Mitochondria are the major organelles that produce reactive oxygen species (ROS) and the main target of ROS-induced damage as observed in various pathological states including aging. Production of NADPH required for the regeneration of glutathione in the mitochondria is critical for scavenging mitochondrial ROS through glutathione reductase and peroxidase systems. We investigated the role of mitochondrial NADP ؉ -dependent isocitrate dehydrogenase (IDPm) in controlling the mitochondrial redox balance and subsequent cellular defense against oxidative damage. We demonstrate in this report that IDPm is induced by ROS and that decreased expression of IDPm markedly elevates the ROS generation, DNA fragmentation, lipid peroxidation, and concurrent mitochondrial damage with a significant reduction in ATP level. Conversely, overproduction of IDPm protein efficiently protected the cells from ROS-induced damage. The protective role of IDPm against oxidative damage may be attributed to increased levels of a reducing equivalent, NADPH, needed for regeneration of glutathione in the mitochondria. Our results strongly indicate that IDPm is a major NADPH producer in the mitochondria and thus plays a key role in cellular defense against oxidative stress-induced damage.Cell damage induced by oxidative stress and reactive oxygen species (ROS) 1 has been implicated in several human diseases including aging, alcohol-mediated organ damage, neurodegenerative diseases, many types of cancers, cardiovascular diseases, and UV-mediated skin disorders (1). As one of the major sources of ROS (2), mitochondria are highly susceptible to oxidative damage. ROS can damage mitochondrial enzymes directly (3), and they can cause mutation in mitochondrial DNAs (4). At the same time, ROS can change the mitochondrial transmembrane potential (⌬m), which is indicative of mitochondrial membrane integrity (5) and precedes cell death induced by various toxic compounds and cytokines (6). Recent reports indicate that mitochondrial ROS cause apoptosis (7, 8) by activating various apoptotic effectors such as cytochrome c release, procaspase-2, procaspase-9, procaspase-3, and latent apoptosis-inducing factor, which is released from the mitochondria during apoptosis (9 -11). Another report also suggested that mitochondrial ROS directly caused apoptosis of T cells (12). It was also reported that tumor necrosis factor ␣ causes a rapid production of mitochondrial ROS (13) and that ceramide, an apoptotic stimulus, also plays a crucial role in tumor necrosis factor ␣-induced mitochondrial ROS generation (14). Furthermore, several other investigators demonstrated that ROS are involved in the signaling pathway of certain growth factors (15) and cytokines (16). In addition, mitochondrial ROS, under hypoxic conditions, activate the transcription of the genes for glycolytic enzymes as well as erythropoietin and vascular endothelial growth factor by upregulating a transcriptional factor, hypoxia-inducible factor 1 (17), suggesting that mitochondrial ROS mediate cross-talk b...

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The differentiated phenotype of chondrocyte is rapidly lost during in vitro culture by a process designated "dedifferentiation." In this study, we investigate the roles of protein kinase C (PKC) and extracellular signalregulated protein kinase (ERK) in the maintenance of the differentiated chondrocyte phenotype. Chondrocytes isolated from rabbit articular cartilage underwent dedifferentiation upon serial monolayer culture with cessation of type II collagen expression and proteoglycan synthesis, which was reversed by culturing dedifferentiated cells in alginate gel. The expression pattern of PKC␣ was essentially the same as that of type II collagen during de-and redifferentiation, in that expression was decreased during dedifferentiation and increased during redifferentiation. In contrast to PKC␣, ERK activity increased 15-fold during dedifferentiation. This enhanced activity was terminated during redifferentiation. Down-regulation of PKC␣ in passage 0 chondrocytes resulted in dedifferentiation. However, overexpression of PKC␣ did not affect type II collagen levels, suggesting that PKC␣ expression is not sufficient to maintain the differentiated phenotype. However, inhibition of ERK by PD98059 enhanced type II collagen expression and proteoglycan synthesis in passage 0 cells, retarded dedifferentiation during monolayer cultures, and reversed dedifferentiation caused by downregulation of PKC. Unlike PKC-dependent ERK regulation of chondrogenesis, PKC and ERK independently modulated chondrocyte dedifferentiation, as confirmed by observations that PKC down-regulation and ERK inhibition did not alter ERK phosphorylation and PKC expression, respectively. In addition, expression of Ncadherin, ␣-catenin, and ␤-catenin, which are oppositely regulated to type II collagen during phenotype alterations, were modulated by PKC and ERK during chondrogenesis but not dedifferentiation, supporting distinct mechanisms for the regulation of chondrocyte differentiation and maintenance of differentiated phenotype by these two protein kinases.

NADPH is an essential cofactor for many enzymatic reactions including glutathione metabolism and fat and cholesterol biosynthesis. We have reported recently an important role for mitochondrial NADP ؉ -dependent isocitrate dehydrogenase in cellular defense against oxidative damage by providing NADPH needed for the regeneration of reduced glutathione. However, the role of cytosolic NADP ؉ -dependent isocitrate dehydrogenase (IDPc) is still unclear. We report here for the first time that IDPc plays a critical role in fat and cholesterol biosynthesis. During differentiation of 3T3-L1 adipocytes, both IDPc enzyme activity and its protein content were increased in parallel in a time-dependent manner. Increased expression of IDPc by stable transfection of IDPc cDNA positively correlated with adipogenesis of 3T3-L1 cells, whereas decreased IDPc expression by an antisense IDPc vector retarded adipogenesis. Furthermore, transgenic mice with overexpressed IDPc exhibited fatty liver, hyperlipidemia, and obesity. In the epididymal fat pads of the transgenic mice, the expressions of adipocyte-specific genes including peroxisome proliferator-activated receptor ␥ were markedly elevated. The hepatic and epididymal fat pad contents of acetylCoA and malonyl-CoA in the transgenic mice were significantly lower, whereas the total triglyceride and cholesterol contents were markedly higher in the liver and serum of transgenic mice compared with those measured in wild type mice, suggesting that the consumption rate of those lipogenic precursors needed for fat biosynthesis must be increased by elevated IDPc activity. Taken together, our findings strongly indicate that IDPc would be a major NADPH producer required for fat and cholesterol synthesis.Abnormal lipid metabolism is frequently associated with obesity and hyperlipidemia. In fat and cholesterol biosynthesis, NADPH is an essential cofactor for numerous enzymes. For instance, 3-L-hydroxylacyl-coenzyme A dehydrogenase and enoyl-coenzyme A reductase in fatty acid synthesis and hydroxymethylglutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol biosynthesis, require NADPH for their enzyme activities. It has been demonstrated that glucose-6-phosphate dehydrogenase (G6PDH), 1 6-phosphogluconate dehydrogenase, and malic enzyme are considered as the major enzymes producing cytosolic NADPH (1). Nevertheless, the activities of these enzymes were markedly lower than that of cytosolic NADP ϩ -dependent isocitrate dehydrogenase (IDPc) in the rat liver (1, 2). Consistent with this observation, McLean and co-workers (3) reported that certain adaptive changes in the pentose phosphate pathway dehydrogenases did not take place in parallel with fat synthesis in adipose tissue and suggested that a major source of NADPH for fat synthesis could be IDPc. It is worthy of note that IDPc is expressed mainly in lipogenic tissues such as liver and adipocytes, whereas G6PDH and 6-phosphogluconate dehydrogenase are expressed ubiquitously (4, 5). These data indicate that NADPH-producing IDPc may ...

Notch activation inhibits neuronal differentiation during development of the nervous system; however, the dynamic role of Notch signaling in individual cell lineages remains poorly understood. We have characterized 3.4 kb 5'-regulatory sequence of a Notch target gene, her4, and used it to drive fluorescent gene expression in transgenic lines where the spatiotemporal pattern of Notch activation can be examined in vivo. The 3.4 kb her4 promoter contains five predicted Su(H) binding sites of which two proximal sites were confirmed to be required for Notch-mediated transcriptional activation. Without Notch, Su(H) effectively represses transcription regulated by the promoter. Analyses of transgenic zebrafish showed that while the expression of proneural genes and Notch activation were both critical for endogenous her4 expression, reporter gene expression was primarily regulated by Notch activity. This study also showed that her4 may be differently regulated in sensory cranial ganglia, where Notch activity is not essential for her4 expression and where Su(H) may repress her4 expression. The establishment of a reporter line with Notch-Su(H)-dependent fluorescent gene expression provides a tool to explore the complex role of Notch signaling in the development of vertebrate nervous system.