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Abstract: Eyes absent 2 (Eya2) is a transcription factor involved in a number of cellular and developmental processes. We have previously shown that Eya2 is up-regulated during regression of cardiac hypertrophy and blocks phenylephrine-induced development of cardiomyocyte hypertrophy in vitro, suggesting that Eya2 is a negative regulator of cardiac hypertrophy. In this study, we generated transgenic mice with cardiac-specific overexpression of Eya2 to elucidate the in vivo function of Eya2 in cardiac remodeling. Mild cardiac hypertrophy developed in Eya2 transgenic mice under baseline conditions with no obvious structural or functional defects. Eya2 overexpression inhibited development of cardiac hypertrophy as judged by the abrogation of increases in heart weight and cross-sectional cell surface areas and re-activation of fetal genes under pressure overload (4 weeks). Eya2 overexpression also prevented wall thinning, ventricular dilation, and deterioration of cardiac function as well as fibrosis and inflammation in the heart under long-term pressure overload (12 weeks). Gene expression profiling using the parametric analysis of gene set enrichment (PAGE) method revealed that gene sets related to mitochondrial biogenesis and metabolism were elevated in the Eya2 transgenic mice. We also observed that the PI3K/Akt/mTOR signaling cascade was preserved in the Eya2 transgenic mice, while it was significantly reduced in the wild type littermates under pressure overload. These results demonstrate that Eya2 prevents adverse cardiac remodeling under pressure overload partly through altering metabolic gene expression and preserving PI3K/Akt/mTOR signaling pathway. [Copyright &y& Elsevier]

Aberrant DNA methylation of various genes has been identified to be associated with disease progression in chronic myeloid leukemia (CML). Our study was intended to investigate DLX4 methylation pattern in different clinical stages of CML and further determine its role in regulating DLX4 expression. Real-time quantitative methylation-specific PCR and bisulfite sequencing PCR were applied to detect DLX4 methylation. 5-aza-2′-deoxycytidine (5-aza-dC) was used for demethylation studies. DLX4 was significantly hypermethylated in CML patients ( P = 0.002) especially in blastic phase (BC) stage ( P < 0.001) as compared with controls. Moreover, DLX4 methylation level in BC stage was significantly higher than in chronic phase (CP) stage ( P < 0.001). DLX4 methylation density was significantly increased during the progression of CML among the tested two patients ( P < 0.001). DLX4 hypermethylation occurred with the highest incidence in BC stage (83%), lower incidence in acute phase (AP) stage (43%), and the lowest incidence in CP stage (26%) ( P = 0.001). Moreover, t(9; 22) with additional alteration cases had significantly higher frequency of DLX4 hypermethylation compared with the other cytogenetics ( P = 0.010). Significantly negative correlation was observed between DLX4 methylation and DLX4-TV2 (the shorter DLX4 isoform) expression (R = −0.382, P = 0.001, n = 78) but not between DLX4 methylation and BP1 (the longer DLX4 isoform) expression (R = 0.134, P = 0.244, n = 78) in CML patients. Both DLX4-TV2 and BP1 mRNA were significantly increased after 5-aza-dC treatment in K562 cell line ( P < 0.001). Our study indicated that hypermethylation of DLX4 correlated with disease progression of CML. Moreover, DLX4 expression was regulated by its methylation in CML. [ABSTRACT FROM AUTHOR]

Abstract: MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules that suppress gene expression via degradation or translational inhibition of their target genes. Many miRNAs are associated with cardiac hypertrophy and heart failure. In this study, we pursued to identify miRNAs that negatively regulate cardiac hypertrophy by utilizing a surgical model for regression of cardiac hypertrophy. Microarray analysis revealed that 15 miRNAs out of the 696 miRNAs tested were specifically up-regulated during the regression period. Among these regression-specific miRNAs, nine microRNAs, which have not been previously reported, were further tested for their effects on phenylephrine (PE)-treated neonatal cardiomyocytes. Consequently, five miRNAs (miR-101b, 142-3p, 181d, 24-2∗, and 450a) completely abrogated PE-induced hypertrophy as determined by measurements of cell size and fetal gene expression. Conversely, antagomers of these miRNAs exacerbated the PE-induced hypertrophy. Collectively, these findings suggest that the five miRNAs newly identified by using our cardiac hypertrophy-regression surgical model negatively regulate cardiac hypertrophy and could be used as potential therapeutic targets for the treatment of heart diseases. [Copyright &y& Elsevier]

Abstract: CCN family members are matricellular proteins with diverse roles in cell function. The differential expression of CCN2 and CCN5 during cardiac remodeling suggests that these two members of the CCN family play opposing roles during the development of cardiac hypertrophy and fibrosis. We aimed to evaluate the role of CCN2 and CCN5 in the development of cardiac hypertrophy and fibrosis. In isolated cardiomyocytes, overexpression of CCN2 induced hypertrophic growth, whereas the overexpression of CCN5 inhibited both phenylephrine (PE)- and CCN2-induced hypertrophic responses. Deletion of the C-terminal (CT) domain of CCN2 transformed CCN2 into a CCN5-like dominant negative molecule. Fusion of the CT domain to the Carboxy-terminus of CCN5 transformed CCN5 into a CCN2-like pro-hypertrophic molecule. CCN2 transgenic (TG) mice did not develop cardiac hypertrophy at baseline but showed significantly increased fibrosis in response to pressure overload. In contrast, hypertrophy and fibrosis were both significantly inhibited in CCN5 TG mice. CCN2 TG mice showed an accelerated deterioration of cardiac function in response to pressure overload, whereas CCN5 TG mice showed conserved cardiac function. TGF-β-SMAD signaling was elevated in CCN2 TG mice, but was inhibited in CCN5 TG mice. CCN2 is pro-hypertrophic and -fibrotic, whereas CCN5 is anti-hypertrophic and -fibrotic. CCN5 lacking the CT domain acts as a dominant negative molecule. CCN5 may provide a novel therapeutic target for the treatment of cardiac hypertrophy and heart failure. [Copyright &y& Elsevier]

Abstract: The long terminal repeats (LTRs) are the control centers for retrovirus gene expression, which possess all of the requisite signals. It has been proved that the LTRs of Moloney murine leukemia virus (MoMLV) could constitutively activate genes in diverse cell types. Recently, a retrovirus-like element, OSP-1 (ovarian-specific promoter 1), was extracted from rat ovary according to the LTRs of MoMLV, whose name was derived from the fact of ovarian-specific transcription. It was reasonable to speculate that the tissue-specificity was acquired through mutations and that there should be abound other mutants, active or inactive. In the present study, we isolated several homologous sequences to OSP-1 and detected their function. Consequently, one of them could also drive target gene expression specifically to ovarian cell lines and was named OSP-2 which shared 98% similarity to OSP-1. On the other hand, we picked up other two closest sequences and proved them inactive, which was 97% and 95% similar to OSP-1, respectively. Sequence analysis revealed the different mutations around/within the binding sites of transcriptional factors that might play important roles in tissue-specificity. In summary, we extracted a novel ovarian-specific promoter as well as other nonfunctional mutants, which in part shed light on the study of ovarian-specific transcription. In addition, it also provided a new tool in cancer gene therapy and to create transgenic animals. [Copyright &y& Elsevier]