Koutaro Yokote

Phosphate-activated glutaminase (GLS2), a p53-inducible regulator of glutamine metabolism and reactive oxygen species

We identified a p53 target gene, phosphate-activated mitochondrial glutaminase (GLS2), a key enzyme in conversion of glutamine to glutamate, and thereby a regulator of glutathione (GSH) synthesis and energy production. GLS2 expression is induced in response to DNA damage or oxidative stress in a p53-dependent manner, and p53 associates with the GLS2 promoter. Elevated GLS2 facilitates glutamine metabolism and lowers intracellular reactive oxygen species (ROS) levels, resulting in an overall decrease in DNA oxidation as determined by measurement of 8-OH-dG content in both normal and stressed cells. Further, siRNA down-regulation of either GLS2 or p53 compromises the GSH-dependent antioxidant system and increases intracellular ROS levels. High ROS levels following GLS2 knockdown also coincide with stimulation of p53-induced cell death. We propose that GLS2 control of intracellular ROS levels and the apoptotic response facilitates the ability of p53 to protect cells from accumulation of genomic damage and allows cells to survive after mild and repairable genotoxic stress. Indeed, overexpression of GLS2 reduces the growth of tumor cells and colony formation. Further, compared with normal tissue, GLS2 expression is reduced in liver tumors. Thus, our results provide evidence for a unique metabolic role for p53, linking glutamine metabolism, energy, and ROS homeostasis, which may contribute to p53 tumor suppressor function.

Mice lacking Smad3 are protected against streptozotocin-induced diabetic glomerulopathy

Abstract Transforming growth factor-β (TGF-β) has been implicated in the development of diabetic glomerulopathy. In order to evaluate a role of Smad3, one of the major signaling molecules downstream of TGF-β, in the pathogenesis of diabetic glomerulopathy, Smad3-null mice were made diabetic with streptozotocin injection and analyzed 4 weeks after induction of diabetes. Electron microscopy revealed that the thickness of glomerular basement membrane (GBM) in wild-type diabetic mice was significantly higher than that in non-diabetic mice, whereas no appreciable GBM thickening was found in Smad3-null diabetic mice. Urinary albumin excretion was dramatically increased in wild-type diabetic mice, whereas Smad3-null diabetic mice did not show any overt albuminuria. Northern blotting revealed that mRNA levels of fibronectin and α3 chain of type IV collagen (α3Col4) in renal cortex of wild-type diabetic mice were approximately twice as much as those of non-diabetic mice, whereas their mRNA levels were not increased in Smad3-null diabetic mice. Real-time polymerase chain reaction (PCR) also confirmed diabetes-induced upregulation of fibronectin and α3Col4 in glomeruli of wild-type mice. Glomerular expression of TGF-β1, as assessed by real-time PCR, was enhanced to a similar degree in wild-type and smad3-null diabetic mice, indicating that the observed differences between wild-type and Smad3-null mice are not attributable to difference in the expression of TGF-β1. These data clearly demonstrate a critical role of Smad3 in the early phase of diabetic glomerulopathy. This may be due at least partly to the present findings that diabetes-induced upregulation of fibronectin and α3Col4 is dependent on Smad3 function.

Ezh2 augments leukemogenicity by reinforcing differentiation blockage in acute myeloid leukemia

EZH2, a catalytic component of the polycomb repressive complex 2, trimethylates histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Although EZH2 is overexpressed in various cancers, including some hematologic malignancies, the role of EZH2 in acute myeloid leukemia (AML) has yet to be examined in vivo. In the present study, we transformed granulocyte macrophage progenitors from Cre-ERT;Ezh2(flox/flox) mice with the MLL-AF9 leukemic fusion gene to analyze the function of Ezh2 in AML. Deletion of Ezh2 in transformed granulocyte macrophage progenitors compromised growth severely in vitro and attenuated the progression of AML significantly in vivo. Ezh2-deficient leukemic cells developed into a chronic myelomonocytic leukemia-like disease with a lower frequency of leukemia-initiating cells compared with the control. Chromatin immunoprecipitation followed by sequencing revealed a significant reduction in the levels of trimethylation at H3K27 in Ezh2-deficient leukemic cells, not only at Cdkn2a, a known major target of Ezh2, but also at a cohort of genes relevant to the developmental and differentiation processes. Overexpression of Egr1, one of the derepressed genes in Ezh2-deficient leukemic cells, promoted the differentiation of AML cells profoundly. Our findings suggest that Ezh2 inhibits differentiation programs in leukemic stem cells, thereby augmenting their leukemogenic activity.

Hepatitis B virus X protein shifts human hepatic transforming growth factor (TGF)-β signaling from tumor suppression to oncogenesis in early chronic hepatitis B†

Hepatitis B virus X (HBx) protein is suspected to participate in oncogenesis during chronic hepatitis B progression. Transforming growth factor β (TGF‐β) signaling involves both tumor suppression and oncogenesis. TGF‐β activates TGF‐β type I receptor (TβRI) and c‐Jun N‐terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become C‐terminally phosphorylated Smad3 (pSmad3C) and linker‐phosphorylated Smad3 (pSmad3L). Reversible shifting of Smad3‐mediated signaling between tumor suppression and oncogenesis in HBx‐expressing hepatocytes indicated that TβRI‐dependent pSmad3C transmitted a tumor‐suppressive TGF‐β signal, while JNK‐dependent pSmad3L promoted cell growth. We used immunostaining, immunoblotting, and in vitro kinase assay to compare pSmad3L‐ and pSmad3C‐mediated signaling in biopsy specimens representing chronic hepatitis, cirrhosis, or hepatocellular carcinoma (HCC) from 90 patients chronically infected with hepatitis B virus (HBV) with signaling in liver specimens from HBx transgenic mice. In proportion to plasma HBV DNA levels, early chronic hepatitis B specimens showed prominence of pSmad3L in hepatocytic nuclei. HBx‐activated JNK/pSmad3L/c‐Myc oncogenic pathway was enhanced, while the TβRI/pSmad3C/p21WAF1 tumor‐suppressive pathway was impaired as human and mouse HBx‐associated hepatocarcinogenesis progressed. Of 28 patients with chronic hepatitis B who showed strong oncogenic pSmad3L signaling, six developed HCC within 12 years; only one of 32 patients showing little pSmad3L developed HCC. In contrast, seven of 30 patients with little Smad3C phosphorylation developed HCC, while no patient who retained hepatocytic tumor‐suppressive pSmad3C developed HCC within 12 years. Conclusion: HBx shifts hepatocytic TGF‐β signaling from the tumor‐suppressive pSmad3C pathway to the oncogenic pSmad3L pathway in early carcinogenic process. Hepatocytic pSmad3L and pSmad3C assessment in HBV‐infected liver specimens should prove clinically useful for predicting risk of HCC. (HEPATOLOGY 2009.)

Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders

Deletion of Ezh2 results in transcriptional repression of developmental regulator genes, derepression of oncogenic polycomb targets, and induction of MDS/MPN-like disease in mice that is exacerbated by concurrent deletion of Tet2.

Enhanced Expression of Osteopontin in Human Diabetic Artery and Analysis of Its Functional Role in Accelerated Atherogenesis

We have previously reported that high glucose stimulates osteopontin (OPN) expression through protein kinase C-dependent pathways as well as hexosamine pathways in cultured rat aortic smooth muscle cells. The finding prompted us to study in vivo expression of OPN in diabetes mellitus. In the present study, we found by immunohistochemistry that medial layers of the carotid arteries of streptozotocin-induced diabetic rats and the forearm arteries of diabetic patients stained positively for OPN antibodies, whereas the staining from arteries of control rats and nondiabetic patients was negative. We also found that OPN stimulated the migration and enhanced platelet-derived growth factor (PDGF)-mediated DNA synthesis of cultured rat aortic smooth muscle cells. OPN and PDGF synergistically activated focal adhesion kinase as well as extracellular signal-regulated kinase; this finding seems to explain the OPN-induced enhancement of PDGF-mediated DNA synthesis. Taken together, our present results raise a possibility that OPN plays a role in the development of diabetic vascular complications.

Multicenter collaborative randomized parallel group comparative study of pitavastatin and atorvastatin in Japanese hypercholesterolemic patients Collaborative study on hypercholesterolemia drug intervention and their benefits for atherosclerosis prevention (CHIBA study)

AIMS To compare the efficacy and safety of pitavastatin and atorvastatin in Japanese patients with hypercholesterolemia. METHODS AND RESULTS Japanese patients with total cholesterol (TC) > or = 220 mg/dL were randomized to receive pitavastatin 2 mg (n=126) or atorvastatin 10 mg (n=125) for 12 weeks. The primary endpoint was percent change from baseline in non-HDL-C level after 12 weeks of treatment. Reduction of non-HDL-C by pitavastatin treatment (39.0%, P=0.456 vs. atorvastatin) was non-inferior to that by atorvastatin (40.3%). Both pitavastatin and atorvastatin also significantly reduced LDL-C by 42.6% and 44.1%, TC by 29.7% and 31.1%, and TG by 17.3% and 10.7%, respectively, at 12 weeks without intergroup differences. HDL-C showed a significant increase at 12 weeks with pitavastatin treatment (3.2%, P=0.033 vs. baseline) but not with atorvastatin treatment (1.7%, P=0.221 vs. baseline). Waist circumference, body weight and BMI were significantly correlated with percent reduction of non-HDL-C in the atorvastatin group, whereas pitavastatin showed consistent reduction of non-HDL-C regardless of the body size. In patients with metabolic syndrome, LDL-C was reduced significantly more in patients receiving pitavastatin when compared with those receiving atorvastatin. AST, ALT and gammaGTP increased significantly in patients receiving atorvastatin but not in those receiving pitavastatin. Both treatments were well tolerated. CONCLUSION Pitavastatin 2 mg and atorvastatin 10 mg are equally effective in improving the lipid profile and were well tolerated in Japanese patients with hypercholesterolemia.

High Glucose-Induced Upregulation of Osteopontin Is Mediated via Rho/Rho Kinase Pathway in Cultured Rat Aortic Smooth Muscle Cells

Objective—Osteopontin is upregulated in the diabetic vascular wall and in vascular smooth muscle cells cultured under high glucose concentration. In the present study, we analyzed the mechanism of high glucose-induced upregulation of osteopontin in cultured rat aortic smooth muscle cells. Methods and Results—We found that an inhibitor of Rho-associated protein kinase, Y-27632, suppressed osteopontin mRNA expression under high glucose concentration. Transfection of cells with a constitutive active Rho mutant, pSR&agr;-myc-RhoDA, enhanced osteopontin mRNA expression. Furthermore, incubation of cells under high glucose concentration activated Rho, indicating that Rho/Rho kinase pathway mediates high-glucose–stimulated osteopontin expression. Treatment of cells with an inhibitor of protein kinase C, GF109203X, and azaserine, an inhibitor of the hexosamine pathway, suppressed high glucose-induced Rho activation. Glucosamine treatment was shown to activate Rho. Treatment of cells with an inhibitor of MEK1, PD98059, suppressed osteopontin mRNA expression under high glucose concentration. Incubation of cells under high glucose concentration activated ERK. Finally, transfection of cells with pSR&agr;-myc-RhoDA also activated ERK. Conclusions—In conclusion, our present findings support a notion that Rho/Rho kinase pathway functions downstream of protein kinase C and the hexosamine pathways and upstream of ERK in mediating high-glucose–induced upregulation of osteopontin expression.

Targeted Disruption of TGF-β–Smad3 Signaling Leads to Enhanced Neointimal Hyperplasia With Diminished Matrix Deposition in Response to Vascular Injury

The role of transforming growth factor (TGF)-&bgr; and its signal in atherogenesis is not fully understood. Here, we examined mice lacking Smad3, a major downstream mediator of TGF-&bgr;, to clarify the precise role of Smad3-dependent signaling in vascular response to injury. Femoral arteries were injured in wild-type and Smad3-null (null) male mice on C57Bl/6 background. Histopathological evaluation of the arteries 1 to 3 weeks after the injury revealed significant enhancement of neointimal hyperplasia in null compared with wild-type mice. Transplantation of null bone marrow to wild-type mice did not enhance neointimal thickening, suggesting that vascular cells in situ play a major role in the response. Null intima contained more proliferating smooth muscle cells (SMC) with less amount of collagen compared with wild-type intima. TGF-&bgr; caused significant inhibition of cellular proliferation in wild-type aortic SMC, whereas the growth of null SMC was only weakly inhibited by TGF-&bgr; in vitro, indicating a crucial role of Smad3 in the growth inhibitory function. On the other hand, Smad3-deficiency did not attenuate chemotaxis of SMC toward TGF-&bgr;. TGF-&bgr; increased transcript level of &agr;2 type I collagen and tissue inhibitor of metalloproteinases-1, and suppressed expression and activity of matrix metalloproteinases in wild-type SMC. However, these effects of TGF-&bgr; were diminished in null SMC. Our findings altogether show that the loss of Smad3 pathway causes enhanced neointimal hyperplasia on injury through modulation of growth and matrix regulation in vascular SMC. These results indicate a vasculoprotective role of endogenous Smad3 in response to injury.

CCN3 Inhibits Neointimal Hyperplasia Through Modulation of Smooth Muscle Cell Growth and Migration

Objective—CCN3 belongs to the CCN family, which constitutes multifunctional secreted proteins that act as matrix cellular regulators. We investigated the pathophysiological roles of CCN3 in the vessels. Methods and Results—We examined the effects of CCN3 on the proliferation and migration of rat vascular smooth muscle cells (VSMC). CCN3 knockout mice were created, and vascular phenotypes and neointimal hyperplasia induced by photochemically induced thrombosis were investigated. CCN3 suppressed the VSMC proliferation induced by fetal bovine serum. The neutralizing antibody for transforming growth factor-&bgr; did not affect the growth inhibitory effect of CCN3. Moreover, CCN3 enhanced the mRNA expression of cyclin-dependent kinase inhibitors, p21 and p15. Gamma secretase inhibitor, an inhibitor of Notch signaling, partially inhibited the enhanced expression of p21 induced by CCN3. CCN3 also inhibited the VSMC migration. Finally, the histopathologic evaluation of the arteries 21 days after the endothelial injury revealed a 6-fold enhancement of neointimal thickening in the null mice compared with the wild-type mice. Conclusion—CCN3 suppresses neointimal thickening through the inhibition of VSMC migration and proliferation. Our findings indicate the involvement of CCN3 in vascular homeostasis, especially on injury, and the potential usefulness of this molecule in the modulation of atherosclerotic vascular disease.