Contribution of HSP90 Cleavage to the Cytotoxic Effect of Suberoylanilide Hydroxamic Acid In Vivo and the Involvement of TXNIP in HSP90 Cleavage.

Heat shock protein (HSP) 90 is expressed in most living organisms, and several client proteins of HSP90 are necessary for cancer cell survival and growth. Previously, we found that HSP90 was cleaved by histone deacetylase (HDAC) inhibitors and proteasome inhibitors, and the cleavage of HSP90 contributes to their cytotoxicity in K562 leukemia cells. In this study, we first established mouse xenograft models with K562 cells expressing the wild-type or cleavage-resistant mutant HSP90ß and found that the suppression of tumor growth by the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was interrupted by the mutation inhibiting the HSP90 cleavage in vivo. Next, we investigated the possible function of thioredoxin interacting protein (TXNIP) in the HSP90 cleavage induced by SAHA. TXNIP is a negative regulator for thioredoxin, an antioxidant protein. SAHA transcriptionally induced the expression of TXNIP in K562 cells. HSP90 cleavage was induced by SAHA also in the thymocytes of normal mice and suppressed by an anti-oxidant and pan-caspase inhibitor. When the thymocytes from the TXNIP knockout mice and their wild-type littermate control mice were treated with SAHA, the HSP90 cleavage was detected in the thymocytes of the littermate controls but suppressed in those of the TXNIP knockout mice suggesting the requirement of TXNIP for HSP90 cleavage. We additionally found that HSP90 cleavage was induced by actinomycin D, ß-mercaptoethanol, and p38 MAPK inhibitor PD169316 suggesting its prevalence. Taken together, we suggest that HSP90 cleavage occurs also in vivo and contributes to the anti-cancer activity of various drugs in a TXNIP-dependent manner.

miR-150-Mediated Foxo1 Regulation Programs CD8+ T Cell Differentiation.

MicroRNA (miR)-150 is a developmental regulator of several immune-cell types, but its role in CD8+ T cells is largely unexplored. Here, we show that miR-150 regulates the generation of memory CD8+ T cells. After acute virus infection, miR-150 knockout (KO) mice exhibited an accelerated differentiation of CD8+ T cells into memory cells and improved production of effector cytokines. Additionally, miR-150 KO CD8+ T cells displayed an enhanced recall response and improved protection against infections with another virus and bacteria. We found that forkhead box O1 (Foxo1) and T cell-specific transcription factor 1 (TCF1) are upregulated during the early activation phase in miR-150 KO CD8+ T cells and that miR-150 directly targets and suppresses Foxo1. These results suggest that miR-150-mediated suppression of Foxo1 regulates the balance between effector and memory cell differentiation, which might aid in the development of improved vaccines and T cell therapeutics.

Thioredoxin-interacting protein mediates hepatic lipogenesis and inflammation via PRMT1 and PGC-1α regulation in vitro and in vivo.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and type 2 diabetes. Thioredoxin-interacting protein (TXNIP) regulates the cellular redox state and metabolism and has been linked to many diseases, including diabetes. Therefore, we examined the role of TXNIP in hepatic steatosis in vitro and in vivo. METHODS: Lipogenic and inflammatory proteins produced by hepatocytes treated with palmitic acid (PA) or transfected with TXNIP or Txnip siRNA were measured by Western blotting. Lipid accumulation was assessed using Oil Red O staining. Protein interactions were assessed by immunoprecipitation and proximity ligation assay. Hepatic protein levels were measured by Western blotting from wild type or Txnip(-/-) mice fed a high-fat diet (HFD) or chow diet. Livers from NAFLD patients were compared with normal liver by immunohistochemistry. RESULTS: PA increased TXNIP, and inflammatory and lipogenic proteins in both AML12 and H4IIE cells. It also increased the peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α), which mediated the expression of lipogenic markers and lipid accumulation. In addition, PA increased protein arginine methyltransferase-1 (PRMT1) and PRMT1 siRNA abolished the increase in lipogenic markers with PGC-1α. Furthermore, TXNIP interacted with PRMT1 in PA-treated hepatocytes. In vivo, levels of lipogenic proteins, inflammatory molecules, PGC-1α, and PRMT1 were increased in the livers of HFD mice compared with those fed a chow diet, and were ameliorated in HFD Txnip(-/-) mice. Moreover, TXNIP, PRMT1, and PGC-1α were elevated in the livers of human NAFLD patients. CONCLUSIONS: TXNIP mediates hepatic lipogenesis via PRMT1 and PGC-1α regulation and inflammation in vitro and in vivo, implying that targeting TXNIP and PRMT1 is a potential therapeutic approach for treatment of NAFLD.

Vitamin D3 up-regulated protein 1 controls the priming phase of liver regeneration.

Vitamin D3 up-regulated protein 1 (VDUP1) is a potent growth suppressor that inhibits tumor cell proliferation and cell cycle progression when overexpressed. In a previous study, we showed that VDUP1 knockout (KO) mice exhibited accelerated liver regeneration because such animals could effectively control the expression of cell cycle regulators that drive the G1-to-S phase progression. In the present study, we further investigated the role played by VDUP1 in initial priming of liver regeneration. To accomplish this, VDUP1 KO and wild-type (WT) mice were subjected to 70% partial hepatectomy (PH) and sacrificed at different times after surgery. The hepatic levels of TNF-α and IL-6 increased after PH, but there were no significant differences between VDUP1 KO and WT mice. Nuclear factor-κB (NF-κB), c-Jun-N-terminal kinase (JNK), and signal transducer and activator of transcription 3 (STAT-3) were activated much earlier and to a greater extent in VDUP1 KO mice after PH. A single injection of TNF-α or IL-6 caused rapid activation of JNK and STAT-3 expression in both mice, but the responses were stronger and more sustained in VDUP1 KO mice. In conclusion, our findings provide evidence that VDUP1 plays a role in initiation of liver regeneration.

Vitamin D3 upregulated protein 1 deficiency promotes N-methyl-N-nitrosourea and Helicobacter pylori-induced gastric carcinogenesis in mice.

OBJECTIVE: Vitamin D(3) upregulated protein 1 (VDUP1) is a potent tumour suppressor whose expression is dramatically reduced in various types of human cancers, including gastric cancer. However, the precise mechanisms underlying tumour development remain unclear. In the present study, the authors examined the effect of VDUP1 on Helicobacter pylori-induced gastric carcinogenesis in mice. DESIGN: Gastric cancer was generated in VDUP1 knockout (KO) and wild-type mice using a combination of N-methyl-N-nitrosourea treatment and H pylori infection. Fifty weeks after treatment, gastric tissues from both types of mice were examined by histopathology, immunohistochemistry and immunoblotting. In vitro tests on the human gastric cancer cell line, AGS, were also performed to identify the underlying mechanisms of cancer development. RESULTS: The overall incidence of gastric cancer was significantly higher in VDUP1 KO mice than in wild-type mice. Similarly, VDUP1 KO mice showed more severe chronic gastritis, glandular atrophy, foveolar hyperplasia, metaplasia and dysplasia. Although no differences in the apoptotic index were apparent, lack of VDUP1 increased the rate of gastric epithelial cell proliferation in non-cancerous stomachs, with corresponding increases in tumour necrosis factor alpha (TNFα) level, nuclear transcription factor kappa B (NF-κB) activation and cyclooxygenase-2 (COX-2) expression. An in vitro study showed that H pylori-associated cell proliferation and induction of TNFα, NF-κB and COX-2 were inhibited in cells transfected with VDUP1. In addition, overexpression of VDUP1 in AGS cells suppressed TNFα-induced NF-κB activation and COX-2 expression. CONCLUSION: Our data show that VDUP1 negatively regulates H pylori-associated gastric carcinogenesis, in part by disrupting cell growth and inhibiting the induction of TNFα, NF-κB and COX-2. These findings provide important insights into the role of VDUP1 in H pylori-associated tumourigenesis.

Vitamin D3 up-regulated protein 1 deficiency accelerates liver regeneration after partial hepatectomy in mice.

BACKGROUND & AIMS: Liver regeneration is a complicated process involving a variety of interacting factors. Vitamin D3 up-regulated protein 1 (VDUP1) is a potent growth suppressor that, upon over-expression, inhibits tumor cell proliferation and cell-cycle progression. Here, we investigated the function of VDUP1 in liver regeneration following hepatectomy in mice. METHODS: Liver regeneration after 70% partial hepatectomy (PH) was compared in VDUP1 knockout (KO) and wild-type (WT) mice, and the activities of proliferative- and cell-cycle-related signaling pathways were measured. RESULTS: Compared with WT mice, liver recovery was significantly accelerated in VDUP1 KO mice during the first day after PH, in association with increased DNA synthesis. Consistent with this observation, the expression levels of key cell-cycle regulatory proteins, including cyclin D, cyclin E, cyclin-dependent kinase 4 (CDK4), p21, and p27, were markedly altered in the livers of VDUP1 KO mice. Induction of growth factors and activation of proliferative signaling pathway components including extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, glycogen synthase kinase 3ß (GSK3ß), mammalian target of rapamycin (mTOR), and p70S6 kinase (p70(S6K)), occurred much earlier and to a greater extent in VDUP1 KO mouse livers. In addition, primary hepatocytes isolated from VDUP1 KO mice displayed increased activation of ERK1/2 and Akt in response to HGF and TGF-α. CONCLUSIONS: Our results reveal an important role for VDUP1 in the regulation of proliferative signaling during liver regeneration. Altered activation of genes involved in ERK1/2 and Akt signaling pathways may explain the accelerated growth responses seen in VDUP1 KO mice.

The role of vitamin D3 upregulated protein 1 in thioacetamide-induced mouse hepatotoxicity.

Thioacetamide (TA) is a commonly used drug that can trigger acute hepatic failure (AHF) through generation of oxidative stress. Vitamin D3 upregulated protein 1 (VDUP1) is an endogenous inhibitor of thioredoxin, a ubiquitous thiol oxidoreductase, that regulates cellular redox status. In this study, we investigated the role of VDUP1 in AHF using a TA-induced liver injury model. VDUP1 knockout (KO) and wild-type (WT) mice were subjected to a single intraperitoneal TA injection, and various parameters of hepatic injury were assessed. VDUP1 KO mice displayed a significantly higher survival rate, lower serum alanine aminotransferase and aspartate aminotransferase levels, and less hepatic damage, compared to WT mice. In addition, induction of apoptosis was decreased in VDUP1 KO mice, with the alteration of caspase-3 and -9 activities, Bax-to-Bcl-2 expression ratios, and mitogen activated protein kinase (MAPK) signaling pathway. Importantly, analysis of TA bioactivation revealed lower plasma clearance of TA and covalent binding of [¹4C]TA to liver macromolecules in VDUP1 KO mice. Furthermore, the level of oxidative stress was significantly less in VDUP1 KO mice than in their WT counterparts, as evident from lipid peroxidation assay. These results collectively indicate that VDUP1 deficiency protects against TA-induced acute liver injury via lower bioactivation of TA and antioxidant effects.