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[This corrects the article DOI: 10.1371/journal.pone.0053831.].

Atrogin-1/MAFbx is an ubiquitin E3 ligase that regulates myocardial structure and function through the ubiquitin-dependent protein modification. However, little is known about the effect of atrogin-1 activation on the gene expression changes in cardiomyocytes. Neonatal rat cardiomyocytes were infected with adenovirus atrogin-1 (Ad-atrogin-1) or GFP control (Ad-GFP) for 24 hours. The gene expression profiles were compared with microarray analysis. 314 genes were identified as differentially expressed by overexpression of atrogin-1, of which 222 were up-regulated and 92 were down-regulated. Atrogin-1 overexpression significantly modulated the expression of genes in 30 main functional categories, most genes clustered around the regulation of cell death, proliferation, inflammation, metabolism and cardiomyoctye structure and function. Moreover, overexpression of atrogin-1 significantly inhibited cardiomyocyte survival, hypertrophy and inflammation under basal condition or in response to lipopolysaccharide (LPS). In contrast, knockdown of atrogin-1 by siRNA had opposite effects. The mechanisms underlying these effects were associated with inhibition of MAPK (ERK1/2, JNK1/2 and p38) and NF-κB signaling pathways. In conclusion, the present microarray analysis reveals previously unappreciated atrogin-1 regulation of genes that could contribute to the effects of atrogin-1 on cardiomyocyte survival, hypertrophy and inflammation in response to endotoxin, and may provide novel insight into how atrogin-1 modulates the programming of cardiac muscle gene expression.

Abdominal aortic aneurysm (AAA) is a life-threatening aortic disease in the elderly. Activation of Notch1 pathway plays a critical role in the development of AAA, but the underlying mechanisms remain poorly understood. In the present study, we explored the mechanisms by which Notch1 activation regulates angiotensin II (Ang II)-induced AAA formation and evaluated the therapeutic potential of a new Notch γ-secretase inhibitor, dibenzazepine (DBZ), for the treatment of AAA. Apolipoprotein E knockout (Apo E(-/-)) mice infused for 4 weeks with Ang II (1000 ng/kg/min, IP) using osmotic mini-pumps were received an intraperitoneal injection of either vehicle or 1 mg/kg/d DBZ. Notch1 signaling was activated in AAA tissue from both Ang II-infused Apo E(-/-) mice and human undergoing AAA repair in vivo, with increased expression of Notch intracellular domain (NICD) and its target gene Hes1, and this effect was effectively blocked by DBZ. Moreover, infusion of Ang II markedly increased the incidence and severity of AAA in Apo E(-/-) mice. In contrast, inhibition of Notch activation by DBZ prevented AAA formation in vivo. Furthermore, DBZ markedly prevented Ang II-stimulated accumulation of macrophages and CD4(+) T cells, and ERK-mediated angiogenesis, simultaneously reversed Th2 response, in vivo. In conclusion, these findings provide new insight into the multiple mechanisms of Notch signaling involved in AAA formation and suggest that γ-secretase inhibitor DBZ might be a novel therapeutic drug for treating AAAS.

Senescence is a recognized mechanism of cardiovascular diseases; however, its contribution to myocardial fibrosis and rupture after infarction and the underlying mechanisms remain unclear. Here we showed that senescent cardiac fibroblasts markedly accumulated in heart after myocardial infarction. The expression of key senescence regulators, especially p53, was significantly up-regulated in the infarcted heart or hypoxia-treated fibroblasts. Furthermore, knockdown of endogenous p53 by siRNA in fibroblasts markedly reduced hypoxia-induced cell senescence, cytokine expression but increased collagen expression, whereas increased expression of p53 protein by adenovirus infection had opposite effects. Consistent with in vitro results in cardiac fibroblasts, p53 deficiency in vivo significantly decreased the accumulation of senescent fibroblasts, the infiltration of macrophages and matrix metalloproteinases, but enhanced collagen deposition after myocardial infarction. In conclusion, these results suggest that the p53-mediated fibroblast senescence limits cardiac collagen production, and inhibition of p53 activity could represent a novel therapeutic target to increase reparative fibrosis and to prevent heart rupture after myocardial infarction.

Although a critical role for caveolae-mediated albumin transcytosis in pulmonary endothelium is well established, considerably less is known about caveolae-independent pathways. In this current study, we confirmed that cultured rat pulmonary microvascular (RPMEC) and pulmonary artery (RPAEC) endothelium endocytosed Alexa488-labeled albumin in a saturable, temperature-sensitive mode and internalization resulted in co-localization by fluorescence microscopy with cholera B toxin and caveolin-1. Although siRNA to caveolin-1 (cav-1) in RPAEC significantly inhibited albumin uptake, a remnant portion of albumin uptake was cav-1-independent, suggesting alternative pathways for albumin uptake. Thus, we isolated and cultured mouse lung endothelial cells (MLEC) from wild type and cav-1(-/-) mice and noted that ~ 65% of albumin uptake, as determined by confocal imaging or live cell total internal reflectance fluorescence microscopy (TIRF), persisted in total absence of cav-1. Uptake of colloidal gold labeled albumin was evaluated by electron microscopy and demonstrated that albumin uptake in MLEC from cav-1(-/-) mice was through caveolae-independent pathway(s) including clathrin-coated pits that resulted in endosomal accumulation of albumin. Finally, we noted that albumin uptake in RPMEC was in part sensitive to pharmacological agents (amiloride [sodium transport inhibitor], Gö6976 [protein kinase C inhibitor], and cytochalasin D [inhibitor of actin polymerization]) consistent with a macropinocytosis-like process. The amiloride sensitivity accounting for macropinocytosis also exists in albumin uptake by both wild type and cav-1(-/-) MLEC. We conclude from these studies that in addition to the well described caveolar-dependent pulmonary endothelial cell endocytosis of albumin, a portion of overall uptake in pulmonary endothelial cells is cav-1 insensitive and appears to involve clathrin-mediated endocytosis and macropinocytosis-like process.

BACKGROUND: Cardiac cell death and generation of oxidative stress contribute to doxorubicin (DOX)-induced cardiac dysfunction. E3 ligase Nrdp1 plays a critical role in the regulation of cell apoptosis, inflammation and production of reactive oxygen species (ROS), which may contribute to heart failure. However, the role of Nrdp1 in DOX-induced cardiac injury remains to be determined. METHODS AND RESULTS: We examined the effect of Nrdp1 overexpression with DOX treatment in rat neonatal cardiomyocytes and mouse heart tissue. Cardiomyocytes were infected with adenovirus containing GFP (Ad-GFP), Nrdp1 wild-type (Ad-Nrdp1) or the dominant-negative form of Nrdp1 (Ad-Dn-Nrdp1), then treated with DOX for 24 hr. DOX treatment increased cell death and apoptosis, with Ad-Nrdp1 infection enhancing these actions but Ad-Dn-Nrdp1 infection attenuating these effects. Furthermore, 5 days after a single injection of DOX (20 mg/kg, intraperitoneally), Nrdp1 transgenic mice (TG) showed decreased cardiac function and increased apoptosis, autophagy and oxidative stress as compared with wild-type (WT) mice (P

Background: Genome-wide association studies (GWAS) in Caucasians have identified fourteen index single nucleotide polymorphisms (iSNPs) that influence colorectal cancer (CRC) risk. Methods: We investigated the role of eleven iSNPs or surrogate SNPs (sSNPs), in high linkage disequilibrium (LD, r²≥0.8) and within 100 kb vicinity of iSNPs, in 2,000 age- and gender-matched Singapore Chinese (SCH) cases and controls. Results: Only iSNP rs6983267 at 8q24.21 and sSNPs rs6695584, rs11986063, rs3087967, rs2059254, and rs7226855 at 1q41, 8q23.3, 11q23.1, 16q22.1 and 18q21.1 respectively showed evidence of association with CRC risk, with odds ratios (OR) ranging from 1.13 to 1.40. sSNP rs827401 at 10p14 was associated with rectal cancer risk (OR = 0.74, 95% CI 0.63-0.88) but not disease prognosis (OR = 0.91, 95% CI 0.69-1.20). Interestingly, sSNP rs3087967 at 11q23.1 was associated with CRC risk in men (OR = 1.34, 95% CI 1.14-1.58) but not women (OR = 1.07, 95% CI: 0.88-1.29), suggesting a gender- specific role. Half of the Caucasian-identified variants, including the recently fine-mapped BMP pathway loci, BMP4, GREM1, BMP2 and LAMA 5, did not show any evidence for association with CRC in SCH (OR ∼1; p-value >0.1). Comparing the results of this study with that of the Northern and Hong Kong Chinese, only variants at chromosomes 8q24.21, 10p14, 11q23.1 and 18q21.1 were replicated in at least two out of the three Chinese studies. Conclusions: The contrasting results between Caucasians and Chinese could be due to different LD patterns and allelic frequencies or genetic heterogeneity. The results suggest that additional common variants contributing to CRC predisposition remained to be identified. [ABSTRACT FROM AUTHOR]

Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could "normalize" the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of "normalized" oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics.