Shuntaro Hara

Transcriptional regulation of thioredoxin reductase 1 expression by cadmium in vascular endothelial cells: Role of NF-E2-related factor-2†

Thioredoxin reductase (TrxR) is a selenoprotein that catalyzes the reduction of the active site disulfide of thioredoxin (Trx), which regulates the redox status of the cells. In the present study, we found that TrxR1, one of the three TrxR isozymes, was induced by cadmium as well as tumor necrosis factor α (TNFα) in bovine arterial endothelial cells (BAEC), and investigated the mechanism of cadmium‐induced TrxR1 expression. We here showed that cadmium, differently from TNFα, enhanced the promoter activity of the 5′‐flanking region of human TrxR1 gene (nucleotides −1692 to +49). Deletion and site‐directed mutation of antioxidant responsive element (ARE) (nucleotides −62 to −48) in this region abolished the response to cadmium. Overexpression of NF‐E2‐related factor‐2 (Nrf2) augmented the TrxR1 promoter activity. In contrast, overexpression of the dominant negative mutant of Nrf2 suppressed cadmium‐induced activation of TrxR1 promoter through the ARE. Chromatin immunoprecipitation (ChIP) assays showed that anti‐Nrf2 antibody precipitated ARE from the chromatin of the cadmium‐treated cells. These results indicated that cadmium‐induced TrxR1 gene expression is mediated by the activation of Nrf2 transcription factor and its binding to ARE in the TrxR1 gene promoter. We further found that in addition to cadmium, the activators of Nrf2, such as diethyl maleate (DEM) and arsenite, induced both TrxR1 and Trx gene expression in BAEC. Nrf2 might play an important role in the regulation of the cellular Trx system consisting of Trx and TrxR.

Human HIF-3α4 is a dominant-negative regulator of HIF-1 and is down-regulated in renal cell carcinoma

A universal response to changes in cellular oxygen tension is governed by a family of heterodimeric transcription factors called hypoxia‐inducible factor (HIF). Tumor hypoxia, as well as various cancer‐causing mutations, has been shown to elevate the level of HIF‐1α, signifying a critical role of the HIF pathway in cancer development. The recently identified third member of the human HIF‐α family, HIF‐3α, produces multiple splice variants that contain extra DNA binding elements and protein‐protein interaction motifs not found in HIF‐1α or HIF‐2α. Here we report the molecular cloning of the alternatively spliced human HIF‐3α variant HIF‐3α4 and show that it attenuates the ability of HIF‐1 to bind hypoxia‐responsive elements located within the enhancer/promoter of HIF target genes. The overexpression of HIF‐3α4 suppresses the transcriptional activity of HIF‐1 and siRNAmediated knockdown of the endogenous HIF‐3α4 increases transcription by hypoxia‐inducible genes. HIF‐3α4 itself is oxygen‐regulated, suggesting a novel feedback mechanism of controlling HIF‐1 activity. Furthermore, the expression of HIF‐3α4 is dramatically down‐regulated in the majority of primary renal carcinomas. These results demonstrate an important dominant‐negative regulation of HIF‐1‐mediated gene transcription by HIF‐3α4 in vivo and underscore its potential significance in renal epithelial oncogenesis. Maynard, M. A., Evans, A. J., Hosomi, T., Hara, S., Jewett, M. A. S., Ohh, M. Human HIF‐3α4 is a dominant‐negative regulator of HIF‐1 and is down‐regulated in renal cell carcinoma. FASEB J. 19, 1396–1406 (2005)

Regulatory role of metallothionein in NF-κB activation

Metallothionein (MT), a low molecular weight, cysteine‐rich metal binding protein, has been associated with cytoprotection from heavy metals and cellular oxidants. As MT has the ability to scavenge hydroxyl radicals, MT may control intracellular redox status. In the present study, we examined whether MT regulates the activity of nuclear factor‐κB (NF‐κB), which is one of the redox‐regulated transcription factors, using the MT null embryonic cell lines established from MT null mice. We first found that tumor necrosis factor (TNF)‐induced activation of the binding of NF‐κB protein to DNA in wild type MT+/+ cells was lower than that in MT−/− cells. The NF‐κB activation in MT‐expressing cells established from MT−/− cells by the transfection of mouse MT‐I gene was also significantly lower than that in MT−/− cells. In addition, transfection of the MT gene inhibited TNF‐induced IκB degradation and suppressed NF‐κB‐dependent gene expression induced by TNF. These results demonstrate that MT may function as a negative regulator of NF‐κB activity.

Modulation of telomerase activity by zinc in human prostatic and renal cancer cells

Because the up-regulation of telomerase in most cancer tissues is considered to be responsible for the unlimited proliferation of cancer cells, suppression of telomerase activity is an attractive potential target for cancer therapy. The mechanism for the activation of telomerase in cancer cells, however, is still unclear. In the present study, we demonstrated that Zn induces an enhancement of telomerase activity in the human renal cell carcinoma (NRC-12) and prostatic cancer (DU145) cell lines. The maximum elevation of the activity was observed 6 hr after treatment with 100 microM Zn; it was diminished by the addition of either metal chelator or cycloheximide. Other metals such as Cd and Cu also enhanced telomerase activity but to a lesser extent, and no correlation between the activation of telomerase and the induction of metallothionein was observed. Our findings provide the first evidence that metals, especially Zn, can modulate telomerase activity in cancer cells.

Overexpression of thioredoxin reductase 1 regulates NF-κB activation

Thioredoxin reductase (TrxR) is a flavoprotein that contains a C‐terminal penultimate selenocysteine (Sec) and has an ability to reduce thioredoxin (Trx), which regulates the activity of NF‐κB. To date, three TrxR isozymes, TrxR1, TrxR2, and TrxR3, have been identified. In the present study, we found that among these isozymes only TrxR1 was induced by tumor necrosis factor‐α (TNFα) in vascular endothelial cells. Furthermore, the overexpression of TrxR1 enhanced TNFα‐induced DNA‐binding activity of NF‐κB and NF‐κB‐dependent gene expression. The catalytic Sec residue of TrxR1, which is essential for reducing Trx, was required for this NF‐κB activation, and aurothiomalate, an inhibitor of TrxR, suppressed TNFα‐induced activation of NF‐κB and the expression of NF‐κB‐targeted proinflammatory genes such as E‐selectin and cyclooxygenase‐2. These results suggest that TrxR1 may act as a positive regulator of NF‐κB and may play an important role in the cellular inflammatory response.

Abundant expression of thromboxane synthase in rat macrophages

The cloned cDNA for rat thromboxane (TX) synthase with a size of 1851 bp contained a 1599‐bp open reading frame which encoded a 533‐amino acid protein sharing 79.7% identity with human TX synthase. RNA blot analysis was carried out with rat cells and tissues. Rat peritoneal macrophages most abundantly expressed mRNA for TX synthase, and its level was not changed by in vivo stimulation of casein. Bone marrow, spleen, lung and thymus also expressed the TX synthase gene. These findings suggest the possibility that TXA2 plays a role in the immune system.

Cyclooxygenase-2 expression and relationship to tumour progression in human renal cell carcinoma

Aims:u2002 Cyclooxygenase (COX), which catalyses the synthesis of prostaglandins from arachidonic acid, has two isoforms; COX‐1 and COX‐2. There is ample evidence to suggest an important role for COX‐2 in cancer. The aim of this study was to evaluate the clinical significance of COX‐2 expression and its localization in the development and progression of human renal cell carcinoma (RCC).

Molecular cloning of cDNAs encoding hypoxia-inducible factor (HIF)-1α and -2α of bovine arterial endothelial cells

Abstract Hypoxia-inducible factor (HIF)-1α and -2α are two basic helix-loop-helix/PAS domain transcriptional factors that mediate hypoxia-induced gene expression. We found that bovine arterial endothelial cells (BAEC) expressed both HIF-1α and -2α by RT-PCR and then isolated cDNAs encoding these two transcriptional factors of BAEC. The deduced amino acid sequences of both HIF-1α and -2α showed high homologies among mammalian species. Northern blot analysis indicated that the mRNAs for HIF-1α and -2α from BAEC showed a size of approx. 5.5 and 6.2 kilobases, respectively and that both mRNAs were constitutively expressed and not induced by hypoxia in BAEC.

Expression Of Cycloxygenase-2 In Human Bladder And Renal Cell Carcinoma

In summary, we have showed that levels of COX-2 are increased in both TCC and RCC derived from urinary tract epithelium as well as gastrointestinal cancer. These results raised the possibility that selective inhibitors of COX-2 may be useful in the prevention or treatment of these diseases.

Selective disappearance of an axonal protein, 440‐kDa ankyrinB, associated with neuronal degeneration induced by methylmercury

The 440‐kDa isoform of brain ankyrin, 440‐kDa ankyrinB, is a neuron‐specific protein and is confined to axons. Cerebellum is one of the areas characteristically altered by methylmercury intoxication both in the adult and during development. When rat cerebellar neurons matured for 7 days in vitro were exposed to methylmercury at 0.03 μM for 48 hr, viability of the cells was unaffected. However, the immunocytochemical staining of 440‐kDa ankyrinB diminished drastically, whereas that of microtubule‐associated protein‐2, which is localized in dendrites and cell bodies, and of glial fibrillary acidic protein (GFAP), a marker for astroglial cells coexisting in the culture, remained unchanged. To confirm these observations, a simplified dot blot assay was established to determine 440‐kDa ankyrinB and several other marker proteins in cultured cell samples. With this assay, we found that methylmercury at a submicromolar range induced a decrease of 440‐kDa ankyrinB and an increase of GFAP in a dose‐dependent manner in cerebellar cells in primary culture. Surprisingly, another axonal protein, tau, remained mostly in its intact molecular sizes even in the presence of 0.3–1.0 μM methylmercury, though its immunocytochemical localization was substantially altered. These results indicate that selective loss of the axonal protein 440‐kDa ankyrinB is associated with the early stage of degeneration of cerebellar neurons induced by methylmercury. Therefore, 440‐kDa ankyrinB should be useful as a specific and sensitive marker for the neurotoxicity of methylmercury.