nodule

There are no accurate data on the diagnostic value of preoperative flexible bronchoscopy (FB) for persistent ground-glass nodule (GGN) of the lung. We evaluated the value of preoperative FB in patients with suspected GGN-type lung cancer. We retrospectively searched a database for subjects who had ‘ground-glass opacity’, ‘non-solid nodule’, ‘part-solid nodule’, or ‘sub-solid nodule’ on chest computed tomography reports between February 2004 and March 2012. Patients who had infiltrative ground-glass opacity lesions, mediastinal lymphadenopathy, or pleural effusion, focal ground-glass opacity lesions >3 cm, and were lost to follow-up were excluded. We assessed the diagnostic value of preoperative FB in patients with persistent GGNs who underwent surgical resection. In total, 296 GGNs were evaluated by FB in 264 patients with persistent GGNs who underwent preoperative FB and surgical resection. The median size of the GGNs was 18 mm; 135 (46%) were pure GGN and 161 (54%) were part-solid GGN. No visible tumor or unsuspected endobronchial metastasis was identified by preoperative FB. Only 3 (1%, 3/208) GGNs were identified preoperatively as malignant by bronchial washing cytology; all were part-solid GGNs. No other etiology was identified by FB. Of the GGNs, 271 (91%) were subsequently confirmed as malignant and 25 (9%) were confirmed as benign at surgical resection. Consequently, the overall diagnostic sensitivity and negative predictive value of preoperative FB on a per-nodule basis was 1% (3/271) and 8% (25/293), respectively. The preoperative FB did not change the surgical strategy. Preoperative FB did not add much to the evaluation of persistent GGNs of the lung. Routine preoperative FB may have limited value in surgical candidates with small persistent pure GGNs.     

Insulin-like growth factor-1 protects H9c2 cardiac myoblasts from oxidative stress-induced apoptosis via phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways

Oxidative stress plays a critical role in cardiac injuries during ischemia/reperfusion. Insulin-like growth factor-1 (IGF-1) promotes cell survival in a number of cell types, but the effect of IGF-1 on the oxidative stress has not been elucidated in cardiac muscle cells. Therefore, we examined the role of IGF-1 signaling pathway in cell survival against H2O2-induced apoptosis in H9c2 cardiac myoblasts. H2O2 treatment induced apoptosis in H9c2 cells, and pretreatment of cells with IGF-1 suppressed apoptotic cell death. The antiapoptotic effect of IGF-1 was blocked by LY294002 (an inhibitor of phosphatidylinositol 3-kinase) and by PD98059 (an inhibitor of extracellular signal-regulated kinase (ERK)). The protective effect of IGF-1 was also blocked by rapamycin (an inhibitor of p70 S6 kinase). Furthermore, H9c2 cells stably transfected with constitutively active PI 3-kinase (H9c2-p110*) and Akt (H9c2-Gag-Akt) constructs were more resistant to H2O2 cytotoxicity than control cells. Although H2O2 activates both p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), IGF-1 inhibited only JNK activation. Activated PI 3-kinase (H9c2-p110*) and pretreatment of cells with IGF-1 down-regulated Bax protein levels compared to control cells. Taken together, our results suggest that IGF-1 transmits a survival signal against oxidative stress-induced apoptosis in H9c2 cells via PI 3-kinase and ERK-dependent pathways and the protective effect of IGF-1 is associated with the inhibition of JNK activation and Bax expression.     

A Statistical Approach for Rare-Variant Association Testing in Affected Sibships

Sequencing and exome-chip technologies have motivated development of novel statistical tests to identify rare genetic variation that influences complex diseases. Although many rare-variant association tests exist for case-control or cross-sectional studies, far fewer methods exist for testing association in families. This is unfortunate, because cosegregation of rare variation and disease status in families can amplify association signals for rare variants. Many researchers have begun sequencing (or genotyping via exome chips) familial samples that were either recently collected or previously collected for linkage studies. Because many linkage studies of complex diseases sampled affected sibships, we propose a strategy for association testing of rare variants for use in this study design. The logic behind our approach is that rare susceptibility variants should be found more often on regions shared identical by descent by affected sibling pairs than on regions not shared identical by descent. We propose both burden and variance-component tests of rare variation that are applicable to affected sibships of arbitrary size and that do not require genotype information from unaffected siblings or independent controls. Our approaches are robust to population stratification and produce analytic p values, thereby enabling our approach to scale easily to genome-wide studies of rare variation. We illustrate our methods by using simulated data and exome chip data from sibships ascertained for hypertension collected as part of the Genetic Epidemiology Network of Arteriopathy (GENOA) study.     

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.     

Systematic reinstatement of Schilbe depressirostris (Peters, 1852), based on differences in DNA barcoding and morphology,from Schilbe intermedius Rüppell, 1832 (Siluriformes,Schilbeidae)

Climate change poses an unprecedented threat to biodiversity worldwide. Consequently, unrecognised taxa may not receive adequate conservation attention to survive. We used molecular and morphological data to address the challenge of species delimitation within the genus Schilbe. The presence or absence of an adipose fin and distribution based on east-flowing, conceivably faster-flowing, or west-flowing, probably more slow-flowing, river systems were considered. Distinctive geographic patterns in genetic variation within southern, eastern, and western African populations were revealed. Particularly, the South African population is distinct from those of Namibia, Botswana and Nigeria. No individuals with rudimentary adipose fins were found at any locality, but specimens from three localities either had or did not have adipose fins. These mixed occurrences are suspected to be a result of human interventions, and that the presence of rudimentary adipose fins in the east African species could be an adaptive feature that serves to stabilise these fish in faster currents. In addition, the genetic divergence observed among African silver catfish from geographically isolated river systems is conceivably the result of micro-evolutionary adaptive responses to different environmental conditions. Collectively, these results distinguish S. depressirostris from S. intermedius.     

STAT3 and NF-kappaB signal pathway is required for IL-23-mediated IL-17 production in spontaneous arthritis animal model IL-1 receptor antagonist-deficient mice

IL-23 is a heterodimeric cytokine composed of a p19 subunit and the p40 subunit of IL-12. IL-23 has proinflammatory activity, inducing IL-17 secretion from activated CD4(+) T cells and stimulating the proliferation of memory CD4(+) T cells. We investigated the pathogenic role of IL-23 in CD4(+) T cells in mice lacking the IL-1R antagonist (IL-1Ra(-/-)), an animal model of spontaneous arthritis. IL-23 was strongly expressed in the inflamed joints of IL-1Ra(-/-) mice. Recombinant adenovirus expressing mouse IL-23 (rAd/mIL-23) significantly accelerated this joint inflammation and joint destruction. IL-1beta further increased the production of IL-23, which induced IL-17 production and OX40 expression in splenic CD4(+) T cells of IL-1Ra(-/-) mice. Blocking IL-23 with anti-p19 Ab abolished the IL-17 production induced by IL-1 in splenocyte cultures. The process of IL-23-induced IL-17 production in CD4(+) T cells was mediated via the activation of Jak2, PI3K/Akt, STAT3, and NF-kappaB, whereas p38 MAPK and AP-1 did not participate in the process. Our data suggest that IL-23 is a link between IL-1 and IL-17. IL-23 seems to be a central proinflammatory cytokine in the pathogenesis of this IL-1Ra(-/-) model of spontaneous arthritis. Its intracellular signaling pathway could be useful therapeutic targets in the treatment of autoimmune arthritis.     

Phenylarsine oxide causes an insulin-dependent, GLUT4-specific degradation in rat adipocytes

An incubation of rat adipocytes with phenylarsine oxide (PAO) and then with insulin caused an inhibition of 3-O-methylglucose equilibrium exchange flux and a parallel reduction in cellular GLUT4 content detected by Western blots. Both the transport inhibition and the GLUT4 reduction were saturable with an increasing concentration of PAO showing essentially an identical Ki value of 35 microM. Both effects were not observed in the absence of insulin or if cells were incubated with insulin first. The reduction was specific to GLUT4; the immunoreactivities of GLUT1, insulin receptor, and clathrin were not affected in these experiments. The GLUT4 reduction occurred only in intact cells and was not observed in homogenized cells or fractionated membranes. GLUT4 in both the microsomal storage pool and the plasma membrane pool were affected with no indication of insulin-induced recruitment impairment. GLUT4 reduction was not observed in the presence of chloroquine or at 18 degrees C suggesting involvement of the lysosomal pathway. Based on these results, we propose that there is a PAO-sensitive protein mechanism that controls an insulin-dependent GLUT4 degradation pathway in adipocytes. This protein mechanism and the GLUT4 degradation pathway may play an important role in determining the steady-state GLUT4 level in the insulin-sensitive peripheral tissues in normal and diseased states.     

5-Aminoimidazole-4-carboxamide riboside suppresses lipopolysaccharide-induced TNF-alpha production through inhibition of phosphatidylinositol 3-kinase/Akt activation in RAW 264.7 murine macrophages

5-Aminoimidazole-4-carboxamide riboside (AICAR) is an adenosine analog and a widely used activator of AMP-activated protein kinase (AMPK). We examined the effect of AICAR on LPS-induced TNF-alpha production in RAW 264.7 and peritoneal macrophages and its molecular mechanism in RAW 264.7 macrophages. Treatment with AICAR inhibited LPS-induced increases in TNF-alpha mRNA and protein levels in these cells. AICAR or LPS did not alter the AMPK activity as well as the phosphorylations of AMPK alpha (Thr172) and ACC (Ser79). Moreover, an adenosine kinase inhibitor 5'-iodotubercidin enhanced the suppressive effect of AICAR on TNF-alpha levels. These results suggest that the effect of AICAR on TNF-alpha suppression in RAW 264.7 cells is independent of AMPK activation. In addition, an adenosine receptor antagonist 8-SPT had no effect on AICAR-induced suppression of TNF-alpha levels. Finally, we observed that AICAR inhibited LPS-induced activation of PI 3-kinase and Akt, whereas it had no effect on the activation of p38 and ERK1/2. Taken together, these results suggest that the anti-inflammatory action of AICAR in RAW 264.7 macrophages is independent of AMPK activation and is associated with inhibition of LPS-induced activation of PI 3-kinase/Akt pathway.