Ayano Naka

Antitumor effects of 2‐oxoglutarate through inhibition of angiogenesis in a murine tumor model

Hypoxia‐inducible factor 1 (HIF‐1) plays essential roles in tumor angiogenesis and growth by regulating the transcription of several key genes in response to hypoxic stress and growth factors. HIF‐1 is a heterodimeric transcriptional activator consisting of inducible α and constitutive β subunits. In oxygenated cells, proteins containing the prolyl hydroxylase domain (PHD) directly sense intracellular oxygen concentrations. PHDs tag HIF‐1α subunits for polyubiquitination and proteasomal degradation by prolyl hydroxylation using 2‐oxoglutarate (2‐OX) and dioxygen. Our recent studies showed that 2‐OX reduces HIF‐1α, erythropoietin, and vascular endothelial growth factor (VEGF) expression in the hepatoma cell line Hep3B when under hypoxic conditions in vitro. Here, we report that similar results were obtained in Lewis lung cancer (LLC) cells in in vitro studies. Furthermore, 2‐OX showed potent antitumor effects in a mouse dorsal air sac assay and a murine tumor xenograft model. In the dorsal air sac assay, 2‐OX reduced the numbers of newly formed vessels induced by LLC cells. In a murine tumor xenograft model, intraperitoneal injection of 2‐OX significantly inhibited tumor growth and angiogenesis in tumor tissues. Moreover, 5‐fluorouracil combined with 2‐OX significantly inhibited tumor growth in this model, which was accompanied by reduction of Vegf gene expression and inhibited angiogenesis in tumor tissues. These results suggest that 2‐OX is a promising anti‐angiogenic therapeutic agent. (Cancer Sci 2009; 100: 1639–1647)

TFE3 Controls Lipid Metabolism in Adipose Tissue of Male Mice by Suppressing Lipolysis and Thermogenesis

Transcription factor E3 (TFE3) is a transcription factor that binds to E-box motifs and promotes energy metabolism-related genes. We previously reported that TFE3 directly binds to the insulin receptor substrate-2 promoter in the liver, resulting in increased insulin response. However, the role of TFE3 in other tissues remains unclear. In this study, we generated adipose-specific TFE3 transgenic (aP2-TFE3 Tg) mice. These mice had a higher weight of white adipose tissue (WAT) and brown adipose tissue than wild-type (WT) mice under fasting conditions. Lipase activity in the WAT in these mice was lower than that in the WT mice. The mRNA level of adipose triglyceride lipase (ATGL), the rate-limiting enzyme for adipocyte lipolysis, was significantly decreased in aP2-TFE3 Tg mice. The expression of Foxo1, which directly activates ATGL expression, was also suppressed in transgenic mice. Promoter analysis confirmed that TFE3 suppressed promoter activities of the ATGL gene. In contrast, G0S2 and Perilipin1, which attenuate ATGL activity, were higher in transgenic mice than in WT mice. These results indicated that the decrease in lipase activity in adipose tissues was due to a decrease in ATGL expression and suppression of ATGL activity. We also showed that thermogenesis was suppressed in aP2-TFE3 Tg mice. The decrease in lipolysis in WAT of aP2-TFE3 Tg mice inhibited the supply of fatty acids to brown adipose tissue, resulting in the inhibition of the expression of thermogenesis-related genes such as UCP1. Our data provide new evidence that TFE3 regulates lipid metabolism by controlling the gene expression related to lipolysis and thermogenesis in adipose tissue.

TFE3 regulates muscle metabolic gene expression, increases glycogen stores, and enhances insulin sensitivity in mice

The role of transcription factor E3 (TFE3), a bHLH transcription factor, in immunology and cancer has been well characterized. Recently, we reported that TFE3 activates hepatic IRS-2 and hexokinase, participates in insulin signaling, and ameliorates diabetes. However, the effects of TFE3 in other organs are poorly understood. Herein, we examined the effects of TFE3 on skeletal muscle, an important organ involved in glucose metabolism. We generated transgenic mice that selectively express TFE3 in skeletal muscles. These mice exhibit a slight acceleration in growth prior to adulthood as well as a progressive increase in muscle mass. In TFE3 transgenic muscle, glycogen stores were more than twofold than in wild-type mice, and this was associated with an upregulation of genes involved in glucose metabolism, specifically glucose transporter 4, hexokinase II, and glycogen synthase. Consequently, exercise endurance capacity was enhanced in this transgenic model. Furthermore, insulin sensitivity was enhanced in transgenic mice and exhibited better improvement after 4 wk of exercise training, which was associated with increased IRS-2 expression. The effects of TFE3 on glucose metabolism in skeletal muscle were different from that in the liver, although they did, in part, overlap. The potential role of TFE3 in regulating metabolic genes and glucose metabolism within skeletal muscle suggests that it may be used for treating metabolic diseases as well as increasing endurance in sport.

TFE3 inhibits myoblast differentiation in C2C12 cells via down-regulating gene expression of myogenin.

Transcription factor E3 (TFE3) belongs to a basic helix-loop-helix family, and is involved in the biology of osteoclasts, melanocytes and their malignancies. We previously reported the metabolic effects of TFE3 on insulin in the liver and skeletal muscles in animal models. In the present study, we explored a novel role for TFE3 in a skeletal muscle cell line. When TFE3 was overexpressed in C2C12 myoblasts by adenovirus before induction of differentiation, myogenic differentiation of C2C12 cells was significantly inhibited. Adenovirus-mediated TFE3 overexpression also suppressed the gene expression of muscle regulatory factors (MRFs), such as MyoD and myogenin, during C2C12 differentiation. In contrast, knockdown of TFE3 using adenovirus encoding short-hairpin RNAi specific for TFE3 dramatically promoted myoblast differentiation associated with significantly increased expression of MRFs. Consistent with these findings, promoter analyses via luciferase reporter assay and electrophoretic mobility shift assay suggested that TFE3 negatively regulated myogenin promoter activity by direct binding to an E-box, E2, in the myogenin promoter. These findings indicated that TFE3 has a regulatory role in myoblast differentiation, and that transcriptional suppression of myogenin expression may be part of the mechanism of action.