Seitaro Nomura

Promotion of CHIP-Mediated p53 Degradation Protects the Heart From Ischemic Injury

Rationale: The number of patients with coronary heart disease, including myocardial infarction, is increasing and novel therapeutic strategy is awaited. Tumor suppressor protein p53 accumulates in the myocardium after myocardial infarction, causes apoptosis of cardiomyocytes, and plays an important role in the progression into heart failure. Objectives: We investigated the molecular mechanisms of p53 accumulation in the heart after myocardial infarction and tested whether anti-p53 approach would be effective against myocardial infarction. Methods and Results: Through expression screening, we found that CHIP (carboxyl terminus of Hsp70-interacting protein) is an endogenous p53 antagonist in the heart. CHIP suppressed p53 level by ubiquitinating and inducing proteasomal degradation. CHIP transcription was downregulated after hypoxic stress and restoration of CHIP protein level prevented p53 accumulation after hypoxic stress. CHIP overexpression in vivo prevented p53 accumulation and cardiomyocyte apoptosis after myocardial infarction. Promotion of CHIP function by heat shock protein (Hsp)90 inhibitor, 17-allylamino-17-demethoxy geldanamycin (17-AAG), also prevented p53 accumulation and cardiomyocyte apoptosis both in vitro and in vivo. CHIP-mediated p53 degradation was at least one of the cardioprotective effects of 17-AAG. Conclusions: We found that downregulation of CHIP level by hypoxia was responsible for p53 accumulation in the heart after myocardial infarction. Decreasing the amount of p53 prevented myocardial apoptosis and ameliorated ventricular remodeling after myocardial infarction. We conclude that anti-p53 approach would be effective to treat myocardial infarction.

Complement C1q-induced activation of β-catenin signalling causes hypertensive arterial remodelling.

Hypertension induces structural remodelling of arteries, which leads to arteriosclerosis and end-organ damage. Hyperplasia of vascular smooth muscle cells (VSMCs) and infiltration of immune cells are the hallmark of hypertensive arterial remodelling. However, the precise molecular mechanisms of arterial remodelling remain elusive. We have recently reported that complement C1q activates β-catenin signalling independent of Wnts. Here, we show a critical role of complement C1-induced activation of β-catenin signalling in hypertensive arterial remodelling. Activation of β-catenin and proliferation of VSMCs were observed after blood-pressure elevation, which were prevented by genetic and chemical inhibition of β-catenin signalling. Macrophage depletion and C1qa gene deletion attenuated the hypertension-induced β-catenin signalling, proliferation of VSMCs and pathological arterial remodelling. Our findings unveil the link between complement C1 and arterial remodelling and suggest that C1-induced activation of β-catenin signalling becomes a novel therapeutic target to prevent arteriosclerosis in patients with hypertension.

CellTree: an R/bioconductor package to infer the hierarchical structure of cell populations from single-cell RNA-seq data.

BackgroundSingle-cell RNA sequencing is fast becoming one the standard method for gene expression measurement, providing unique insights into cellular processes. A number of methods, based on general dimensionality reduction techniques, have been suggested to help infer and visualise the underlying structure of cell populations from single-cell expression levels, yet their models generally lack proper biological grounding and struggle at identifying complex differentiation paths.ResultsHere we introduce cellTree: an R/Bioconductor package that uses a novel statistical approach, based on document analysis techniques, to produce tree structures outlining the hierarchical relationship between single-cell samples, while identifying latent groups of genes that can provide biological insights.ConclusionsWith cellTree, we provide experimentalists with an easy-to-use tool, based on statistically and biologically-sound algorithms, to efficiently explore and visualise single-cell RNA data. The cellTree package is publicly available in the online Bionconductor repository at: http://bioconductor.org/packages/cellTree/.

Hypoxia-Inducible Factor-1α Activates the Transforming Growth Factor-β/SMAD3 Pathway in Kidney Tubular Epithelial Cells.

Background: Kidney injury, including chronic kidney disease and acute kidney injury, is a worldwide health problem. Hypoxia and transforming growth factor-β (TGF-β) are well-known factors that promote kidney injury. Hypoxia-inducible factor (HIF) and SMAD3 are their main downstream transcriptional factors. Hypoxia-HIF pathway and TGF-β/SMAD3 pathway play a crucial role in the progression of kidney injury. However, reports on their interactions are limited, and the global transcriptional regulation under their control is almost unknown. Methods: Kidney tubular epithelial cells were cultured and stimulated by hypoxia and TGF-β. We detected global binding sites of HIF-1α and SMAD3 in cells using chromatin immunoprecipitation-sequencing (ChIP-Seq), and measured the gene expression using RNA-sequencing (RNA-Seq). ChIP-quantitative PCR (qPCR) was used to quantitatively evaluate bindings of SMAD3. Results: ChIP-Seq revealed that 2,065 and 5,003 sites were bound by HIF-1α and SMAD3, respectively, with 614 sites co-occupied by both factors. RNA-Seq showed that hypoxia and TGF-β stimulation causes synergistic upregulation of 249 genes, including collagen type I alpha 1 (COL1A1) and serpin peptidase inhibitor, clade E, member 1, which are well-known to be involved in fibrosis. Ontology of the 249 genes implied that the interaction of HIF-1α and SMAD3 is related to biological processes such as fibrosis. ChIP-qPCR of SMAD3 at HIF-1α binding sites near COL1A1 and SERPINE1 indicated that HIF-1α promotes the bindings of SMAD3, which is induced by TGF-β. Conclusions: These findings suggest that HIF-1α induced by hypoxia activates the TGF-β/SMAD3 pathway. This mechanism may promote kidney injury, especially by upregulating genes related to fibrosis.

Wnt/β-Catenin Signaling Contributes to Skeletal Myopathy in Heart Failure via Direct Interaction With Forkhead Box O

Background—There are changes in the skeletal muscle of patients with chronic heart failure (CHF), such as volume reduction and fiber type shift toward fatigable type IIb fiber. Forkhead box O (FoxO) signaling plays a critical role in the development of skeletal myopathy in CHF, and functional interaction between FoxO and the Wnt signal mediator &bgr;-catenin was previously demonstrated. We have recently reported that serum of CHF model mice activates Wnt signaling more potently than serum of control mice and that complement C1q mediates this activation. We, therefore, hypothesized that C1q-induced activation of Wnt signaling plays a critical role in skeletal myopathy via the interaction with FoxO. Methods and Results—Fiber type shift toward fatigable fiber was observed in the skeletal muscle of dilated cardiomyopathy model mice, which was associated with activation of both Wnt and FoxO signaling. Wnt3a protein activated FoxO signaling and induced fiber type shift toward fatigable fiber in C2C12 cells. Wnt3a-induced fiber type shift was inhibited by suppression of FoxO1 activity, whereas Wnt3a-independent fiber type shift was observed by overexpression of constitutively active FoxO1. Serum of dilated cardiomyopathy mice activated both Wnt and FoxO signaling and induced fiber type shift toward fatigable fiber in C2C12 cells. Wnt inhibitor and C1-inhibitor attenuated FoxO activation and fiber type shift both in C2C12 cells and in the skeletal muscle of dilated cardiomyopathy mice. Conclusions—C1q-induced activation of Wnt signaling contributes to fiber type shift toward fatigable fiber in CHF. Wnt signaling may be a novel therapeutic target to prevent skeletal myopathy in CHF.

Dysbiosis and compositional alterations with aging in the gut microbiota of patients with heart failure

Emerging evidence has suggested a potential impact of gut microbiota on the pathophysiology of heart failure (HF). However, it is still unknown whether HF is associated with dysbiosis in gut microbiota. We investigated the composition of gut microbiota in patients with HF to elucidate whether gut microbial dysbiosis is associated with HF. We performed 16S ribosomal RNA gene sequencing of fecal samples obtained from 12 HF patients and 12 age-matched healthy control (HC) subjects, and analyzed the differences in gut microbiota. We further compared the composition of gut microbiota of 12 HF patients younger than 60 years of age with that of 10 HF patients 60 years of age or older. The composition of gut microbial communities of HF patients was distinct from that of HC subjects in both unweighted and weighted UniFrac analyses. Eubacterium rectale and Dorea longicatena were less abundant in the gut microbiota of HF patients than in that of HC subjects. Compared to younger HF patients, older HF patients had diminished proportions of Bacteroidetes and larger quantities of Proteobacteria. The genus Faecalibacterium was depleted, while Lactobacillus was enriched in the gut microbiota of older HF patients. These results suggest that patients with HF harbor significantly altered gut microbiota, which varies further according to age. New concept of heart-gut axis has a great potential for breakthroughs in the development of novel diagnostic and therapeutic approach for HF.

Activation of endothelial β-catenin signaling induces heart failure

Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure.

DNA single-strand break-induced DNA damage response causes heart failure

The DNA damage response (DDR) plays a pivotal role in maintaining genome integrity. DNA damage and DDR activation are observed in the failing heart, however, the type of DNA damage and its role in the pathogenesis of heart failure remain elusive. Here we show the critical role of DNA single-strand break (SSB) in the pathogenesis of pressure overload-induced heart failure. Accumulation of unrepaired SSB is observed in cardiomyocytes of the failing heart. Unrepaired SSB activates DDR and increases the expression of inflammatory cytokines through NF-κB signalling. Pressure overload-induced heart failure is more severe in the mice lacking XRCC1, an essential protein for SSB repair, which is rescued by blocking DDR activation through genetic deletion of ATM, suggesting the causative role of SSB accumulation and DDR activation in the pathogenesis of heart failure. Prevention of SSB accumulation or persistent DDR activation may become a new therapeutic strategy against heart failure.

Echinomycin inhibits adipogenesis in 3T3-L1 cells in a HIF-independent manner

Obesity is a risk factor for many diseases including diabetes, cancer, cardiovascular disease, and chronic kidney disease. Obesity is characterized by the expansion of white adipose tissue (WAT). Hypertrophy and hyperplasia of adipocytes cause tissue hypoxia followed by inflammation and fibrosis. Its trigger, preadipocyte differentiation into mature adipocytes, is finely regulated by transcription factors, signal molecules, and cofactors. We found that echinomycin, a potent HIF-1 inhibitor, completely inhibited adipogenesis in 3T3-L1 WAT preadipocytes by affecting the early phase of mitotic clonal expansion. The dose required to exert the effect was surprisingly low and the time was short. Interestingly, its inhibitory effect was independent of HIF-1 pathways. Time-course DNA microarray analysis of drug-treated and untreated preadipocytes extracted a major transcription factor, CCAAT/enhancer-protein β, as a key target of echinomycin. Echinomycin also inhibited adipogenesis and body weight gain in high fat diet mice. These findings highlight a novel role of echinomycin in suppressing adipocyte differentiation and offer a new therapeutic strategy against obesity and diabetes.

Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors

Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.