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We previously showed that the matricellular protein CCN5 reverses established cardiac fibrosis (CF) through inducing apoptosis in myofibroblasts (MyoFBs) but not in cardiomyocytes or fibroblasts (FBs). In this study, we set out to elucidate the molecular mechanisms underlying CCN5-mediated selective apoptosis of MyoFBs. We first observed that the apoptotic protein p53 and the anti-apoptotic protein NFκB are simultaneously induced in MyoFBs. When the expression level of p53 was suppressed using a siRNA, CCN5 did not induce apoptosis in MyoFBs. By contrast, when NFκB signaling was inhibited using IKK VII, an IκB inhibitor, MyoFBs underwent apoptosis even in the absence of CCN5. SMAD7 is one of the downstream targets of CCN5 and it was previously shown to potentiate apoptosis in epithelial cells through inhibition of NFκB. In accordance with these reports, when the expression of SMAD7 was suppressed using a siRNA, NFκB signaling was enhanced, and CCN5 did not induce apoptosis. Lastly, we used a luciferase reporter construct to show that CCN5 positively regulated SMAD7 expression at the transcriptional level. Collectively, our data suggest that a delicate balance between the two mutually antagonistic proteins p53 and NFκB is maintained for MyoFBs to survive, and CCN5 tips the balance in favor of the apoptotic protein p53. This study provides insight into the anti-fibrotic activity of CCN5 during the regression of CF. [ABSTRACT FROM AUTHOR]

Atrial structural remodelling including atrial hypertrophy and fibrosis is a key mediator of atrial fibrillation (AF). We previously demonstrated that the matricellular protein CCN5 elicits anti‐fibrotic and anti‐hypertrophic effects in left ventricles under pressure overload. We here determined the utility of CCN5 in ameliorating adverse atrial remodelling and arrhythmias in a murine model of angiotensin II (AngII) infusion. Advanced atrial structural remodelling was induced by AngII infusion in control mice and mice overexpressing CCN5 either through transgenesis (CCN5 Tg) or AAV9‐mediated gene transfer (AAV9‐CCN5). The mRNA levels of pro‐fibrotic and pro‐inflammatory genes were markedly up‐regulated by AngII infusion, which was significantly normalized by CCN5 overexpression. In vitro studies in isolated atrial fibroblasts demonstrated a marked reduction in AngII‐induced fibroblast trans‐differentiation in CCN5‐treated atria. Moreover, while AngII increased the expression of phosphorylated CaMKII and ryanodine receptor 2 levels in HL‐1 cells, these molecular features of AF were prevented by CCN5. Electrophysiological studies in ex vivo perfused hearts revealed a blunted susceptibility of the AAV9‐CCN5–treated hearts to rapid atrial pacing‐induced arrhythmias and concomitant reversal in AngII‐induced atrial action potential prolongation. These data demonstrate the utility of a gene transfer approach targeting CCN5 for reversal of adverse atrial structural and electrophysiological remodelling. [ABSTRACT FROM AUTHOR]

Retinal pigment epithelium (RPE) plays an essential role in maintaining retinal function, and its defect is thought to be critically implicated in various ocular disorders. This study demonstrated that the matricellular protein CCN5 was down-regulated in ARPE-19 cells treated with the pro-fibrotic agent transforming growth factor (TGF)-β. A recombinant adenovirus expressing CCN5 (AdCCN5) was used to restore the level of CCN5 in these cells. AdCCN5 prevented TGF-β-induced fibrotic changes, including disruption of tight junctions, up-regulation of mesenchymal marker proteins, and down-regulation of epithelial marker proteins. In addition, AdCCN5 prevented TGF-β-induced functional defects, including increased migratory activity and reduced phagocytic activity. Notably, AdCCN5 reversed morphological and functional defects pre-established by TGF-β prior to viral infection. The CCN5 level was down-regulated in RPE of 18-month-old Ccl2-/- mice, which exhibited retinal defects. Restoration of the CCN5 level via intravitreal injection of a recombinant adeno-associated virus expressing CCN5 (AAV9-CCN5) normalized the altered expression of mesenchymal, epithelial, and functional marker proteins, as assessed by western blotting and immunohistochemistry. Taken together, these data suggest that down-regulation of CCN5 is associated with fibrotic deformation of RPE under pathological conditions and that restoration of the CCN5 level effectively promotes recovery of deformed RPE. [ABSTRACT FROM AUTHOR]

Pressure overload in the heart induces pathological hypertrophy and is associated with cardiac dysfunction. Apoptosis and fibrosis signaling initiated by the endoplasmic reticulum stress (ERS) is known to contribute to these maladaptive effects. The aim of this study was to investigate whether reduction of ERS by a known chemical chaperone, tauroursodeoxycholic acid (TUDCA) can attenuate pressure overload-induced cardiac remodeling in a mouse model of transverse aortic constriction (TAC). Oral administration of TUDCA at a dose of 300 mg/kg body weight (BW) in the TUDCA-TAC group reduced ERS markers (GRP78, p-PERK, and p-eIf2α), compared to the Vehicle (Veh)-TAC group. TUDCA administration, for 4 weeks after TAC significantly reduced cardiac hypertrophy as shown by the reduced heart weight (HW) to BW ratio, and expression of hypertrophic marker genes (ANF, BNP, and α-SKA). Masson's trichrome staining showed that myocardial fibrosis and collagen deposition were also significantly reduced in the TUDCA-TAC group. We also found that TUDCA significantly decreased expression of TGF-β signaling proteins and collagen isoforms. TUDCA administration also reduced cardiac apoptosis and the related proteins in the TUDCA-TAC group. Microarray analysis followed by gene ontology (GO) and pathway analysis demonstrated that extracellular matrix genes responsible for hypertrophy and fibrosis, and mitochondrial genes responsible for apoptosis and fatty acid metabolism were significantly altered in the Veh-TAC group, but the alterations were normalized in the TUDCA-TAC group, suggesting potential of TUDCA in treatment of heart diseases related to pressure-overload. [ABSTRACT FROM AUTHOR]

Cytokine-like 1 (Cytl1) is a secreted protein that is involved in diverse biological processes. A comparative modeling study indicated that Cytl1 is structurally and functionally similar to monocyte chemoattractant protein 1 (MCP-1). As MCP-1 plays an important role in cardiac fibrosis (CF) and heart failure (HF), we investigated the role of Cytl1 in a mouse model of CF and HF. Cytl1 was upregulated in the failing mouse heart. Pressure overload-induced CF was significantly attenuated in cytl1 knock-out (KO) mice compared to that from wild-type (WT) mice. By contrast, adeno-associated virus (AAV)-mediated overexpression of cytl1 alone led to the development of CF in vivo. The endothelial-mesenchymal transition (EndMT) and the transdifferentiation of fibroblasts (FBs) to myofibroblasts (MFBs) have been suggested to contribute considerably to CF. Adenovirus-mediated overexpression of cytl1 was sufficient to induce these two critical CF-related processes in vitro, which were completely abrogated by co-treatment with SB-431542, an antagonist of TGF-β receptor 1. Cytl1 induced the expression of TGF-β2 both in vivo and in vitro. Antagonizing the receptor for MCP-1, C-C chemokine receptor type 2 (CCR2), with CAS 445479-97-0 did not block the pro-fibrotic activity of Cytl1 in vitro. Collectively, our data suggest that Cytl1 plays an essential role in CF likely through activating the TGF-β-SMAD signaling pathway. Although the receptor for Cyt1l remains to be identified, Cytl1 provides a novel platform for the development of anti-CF therapies. [ABSTRACT FROM AUTHOR]

The calcium-transporting ATPase ATP2A2, also known as SERCA2a, is a critical ATPase responsible for Ca2+ re-uptake during excitation-contraction coupling. Impaired Ca2+ uptake resulting from decreased expression and reduced activity of SERCA2a is a hallmark of heart failure. Accordingly, restoration of SERCA2a expression by gene transfer has proved to be effective in improving cardiac function in heart-failure patients, as well as in animal models. The small ubiquitin-related modifier (SUMO) can be conjugated to lysine residues of target proteins, and is involved in many cellular processes. Here we show that SERCA2a is SUMOylated at lysines 480 and 585 and that this SUMOylation is essential for preserving SERCA2a ATPase activity and stability in mouse and human cells. The levels of SUMO1 and the SUMOylation of SERCA2a itself were greatly reduced in failing hearts. SUMO1 restitution by adeno-associated-virus-mediated gene delivery maintained the protein abundance of SERCA2a and markedly improved cardiac function in mice with heart failure. This effect was comparable to SERCA2A gene delivery. Moreover, SUMO1 overexpression in isolated cardiomyocytes augmented contractility and accelerated Ca2+ decay. Transgene-mediated SUMO1 overexpression rescued cardiac dysfunction induced by pressure overload concomitantly with increased SERCA2a function. By contrast, downregulation of SUMO1 using small hairpin RNA (shRNA) accelerated pressure-overload-induced deterioration of cardiac function and was accompanied by decreased SERCA2a function. However, knockdown of SERCA2a resulted in severe contractile dysfunction both in vitro and in vivo, which was not rescued by overexpression of SUMO1. Taken together, our data show that SUMOylation is a critical post-translational modification that regulates SERCA2a function, and provide a platform for the design of novel therapeutic strategies for heart failure. [ABSTRACT FROM AUTHOR]

We have previously reported a genetic assay that is suitable for the study of substrate specificity of a protease in vivo, and herein present a simplified version of the method. In this procedure, expressed in Saccharomyces cerevisiae by using the constitutive alcohol dehydrogenase promoter is a fusion protein in which a transcription factor is linked to the intracellular domain of an integral membrane protein by a protease substrate sequence. Following this, a protease is expressed by using the inducible GAL promoter in the same yeast cells. The cleavage of the substrate sequence by the specific protease results in the release of the transcription factor and subsequent activation of reporter genes in nucleus. Since the expression of a protease is strictly under the control of the inducible GAL promoter, false substrate sequences that are cleaved by endogenous yeast proteases can be easily recognized and eliminated from further characterization. This suggests that the modified strategy provides an efficient tool for the analysis of substrate sequences of a protease in vivo. [ABSTRACT FROM AUTHOR]

Background: Cardiac fibrosis (CF) is associated with increased ventricular stiffness and diastolic dysfunction and is an independent predictor of long-term clinical outcomes of patients with heart failure (HF). We previously showed that the matricellular CCN5 protein is cardioprotective via its ability to inhibit CF and preserve cardiac contractility.Objectives: This study examined the role of CCN5 in human heart failure and tested whether CCN5 can reverse established CF in an experimental model of HF induced by pressure overload.Methods: Human hearts were obtained from patients with end-stage heart failure. Extensive CF was induced by applying transverse aortic constriction for 8 weeks, which was followed by adeno-associated virus-mediated transfer of CCN5 to the heart. Eight weeks following gene transfer, cellular and molecular effects were examined.Results: Expression of CCN5 was significantly decreased in failing hearts from patients with end-stage heart failure compared to nonfailing hearts. Trichrome staining and myofibroblast content measurements revealed that the established CF had been reversed by CCN5 gene transfer. Anti-CF effects of CCN5 were associated with inhibition of the transforming growth factor beta signaling pathway. CCN5 significantly inhibited endothelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation, which are 2 critical processes for CF progression, both in vivo and in vitro. In addition, CCN5 induced apoptosis in myofibroblasts, but not in cardiomyocytes or fibroblasts, both in vivo and in vitro. CCN5 provoked the intrinsic apoptotic pathway specifically in myofibroblasts, which may have been due the ability of CCN5 to inhibit the activity of NFκB, an antiapoptotic molecule.Conclusions: CCN5 can reverse established CF by inhibiting the generation of and enhancing apoptosis of myofibroblasts in the myocardium. CCN5 may provide a novel platform for the development of targeted anti-CF therapies. [ABSTRACT FROM AUTHOR]

Luteolin (3,4,5,7-tetrahydroxyflavones), a major dietary flavone, regulates a variety of biological effects including cancer progression, insulin resistance and inflammation. However, its exact actions on adipogenesis and osteogenesis and the underlying molecular mechanisms are yet to be clarified. In this study, we show that luteolin suppresses lipid accumulation but increases osteoblast differentiation. In mechanism studies, luteolin increases the expression of the heat shock proteins (Hsp) 40 (Dnajb1) and Hsp90 (Hsp90b1), but not those of other heat shock proteins including Hsp20, Hsp27, Hsp47, Hsp70, Hsp72, and Hsp90, and another type of Hsp40 (Dnaja1). Silencing Dnajb1 by using small interfering RNAs (siRNAs), but not against Hsp90b1, recapitulates the effects of luteolin in adipocyte and osteoblast differentiation. Consistently, the forced expression of Dnajb1 decreases the lipid accumulation and stimulates alkaline phosphatase (ALPL) activity. The antiadipogenic and proosteogenic effects of luteolin are significantly blunted in Dnajb1-deficient cells, further suggesting that Dnajb1 is, at least in part, required for luteolin's dual actions in adipogenesis and osteogenesis. Together, our data implicate luteolin as an ingredient and Dnajb1 as a molecular target for the development of functional foods and drugs in metabolic and bone-related diseases. [ABSTRACT FROM AUTHOR]

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: A protein complex present at the junctional sarcoplasmic reticulum (SR) membrane is implicated in the Ca2+ release process during muscle contraction. The histidine-rich Ca2+-binding protein (HRC) is an emerging component associated into the SR protein complex. We cloned cDNAs for rat and monkey HRCs, showing a conserved sequence organization in common with other mammalian HRCs. Genomic analysis revealed that each mammalian HRC gene is present as a single copy in the genome, consisting of 6 exons and 5 introns. Developmental expression analysis using mouse embryos and postnatal hearts demonstrated that Hrc transcription begins at 12.5 days postcoitum and its level increases gradually, reaching an adult level in the range 5–20 days after birth. Comparing the Hrc gene and other SR genes, we found that the timing and pattern of gene expression vary among the SR genes and the full-level expression of these genes is achieved in the heart after postnatal day 20. Collectively, our study provides comprehensive information about the structure and expression of the mammalian HRC gene, together with the comparative expression data of the related SR genes. [Copyright &y& Elsevier]