Kosaku Okubo

Bio: Kosaku Okubo is an academic researcher from Osaka University. The author has contributed to research in topics: High endothelial venules & Endothelial stem cell. The author has an hindex of 5, co-authored 5 publications receiving 4959 citations.

Isolation and characterization of human cathepsin V : A major proteinase in corneal epithelium

Abstract: PURPOSE. To isolate and characterize a novel cathepsin gene, as part of the systematic isolation of genes uniquely active in corneal epithelium. METHODS. For the isolation of a full-length cDNA clone, a probe was selected from a set of expressed sequence tag clones classified as unique to corneal epithelium. Inserted cDNA was introduced into insect cells using a baculovirus expression system, and the secretion of recombinant protein was identified using antisera against a synthetic peptide. Proteolytic activity was determined using bovine serum albumin (BSA) as substrate. The expressions of the novel cathepsin in human cornea and other tissues were examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS. The full-length cDNA clone encoded a peptide of 334 amino acids with 82% identity with bovine cathepsin L and 77% identity with human cathepsin L when aligned. The recombinant protein produced in the baculovirus expression system cleaves BSA, and its activity was inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, but not by pepstatin A, phenylmethylsulfonyl fluoride, and EDTA. By RT-PCR, a low level of expression was observed in some other epithelial tissues of ectodermal origin, but only in cornea was it higher than cathepsin L, which is known to be a general lysosomal cathepsin. Cathepsin V protein was detected in human corneal epithelium by western blot analysis, but not in tear fluid. CONCLUSIONS. The amino acid homology and proteolytic activity of the recombinant protein indicate that the novel gene is a new member of the cathepsins that have features of cysteine proteinase. Its uniquely high expression in corneal epithelium strongly implies an important role in corneal physiology.

Gene Expression Profiling of Mucosal Addressin Cell Adhesion Molecule-1+ High Endothelial Venule Cells (HEV) and Identification of a Leucine-Rich HEV Glycoprotein as a HEV Marker

Abstract: High endothelial venule (HEV) cells support lymphocyte migration from the peripheral blood into secondary lymphoid tissues. Using gene expression profiling of mucosal addressin cell adhesion molecule-1+ mesenteric lymph node HEV cells by quantitative 3′-cDNA collection, we have identified a leucine-rich protein, named leucine-rich HEV glycoprotein (LRHG) that is selectively expressed in these cells. Northern blot analysis revealed that LRHG mRNA is ∼1.3 kb and is expressed in lymph nodes, liver, and heart. In situ hybridization analysis demonstrated that the mRNA expression in lymph nodes is strictly restricted to the HEV cells, and immunofluorescence analysis with polyclonal Abs against LRHG indicated that the LRHG protein is localized mainly to HEV cells and possibly to some lymphoid cells surrounding the HEVs. LRHG cDNA encodes a 342-aa protein containing 8 tandem leucine-rich repeats of 24 aa each and has high homology to human leucine-rich α2-glycoprotein. Similar to some other leucine-rich repeat protein family members, LRHG can bind extracellular matrix proteins that are expressed on the basal lamina of HEVs, such as fibronectin, collagen IV, and laminin. In addition, LRHG binds TGF-β. These results suggest that LRHG is likely to be multifunctional in that it may capture TGF-β and/or other related humoral factors to modulate cell adhesion locally and may also be involved in the adhesion of HEV cells to the surrounding basal lamina.

Expression profile of active genes in mouse lymph node high endothelial cells.

Abstract: High endothelial venules (HEV) allow rapid and selective lymphocyte trafficking from the blood into secondary lymphoid tissues. Here we report the expression profile of active genes in mouse high endothelial cells (HEC). HEC were first purified from mouse lymph nodes (LN) by magnetic cell sorting with MECA-79 mAb and a 3'-directed cDNA library that faithfully represents the composition of mRNA was constructed. A total of 1495 cDNA sequences were obtained from randomly selected clones. Based on their sequence identity, they were grouped into 754 different species [gene signatures (GS)] of which 335 GS were identified in GenBank. Among the previously identified genes, expression of several endothelial cell surface molecules including endoglin and ICAM-1 was detected in HEC. Comparison of the gene expression profile with that of purified CD31(+) flat endothelial cells identified several molecules, such as KC chemokine and Duffy antigen/receptor for chemokines, that are known to be selectively expressed in activated endothelial cells or post-capillary venules. Interestingly, mac25/TAF, which is known to be expressed specifically in tumor vessels and implicated in the regulation of cell adhesion, was highly and selectively expressed in HEC in mouse LN, suggesting that it may participate in regulating HEC-specific functions. Comparison with the expression profiles obtained from 35 different cell types showed at least 22 GS that were apparently specific to HEC. Our results illustrate the expression differences between HEC and CD31(+) flat endothelial cells, and will be useful for the identification and characterization of genes specific for HEC.

Obesity is associated with macrophage accumulation in adipose tissue

Abstract: Obesity alters adipose tissue metabolic and endocrine function and leads to an increased release of fatty acids, hormones, and proinflammatory molecules that contribute to obesity associated complications. To further characterize the changes that occur in adipose tissue with increasing adiposity, we profiled transcript expression in perigonadal adipose tissue from groups of mice in which adiposity varied due to sex, diet, and the obesity-related mutations agouti (Ay) and obese (Lepob). We found that the expression of 1,304 transcripts correlated significantly with body mass. Of the 100 most significantly correlated genes, 30% encoded proteins that are characteristic of macrophages and are positively correlated with body mass. Immunohistochemical analysis of perigonadal, perirenal, mesenteric, and subcutaneous adipose tissue revealed that the percentage of cells expressing the macrophage marker F4/80 (F4/80+) was significantly and positively correlated with both adipocyte size and body mass. Similar relationships were found in human subcutaneous adipose tissue stained for the macrophage antigen CD68. Bone marrow transplant studies and quantitation of macrophage number in adipose tissue from macrophage-deficient (Csf1op/op) mice suggest that these F4/80+ cells are CSF-1 dependent, bone marrow-derived adipose tissue macrophages. Expression analysis of macrophage and nonmacrophage cell populations isolated from adipose tissue demonstrates that adipose tissue macrophages are responsible for almost all adipose tissue TNF-alpha expression and significant amounts of iNOS and IL-6 expression. Adipose tissue macrophage numbers increase in obesity and participate in inflammatory pathways that are activated in adipose tissues of obese individuals.

Inflammation in atherosclerosis

Abstract: Abundant data link hypercholesterolaemia to atherogenesis. However, only recently have we appreciated that inflammatory mechanisms couple dyslipidaemia to atheroma formation. Leukocyte recruitment and expression of pro-inflammatory cytokines characterize early atherogenesis, and malfunction of inflammatory mediators mutes atheroma formation in mice. Moreover, inflammatory pathways promote thrombosis, a late and dreaded complication of atherosclerosis responsible for myocardial infarctions and most strokes. The new appreciation of the role of inflammation in atherosclerosis provides a mechanistic framework for understanding the clinical benefits of lipid-lowering therapies. Identifying the triggers for inflammation and unravelling the details of inflammatory pathways may eventually furnish new therapeutic targets.

The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity

Abstract: Adiponectin is an adipocyte-derived hormone. Recent genome-wide scans have mapped a susceptibility locus for type 2 diabetes and metabolic syndrome to chromosome 3q27, where the gene encoding adiponectin is located. Here we show that decreased expression of adiponectin correlates with insulin resistance in mouse models of altered insulin sensitivity. Adiponectin decreases insulin resistance by decreasing triglyceride content in muscle and liver in obese mice. This effect results from increased expression of molecules involved in both fatty-acid combustion and energy dissipation in muscle. Moreover, insulin resistance in lipoatrophic mice was completely reversed by the combination of physiological doses of adiponectin and leptin, but only partially by either adiponectin or leptin alone. We conclude that decreased adiponectin is implicated in the development of insulin resistance in mouse models of both obesity and lipoatrophy. These data also indicate that the replenishment of adiponectin might provide a novel treatment modality for insulin resistance and type 2 diabetes.

Increased oxidative stress in obesity and its impact on metabolic syndrome

Abstract: Obesity is a principal causative factor in the development of metabolic syndrome. Here we report that increased oxidative stress in accumulated fat is an important pathogenic mechanism of obesity-associated metabolic syndrome. Fat accumulation correlated with systemic oxidative stress in humans and mice. Production of ROS increased selectively in adipose tissue of obese mice, accompanied by augmented expression of NADPH oxidase and decreased expression of antioxidative enzymes. In cultured adipocytes, elevated levels of fatty acids increased oxidative stress via NADPH oxidase activation, and oxidative stress caused dysregulated production of adipocytokines (fat-derived hormones), including adiponectin, plasminogen activator inhibitor-1, IL-6, and monocyte chemotactic protein-1. Finally, in obese mice, treatment with NADPH oxidase inhibitor reduced ROS production in adipose tissue, attenuated the dysregulation of adipocytokines, and improved diabetes, hyperlipidemia, and hepatic steatosis. Collectively, our results suggest that increased oxidative stress in accumulated fat is an early instigator of metabolic syndrome and that the redox state in adipose tissue is a potentially useful therapeutic target for obesity-associated metabolic syndrome.

Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase

Abstract: Adiponectin (Ad) is a hormone secreted by adipocytes that regulates energy homeostasis and glucose and lipid metabolism. However, the signaling pathways that mediate the metabolic effects of Ad remain poorly identified. Here we show that phosphorylation and activation of the 5'-AMP-activated protein kinase (AMPK) are stimulated with globular and full-length Ad in skeletal muscle and only with full-length Ad in the liver. In parallel with its activation of AMPK, Ad stimulates phosphorylation of acetyl coenzyme A carboxylase (ACC), fatty-acid oxidation, glucose uptake and lactate production in myocytes, phosphorylation of ACC and reduction of molecules involved in gluconeogenesis in the liver, and reduction of glucose levels in vivo. Blocking AMPK activation by dominant-negative mutant inhibits each of these effects, indicating that stimulation of glucose utilization and fatty-acid oxidation by Ad occurs through activation of AMPK. Our data may provide a novel paradigm that an adipocyte-derived antidiabetic hormone, Ad, activates AMPK, thereby directly regulating glucose metabolism and insulin sensitivity in vitro and in vivo.