Yuichi Oike

DC-STAMP is essential for cell–cell fusion in osteoclasts and foreign body giant cells

Osteoclasts are bone-resorbing cells that play a pivotal role in bone remodeling. Osteoclasts form large multinuclear giant cells by fusion of mononuclear osteoclasts. How cell fusion is mediated, however, is unclear. We identify the dendritic cell–specific transmembrane protein (DC-STAMP), a putative seven-transmembrane protein, by a DNA subtraction screen between multinuclear osteoclasts and mononuclear macrophages. DC-STAMP is highly expressed in osteoclasts but not in macrophages. DC-STAMP–deficient mice were generated, and osteoclast cell fusion was completely abrogated in homozygotes despite normal expression of osteoclast markers and cytoskeletal structure. As osteoclast multinucleation was restored by retroviral introduction of DC-STAMP, loss of cell fusion was directly attributable to a lack of DC-STAMP. Defects in osteoclast multinucleation reduce bone-resorbing activity, leading to osteopetrosis. Similar to osteoclasts, foreign body giant cell formation by macrophage cell fusion was also completely abrogated in DC-STAMP–deficient mice. We have thus identified an essential regulator of osteoclast and macrophage cell fusion, DC-STAMP, and an essential role of osteoclast multinucleation in bone homeostasis.

Adiponectin and AdipoR1 regulate PGC-1α and mitochondria by Ca2+ and AMPK/SIRT1

Adiponectin is an anti-diabetic adipokine. Its receptors possess a seven-transmembrane topology with the amino terminus located intracellularly, which is the opposite of G-protein-coupled receptors. Here we provide evidence that adiponectin induces extracellular Ca2+ influx by adiponectin receptor 1 (AdipoR1), which was necessary for subsequent activation of Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ), AMPK and SIRT1, increased expression and decreased acetylation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and increased mitochondria in myocytes. Moreover, muscle-specific disruption of AdipoR1 suppressed the adiponectin-mediated increase in intracellular Ca2+ concentration, and decreased the activation of CaMKK, AMPK and SIRT1 by adiponectin. Suppression of AdipoR1 also resulted in decreased PGC-1α expression and deacetylation, decreased mitochondrial content and enzymes, decreased oxidative type I myofibres, and decreased oxidative stress-detoxifying enzymes in skeletal muscle, which were associated with insulin resistance and decreased exercise endurance. Decreased levels of adiponectin and AdipoR1 in obesity may have causal roles in mitochondrial dysfunction and insulin resistance seen in diabetes.

Tumour necrosis factor α signalling through activation of Kupffer cells plays an essential role in liver fibrosis of non-alcoholic steatohepatitis in mice

Background: While tumour necrosis factor α (TNF-α) appears to be associated with the development of non-alcoholic steatohepatitis (NASH), its precise role in the pathogenesis of NASH is not well understood. Methods: Male mice deficient in both TNF receptors 1 (TNFR1) and 2 (TNFR2) (TNFRDKO mice) and wild-type mice were fed a methionine and choline deficient (MCD) diet or a control diet for eight weeks, maintaining isoenergetic intake. Results: MCD dietary feeding of TNFRDKO mice for eight weeks resulted in attenuated liver steatosis and fibrosis compared with control wild-type mice. In the liver, the number of activated hepatic Kupffer cells recruited was significantly decreased in TNFRDKO mice after MCD dietary feeding. In addition, hepatic induction of TNF-α, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 was significantly suppressed in TNFRDKO mice. While in control animals MCD dietary feeding dramatically increased mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) in both whole liver and hepatic stellate cells, concomitant with enhanced activation of hepatic stellate cells, both factors were significantly lower in TNFRDKO mice. In primary cultures, TNF-α administration enhanced TIMP-1 mRNA expression in activated hepatic stellate cells and suppressed apoptotic induction in activated hepatic stellate cells. Inhibition of TNF induced TIMP-1 upregulation by TIMP-1 specific siRNA reversed the apoptotic suppression seen in hepatic stellate cells. Conclusions: Enhancement of the TNF-α/TNFR mediated signalling pathway via activation of Kupffer cells in an autocrine or paracrine manner may be critically involved in the pathogenesis of liver fibrosis in this NASH animal model.

Impaired Insulin Signaling in Endothelial Cells Reduces Insulin-Induced Glucose Uptake by Skeletal Muscle

In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.

Mouse Fbw7/Sel-10/Cdc4 Is Required for Notch Degradation during Vascular Development

Mammalian Fbw7 (also known as Sel-10, hCdc4, or hAgo) is the F-box protein component of an SCF (Skp1-Cul1-F-box protein-Rbx1)-type ubiquitin ligase, and the mouse Fbw7 is expressed prominently in the endothelial cell lineage of embryos. We generated mice deficient in Fbw7 and found that the embryos died in utero at embryonic day 10.5-11.5, manifesting marked abnormalities in vascular development. Vascular remodeling was impaired in the brain and yolk sac, and the major trunk veins were not formed. In vitro para-aortic splanchnopleural explant cultures from Fbw7-/- embryos also manifested an impairment of vascular network formation. Notch4, which is the product of the proto-oncogene Int3 and an endothelial cell-specific mammalian isoform of Notch, accumulated in Fbw7-/- embryos, resulting in an increased expression of Hey1, which encodes a transcriptional repressor that acts downstream of Notch signaling and is implicated in vascular development. Expression of Notch1, -2, or -3 or of cyclin E was unaffected in Fbw7-/- embryos. Mammalian Fbw7 thus appears to play an indispensable role in negative regulation of the Notch4-Hey1 pathway and is required for vascular development.

Angiopoietin-like Protein 2 Promotes Chronic Adipose Tissue Inflammation and Obesity-Related Systemic Insulin Resistance

Recent studies of obesity have provided new insights into the mechanisms underlying insulin resistance and metabolic dysregulation. Numerous efforts have been made to identify key regulators of obesity-linked adipose tissue inflammation and insulin resistance. We found that angiopoietin-like protein 2 (Angptl2) was secreted by adipose tissue and that its circulating level was closely related to adiposity, systemic insulin resistance, and inflammation in both mice and humans. Angptl2 activated an inflammatory cascade in endothelial cells via integrin signaling and induced chemotaxis of monocytes/macrophages. Constitutive Angptl2 activation in vivo induced inflammation of the vasculature characterized by abundant attachment of leukocytes to the vessel walls and increased permeability. Angptl2 deletion ameliorated adipose tissue inflammation and systemic insulin resistance in diet-induced obese mice. Conversely, Angptl2 overexpression in adipose tissue caused local inflammation and systemic insulin resistance in nonobese mice. Thus, Angptl2 is a key adipocyte-derived inflammatory mediator that links obesity to systemic insulin resistance.

Angiogenic Role of LYVE-1–Positive Macrophages in Adipose Tissue

Here we report the discovery of a characteristic dense vascular network (DVN) in the tip portion of epididymal adipose tissue in adult mice. The DVN is formed by angiogenesis rather than by vasculogenesis, and has functional blood circulation. This DVN and its subsequent branching may provide a new functional route for adipogenesis. The recruitment, infiltration, and accumulation of bone marrow-derived LYVE-1+ macrophages in the tip region are crucial for the formation of the DVN. Matrix metalloproteinases (MMPs) and the VEGF-VEGFR2 system are responsible not only for the formation of the DVN, but also for the recruitment and infiltration of LYVE-1+ macrophages into the epididymal adipose tissue tip region. SDF-1, but not the MCP-1-CCR2 system, is a critical factor in recruitment and ongoing retention of macrophages in this area. We also demonstrate that the tip region of epididymal adipose tissue is highly hypoxic, and thus provides a microenvironment conducive to the high expression and enhanced activities of VEGF, VEGFR2, MMPs, and SDF-1 in autocrine and paracrine manners, to create an ideal niche for the recruitment, retention, and angiogenic action of macrophages. These findings shed light on the complex interplay between macrophage infiltration, angiogenesis, and adipogenesis in the tip region of adult epididymal adipose tissue, and provide novel insight into the regulation of alternative outgrowth of adipose tissue.

Oncogenic transcription factor Evi1 regulates hematopoietic stem cell proliferation through GATA‐2 expression

The ecotropic viral integration site‐1 (Evi1) is an oncogenic transcription factor in murine and human myeloid leukemia. We herein show that Evi1 is predominantly expressed in hematopoietic stem cells (HSCs) in embryos and adult bone marrows, suggesting a physiological role of Evi1 in HSCs. We therefore investigate the role and authentic target genes of Evi1 in hematopoiesis using Evi1−/− mice, which die at embryonic day 10.5. HSCs in Evi1−/− embryos are markedly decreased in numbers in vivo with defective self‐renewing proliferation and repopulating capacity. Notably, expression rate of GATA‐2 mRNA, which is essential for proliferation of definitive HSCs, is profoundly reduced in HSCs of Evi1−/− embryos. Restoration of the Evi1 or GATA‐2 expression in Evi1−/− HSCs could prevent the failure of in vitro maintenance and proliferation of HSC through upregulation of GATA‐2 expression. An analysis of the GATA‐2 promoter region revealed that Evi1 directly binds to GATA‐2 promoter as an enhancer. Our results reveal that GATA‐2 is presumably one of critical targets for Evi1 and that transcription factors regulate the HSC pool hierarchically.

Angiopoietin-related growth factor antagonizes obesity and insulin resistance

Angiopoietin-related growth factor (AGF), a member of the angiopoietin-like protein (Angptl) family, is secreted predominantly from the liver into the systemic circulation. Here, we show that most (>80%) of the AGF-deficient mice die at about embryonic day 13, whereas the surviving AGF-deficient mice develop marked obesity, lipid accumulation in skeletal muscle and liver, and insulin resistance accompanied by reduced energy expenditure relative to controls. In parallel, mice with targeted activation of AGF show leanness and increased insulin sensitivity resulting from increased energy expenditure. They are also protected from high-fat diet–induced obesity, insulin resistance and nonadipose tissue steatosis. Hepatic overexpression of AGF by adenoviral transduction, which leads to an approximately 2.5-fold increase in serum AGF concentrations, results in a significant (P < 0.01) body weight loss and increases insulin sensitivity in mice fed a high-fat diet. This study establishes AGF as a new hepatocyte-derived circulating factor that counteracts obesity and related insulin resistance.

Genetic variants at the 9p21 locus contribute to atherosclerosis through modulation of ANRIL and CDKN2A/B

UNLABELLED Genome-wide association studies (GWAS) have identified genetic variants contributing to the risk of cardiovascular disease (CVD) at the chromosome 9p21 locus. The CVD-associated region is adjacent to the two cyclin dependent kinase inhibitors (CDKN)2A and 2B and the last exons of the non-coding RNA, ANRIL. It is still not clear which of or how these transcripts are involved in the pathogenesis of atherosclerosis. OBJECTIVE We assessed the hypothesis that 9p21 locus polymorphisms influence the expression of the transcripts in the region (ANRIL, CDKN2A/B) and that these transcripts contribute to atherogenesis through the modulation of proliferation in VSMC. METHODS We genotyped 18 SNPs (r(2)<0.8 and MAF>0.05) across the region of interest: CDKN2A/B and ANRIL, encompassing the CVD-associated region. RNA and DNA were extracted from the blood of 57 volunteers (69-72 years old). Carotid ultrasound was performed in 56 subjects. CDKN2A/B and ANRIL (exons 1-2 and 17-18) expression was measured employing RT-PCR. Gene expression and cell growth were evaluated in cultured VSMC after the siRNA-mediated knock-down of ANRIL. RESULTS The risk alleles for atherosclerosis-related phenotypes were consistently associated with a lower expression of ANRIL when evaluating exons 1-2. Common carotid artery stenosis was associated with a significantly lower (P<0.01) expression of ANRIL (exons 1-2). ANRIL knock-down in VSMC caused significant variation in expression of CDKN2A/B (P<0.05) and reduction of cell growth (P<0.05) in vitro. CONCLUSION Disease-associated SNPs at the 9p21 locus predominantly affect the expression of ANRIL. Overall, our results suggest that several CVD-associated SNPs in the 9p21 locus affect the expression of ANRIL, which, in turn modulate cell growth, possibly via CDKN2A/B regulation.