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 ...

Basic studies of oncogenesis have demonstrated that either the elevated production of particular oncogene proteins or the occurrence of qualitative abnormalities in oncogenes can contribute to neoplastic cellular transformation. The purpose of this study was to identify unique oncogenes that are differentially expressed in human cancers and characterize their functions in tumorigenesis. To discover new putative oncogenes, the differential display RT-PCR method was applied using normal cervical tissues, cervical cancer cell lines, cervical cancer tissues, and metastatic tissues. We identified a new human cervical cancer oncogene HCCR-2 that was overexpressed in various human tumors including leukemia, lymphoma, and carcinomas of the breast, kidney, ovary, stomach, colon, and uterine cervix. Ectopic expression of HCCR-2 resulted in direct tumorigenic conversions of NIH/3T3 and Rat1 fibroblasts. Nude mice injected with NIH/3T3 cells stably transfected with HCCR-2 formed tumors in 4 weeks. The resultant tumors display characteristics of an epithelial carcinoma. In HCCR-2 transfected NCI-H460 cells and RKO cells, stabilization of the p53 tumor suppressor occurred without genetic mutation and correlated with functional impairment, as indicated by the defective induction of p53-induced p21 WAF1 , MDM2, and bax. These results indicate that HCCR-2 probably represents a new oncogene that is related to tumorigenesis, functioning as a negative regulator of the p53 tumor suppressor.

To identify novel molecules regulating chondrogenesis and cartilage development, we screened a cartilage-specific expressed sequence tag data base. Cytokine-like 1 (Cytl1), a possible cytokine candidate with unknown function that was originally identified in bone marrow-derived CD34-positive cells, was selected for functional characterization. In view of the initial observation that Cytl1 is predominantly expressed in chondrocytes and cartilage, we investigated its possible role in chondrogenesis and hypertrophic maturation of chondrocytes. Cytl1 expression was very low in mesenchymal cells, dramatically increased during chondrogenesis, and decreased during hypertrophic maturation, both in vivo and in vitro. The role of Cytl1 in chondrogenesis and hypertrophic maturation was examined by treating chondrifying mesenchymal cells with exogenous Cytl1 or ectopic expression of Cytl1. Notably, exogenous Cytl1 caused chondrogenic differentiation of mouse limb bud mesenchymal cells during micromass culture. Lentivirus-mediated overexpression of Cytl1 additionally induced chondrogenic differentiation of mesenchymal cells. However, Cytl1 did not affect the hypertrophic maturation of chondrocytes. Cytl1 exerted its chondrogenic effect via stimulation of Sox9 transcriptional activity. In addition, Cytl1 caused expression of insulin-like growth factor 1, which has a capacity to induce chondrogenesis. Thus, our results collectively suggest that chondrocyte-specific Cytl1 regulates chondrogenesis as a novel autocrine factor, but not hypertrophic maturation of chondrocytes during cartilage development.Cartilage formation during embryonic development begins with the aggregation of mesenchymal cells, which ultimately differentiate into chondrocytes. Differentiated chondrocytes proliferate rapidly and secrete a cartilage-specific extracellular matrix such as type II collagen and sulfated proteoglycan to form cartilage. The cartilage serves as a template for endochondral ossification, which requires the maturation of hypertrophic chondrocytes (1-4). These sequential events during chondrogenesis and cartilage formation are precisely regulated by various growth factors released from cartilage elements and perichondrium. Secreted growth factors exert their effects by modulating intracellular signaling (1, 2). Although several regulatory growth factors have been identified, including bone morphogenetic proteins, fibroblast growth factors, insulin-like growth factor-1 (IGF-1), 2 transforming growth factor-␤, and parathyroid hormone-related peptide, the precise mechanisms of regulation of chondrogenesis and cartilage development remain to be elucidated. In this study, we analyze a cartilagespecific expressed sequence tag (EST) data base in an attempt to identify novel molecules that modulate chondrogenesis and cartilage development.The EST data base provides important information on novel genes displaying tissue-specific expression profiles (5-7). We analyzed the human cartilage UniGene library (8), and selected Cytl1 as a possible candid...

Phospholipase D (PLD) has been suggested to play an important role in a variety of cellular functions. PLD activity has been shown to be significantly elevated in many tumours and transformed cells, suggesting the possibility that PLD might be involved in tumorigenesis. In this study, we have established stable cell lines overexpressing PLD1 and PLD2 from fibroblast cells. These cells, but not control cells, showed altered growth properties and anchorage-independent growth in soft agar. Both PLD1 and PLD2 also induced an up-regulation of the activity of matrix metalloprotease-9 as detected by zymograms. Furthermore, both PLD1 and PLD2 transformants, but not vector-transfectants, induced undifferentiated sarcoma when transplanted into nude mice. Both PLD1- and PLD2-mediated cell cycle distributions in stable cell lines revealed an increased fraction of cells in the S phase compared with control cells. Interestingly, the level of cyclin D3 protein, known as an activator of G(1) to S phase transition in the cell cycle, was aberrantly high in cells overexpressing PLD1 and PLD2 compared with control cells. These results suggest that overexpression of PLD isozymes may play an important role in neoplastic transformation.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. Despite progress in developing chemotherapeutics for the treatment of NSCLC, primary and secondary resistance limits therapeutic success. NSCLC cells exhibit multiple mutations in the epidermal growth factor receptor (EGFR), which cause aberrant activation of diverse cell signaling pathways. Therefore, suppression of the inappropriate amplification of EGFR downstream signaling cascades is considered to be a rational therapeutic and preventive strategy for the management of NSCLC. Our initial molecular target-oriented virtual screening revealed that the ginger components, including [6]-shogaol, [6]-paradol and [6]-gingerol, seem to be potential candidates for the prevention and treatment of NSCLC. Among the compounds, [6]-shogaol showed the greatest inhibitory effects on the NSCLC cell proliferation and anchorage-independent growth. [6]-Shogaol induced cell cycle arrest (G1 or G2/M) and apoptosis. Furthermore, [6]-shogaol inhibited Akt kinase activity, a downstream mediator of EGFR signaling, by binding with an allosteric site of Akt. In NCI-H1650 lung cancer cells, [6]-shogaol reduced the constitutive phosphorylation of signal transducer and activator of transcription-3 (STAT3) and decreased the expression of cyclin D1/3, which are target proteins in the Akt signaling pathway. The induction of apoptosis in NCI-H1650 cells by [6]-shogaol corresponded with the cleavage of caspase-3 and caspase-7. Moreover, intraperitoneal administration of [6]-shogaol inhibited the growth of NCI-H1650 cells as tumor xenografts in nude mice. [6]-Shogaol suppressed the expression of Ki-67, cyclin D1 and phosphorylated Akt and STAT3 and increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positivity in xenograft tumors. The current study clearly indicates that [6]-shogaol can be exploited for the prevention and/or treatment of NSCLC.

Atopic dermatitis (AD) is a chronically relapsing, non contagious pruritic skin disease with two phases: acute and chronic. Cysteine protease cathepsin S (CTSS) is involved in inflammatory processes, possibly leading to atherosclerosis and asthma. Recently, it has been reported that CTSS can arouse a predominant sensation of itch accompanied by classical ligand–receptor signaling [corrected]. Recently, CTSS was shown to be a ligand for proteinase-activated receptor-2 (PAR-2), which is associated with itching. In this study, we show that CTSS-overexpressing transgenic (TG) mice spontaneously develop a skin disorder similar to chronic AD. The results of this study suggest that CTSS overexpression triggers PAR-2 expression in dendritic cells (DCs), resulting in the promotion of CD4(+) differentiation, which is involved in major histocompatibility complex (MHC) class II expression. In addition, we investigated mast cells and macrophages and found significantly higher mean levels of T helper type 1 (Th1) cell-associated cytokines than T helper type 2 (Th2) cell-associated cytokines in CTSS-overexpressing TG mice. These results suggest that increased PAR-2 expression in DCs as a result of CTSS overexpression induces scratching behavior and Th1 cell-associated cytokine expression, and can trigger chronic AD symptoms.

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House dust mites have been implicated in the etiology and exacerbation of atopic dermatitis. Diverse factors contribute to house dust mite allergenicity through the activation of innate immunity. We investigated whether Dermatophagoides farinae extract (DFE) allergens mediate innate immune activation through specific toll-like receptors (TLRs) in epidermal keratinocytes, a DFE-induced murine atopic dermatitis model, and human atopic dermatitis lesions. DFE activated the expression of TLR1, TLR6, IL-25, and IL-33 in human primary keratinocytes and HaCaT cells. Knockdown of TLR6 inhibited DFE-induced upregulation of IL-25 or IL-33. In addition, the suppression of TLR1 inhibited the release of IL-33. DFE induced the expression of IL-25 and IL-33 by upregulation of IL-1 receptor-associated kinase 1, transforming growth factor-β activated kinase-1, IκB kinase, and NF-κB pathways. Tlr6 mice did not show DFE-induced upregulation of IL-25 and IL-33. Furthermore, DFE-induced upregulation of IL-25 was not induced in Tlr1 mice. We also identified upregulated mRNA and protein expression of TLR1, TLR6, IL-25, and IL-33 in human atopic dermatitis skin lesions with high house dust mite sensitization. We found that DFE-induced activation of TLR1 and TLR6 may cause polarization toward a T helper type 2 immune response via the release of IL-25 and IL-33.