👤 Toshiyuki Nakagawa

The carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in converting excess carbohydrate to storage fat in liver. In response to changing glucose levels, ChREBP activity is regulated by nucleo-cytoplasmic shuttling of ChREBP via interactions with 14-3-3 proteins and importins. The nuclear/cytosol trafficking is regulated partly by phosphorylation/dephosphorylation of serine 196 mediated by cAMP-dependent protein kinase and protein phosphatase. We show here that protein-free extracts of starved and high fat-fed livers contain metabolites that activate interaction of ChREBP·14-3-3 and inhibit the ChREBP/importin α interaction, resulting in cytosolic localization. These metabolites were identified as β-hydroxybutyrate and acetoacetate. Nuclear localization of GFP-ChREBP is rapidly inhibited in hepatocytes incubated in β-hydroxybutyrate or fatty acids, and the observed inhibition is closely correlated with the production of ketone bodies. These observations show that ketone bodies play an important role in the regulation of ChREBP activity by restricting ChREBP localization to the cytoplasm, thus inhibiting fat synthesis during periods of ketosis. Show less

The mass spectrometry-based peptidomics approaches have proven its usefulness in several areas such as the discovery of physiologically active peptides or biomarker candidates derived from various biological fluids including blood and cerebrospinal fluid. However, to identify biomarkers that are reproducible and clinically applicable, development of a novel technology, which enables rapid, sensitive, and quantitative analysis using hundreds of clinical specimens, has been eagerly awaited. Here we report an integrative peptidomic approach for identification of lung cancer-specific serum peptide biomarkers. It is based on the one-step effective enrichment of peptidome fractions (molecular weight of 1,000-5,000) with size exclusion chromatography in combination with the precise label-free quantification analysis of nano-LC/MS/MS data set using Expressionist proteome server platform. We applied this method to 92 serum samples well-managed with our SOP (standard operating procedure) (30 healthy controls and 62 lung adenocarcinoma patients), and quantitatively assessed the detected 3,537 peptide signals. Among them, 118 peptides showed significantly altered serum levels between the control and lung cancer groups (p<0.01 and fold change >5.0). Subsequently we identified peptide sequences by MS/MS analysis and further assessed the reproducibility of Expressionist-based quantification results and their diagnostic powers by MRM-based relative-quantification analysis for 96 independently prepared serum samples and found that APOA4 273-283, FIBA 5-16, and LBN 306-313 should be clinically useful biomarkers for both early detection and tumor staging of lung cancer. Our peptidome profiling technology can provide simple, high-throughput, and reliable quantification of a large number of clinical samples, which is applicable for diverse peptidome-targeting biomarker discoveries using any types of biological specimens. Show less

Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. Circulating blood glucose levels affect ChREBP activity in hepatocytes largely by post-translational mechanisms that include phosphorylation-dependent subcellular localization. Previously, we showed that ChREBP is retained in the cytosol by phosphorylation-dependent binding to 14-3-3 protein dimers and identified the α2 helix (residues 125-135) phospho-Ser(140) domain as the primary 14-3-3 binding site (Sakiyama, H., Wynn, R. M., Lee, W. R., Fukasawa, M., Mizuguchi, H., Gardner, K. H., Repa, J. J., and Uyeda, K. (2008) J. Biol. Chem. 283, 24899-24908). To enter the nucleus in response to high glucose, ChREBP must bind importin-α; this heterodimer then forms a complex with importin-β to interact with the nuclear pore complex. In this work, we recharacterized the importin-α binding nuclear localization signal (NLS) of rat ChREBP, identifying it as an extended classical bipartite NLS encompassing minimally residues 158-190. Replacing Lys(159)/Lys(190) residues of ChREBP with alanine resulted in loss of importin-α binding, glucose-stimulated transcriptional activity and nuclear localization. A secondary 14-3-3 protein binding site also was identified, the α3 helix (residues 170-190) phospho-Ser(196) domain. Importin-α and 14-3-3 were found to bind competitively to this secondary site. These results suggest an important mechanism by which importin-α and 14-3-3 control movement of ChREBP in and out of the nucleus in response to changes in glucose levels in liver and thus further suggest that the extended NLS of ChREBP is a critical glucose-sensing, glucose-responsive site. Show less

The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31. Show less

Mammalian sirtuins have diverse roles in aging, metabolism and disease. Recently we reported a new function for SIRT5 in urea cycle regulation. Our study uncovered that SIRT5 localized to mitochondria matrix and deacetylates carbamoyl phosphate synthetase 1 (CPS1), an enzyme which is the first and rate-limiting step of urea cycle. Deacetylation of CPS1 by SIRT5 resulted in activation of CPS1 enzymatic activity. Indeed, SIRT5-deficient mice failed to up-regulate CPS1 activity and showed hyper ammonemia during fasting. Similar effects are also observed on high protein diet or calorie restriction. These data indicate SIRT5 also has an emerging role in the metabolic adaptation to fasting, high protein diet and calorie restriction. Show less

Sirtuins are NAD-dependent protein deacetylases that connect metabolism and aging. In mammals, there are seven sirtuins (SIRT1-7), three of which are associated with mitochondria. Here, we show that SIRT5 localizes in the mitochondrial matrix and interacts with carbamoyl phosphate synthetase 1 (CPS1), an enzyme, catalyzing the initial step of the urea cycle for ammonia detoxification and disposal. SIRT5 deacetylates CPS1 and upregulates its activity. During fasting, NAD in liver mitochondria increases, thereby triggering SIRT5 deacetylation of CPS1 and adaptation to the increase in amino acid catabolism. Indeed, SIRT5 KO mice fail to upregulate CPS1 activity and show elevated blood ammonia during fasting. Similar effects occur during long-term calorie restriction or a high protein diet. These findings demonstrate SIRT5 plays a pivotal role in ammonia detoxification and disposal by activating CPS1. Show less

Human ATP-binding cassette (ABC) transporter ABCC2 (cMOAT/MRP2) plays a crucial role in the hepatobiliary transport of sulfate-, glucuronide-, and glutathione-conjugated metabolites as well as a variety of amphiphilic organic anions derived from hepatic metabolism. Molecular mechanisms underlying the induction of this hepatic ABC transporter are of great interest to understand the transport-metabolism interplay in vivo. In the present study, to gain insight into the mechanism of ABCC2 induction, we tested a total of 46 structurally diverse compounds, including nuclear receptor ligands, antibiotics, bile salts, phytochemicals, and anticancer drugs. Among them, we found that LXRalpha ligands, i.e., T0901317, paxilline, and 22(R)-hydroxycholesterol, acted potently to induce the expression of ABCC2 at both mRNA and protein levels in human hepatocellular carcinoma HepG2 cells. The ABCC2 induction by T0901317 was dose- and time-dependent, where the induction pattern of ABCC2 was very similar to that of ABCG1, one of the target genes of LXRalpha. The ABCC2 induction by T0901317 was more strongly elicited when the LXRalpha gene was transiently transfected into HepG2 cells. In contrast, ABCC2 induction by T0901317 was attenuated by transient transfection of a dominant negative LXRalpha variant, suggesting that LXRalpha is involved in ABCC2 induction. Interestingly, RXR, a heterodimer partner of LXRalpha, affected the mRNA levels of ABCC2 and ABCG1 differently. ABCC2 induction by T0901317 was enhanced by RXR siRNA treatment, whereas ABCG1 induction was suppressed by the same treatment. This is the first report demonstrating that LXRalpha is potentially involved in ABCC2 induction. Show less

The basic helix-loop-helix transcriptional repressor Hairy-related transcription factor 2 (Hrt2) is expressed in ventricular, but not atrial, cardiomyocytes, and in endothelial and vascular smooth muscle cells. Mice homozygous for a null mutation of Hrt2 die perinatally from a spectrum of cardiac abnormalities, raising questions about the specific functions of this transcriptional regulator in individual cardiac cell lineages. Using a conditional Hrt2 null allele, we show that cardiomyocyte-specific deletion of Hrt2 in mice results in ectopic activation of atrial genes in ventricular myocardium with an associated impairment of cardiac contractility and a unique distortion in morphology of the right ventricular chamber. Consistent with the atrialization of ventricular gene expression in Hrt2 mutant mice, forced expression of Hrt2 in atrial cardiomyocytes is sufficient to repress atrial cardiac genes. These findings reveal a ventricular myocardial cell-autonomous function for Hrt2 in the suppression of atrial cell identity and the maintenance of postnatal cardiac function. Show less

The novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. The role of RGPR-p117 in cell function has not been fully clarified. This study was undertaken to determine whether overexpression of RGPR-p117 regulates various types of signaling factor-induced apoptotic cell death in the cloned normal rat kidney proximal tubular epithelial NRK52E cells. NRK52E cells (wild-type) or stable RGPR-p117/phCMV2-transfected cells (transfectant) were cultured in Dulbecco's modified Eagle's medium containing 5% bovine serum (BS). NRK52E cells with subconfluent monolayers were cultured for 24-72 h in a medium without BS. The presence of tumor necrosis factor-alpha (TNF-alpha; 1.0 or 10 ng/ml of medium), lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml), Bay K 8644 (10(-6) or 10(-5) M), or thapsigargin (10(-8) or 10(-7) M) caused a significant decrease in the number of NRK52E wild-type cells or phCMV2-transfected (mock-type) cells. The effect of TNF-alpha, LPS, Bay K 8644, or thapsigargin in decreasing cell number was significantly suppressed in the presence of the caspase-3 inhibitor (10(-8) M) in wild-type cells cultured for 48 h. The effect of TNF-alpha, LPS, or Bay K 8644 in decreasing cell number was significantly inhibited in the transfectants, while the effect of thapsigargin on cell death was not inhibited in the transfectants. Culture with TNF-alpha or LPS caused DNA fragmentation in wild-type cells. These effects were significantly suppressed in the transfectants. The result of reverse transcription-polymerase chain reaction analysis using specific primers for the genes of apoptotic cell death-related proteins showed that IAP-1, FADD, caspase-8, caspase-9, and caspase-3 mRNA levels were significantly decreased in the transfectants, while Akt-1, Bid, Apaf-1, and glyceroaldehyde-3-phosphate dehydrogenase mRNA levels were not significantly altered in the transfectants. Culture with TNF-alpha, LPS, Bay K 8644, or thapsigargin caused a significant increase in Apaf-1 or caspase-3 mRNA levels. Such an effect was not seen in the transfectants. This study demonstrates that overexpression of RGPR-p117 has a suppressive effect on cell death and apoptosis induced by TNF-alpha, LPS, or Bay K 8644 whose actions are mediated through intracellular signaling pathways. This study also demonstrates that RGPR-p117 regulates the gene expression of apoptosis-related proteins. Show less

Embryonic stem (ES) cells are highlighted as promising cell sources for regenerative medicine. Here, we focused on providing the platform that forced ES cells to reproduce the vascular organization process, leading to efficiency and safety evaluation as preclinical testing of biological agents. Murine ES cell-derived embryoid bodies on matrigel, but not collagen or gelatin, could be differentiated into sprouting blood vessels without the addition of growth factors. The expression of endothelial cell marker CD31 and smooth muscle marker alpha-smooth muscle actin was partially colocalized and started to increase 7 days after culture on matrigel, accompanied by the induction of a number of growth factors, such as vascular endothelial growth factor, fibroblast growth factor-2, hepatocyte growth factor, transforming growth factor-beta, and angiopoietin-1. Moreover, notch-related genes, such as Del1 or Del4 (delta-like 1/4) and hey1 or hey2 (hairy/enhancer of split related TRPW motif 1/2), were upregulated in a similar time course. The treatment of neutralizing antibodies against these growth factors failed to inhibit the differentiation into the sprouting blood vessels, whereas arginine-glycine-aspartic peptide, a selective inhibitor for the alphavbeta3-integrins, did inhibit differentiation. An anticancer drug to inhibit angiogenesis, TNP-470, also blocked the vascular formation in this model. ES cells could reproduce the vascular organization process on the biosynthetic scaffolds, such as matrigel, without the addition of growth factors. In the future, a human ES-based tissue model would be an optional tool for the screening of pharmaceutical drugs for vascular disease. Show less

gp130-dependent signaling is known to play a critical role in the onset of heart failure. In that regard, cardiotrophin-1 (CT-1) activates several signaling pathways via gp130, and induces hypertrophy in neonatal rat cardiomyocytes. Among the mediators activated by CT-1, STAT3 is thought to be important for induction of cell hypertrophy, though its precise function in the CT-1 signaling pathway is not fully understood. In the present study, therefore, to better understand the significance of STAT3 activity in CT-1 signaling, we infected cultured cardiomyocytes with adenoviral vectors harboring a dominant-negative STAT3 mutant or one of two endogenous negative regulators of cytokine signaling via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways [suppressor of cytokine signaling (SOCS) 1 and 3] and then examined their effects on three indexes of CT-1-induced cell hypertrophy: protein synthesis, secretion of brain natriuretic peptide and changes in cell surface area. In control cells, CT-1-induced both STAT3 phosphorylation and cell hypertrophy. Overexpression of dominant-negative STAT3 mutant suppressed CT-1-induced STAT3 phosphorylation, but did not affect cell hypertrophy. On the other hand overexpression of SOCS1 or SOCS3 inhibited both CT-1-induced STAT3 phosphorylation and cell hypertrophy. CT-1 also induced phosphorylations of ERK1/2 and ERK5 in cardiomyocytes, and those, too, were suppressed by overexpression of SOCSs. CT-1-induced cell hypertrophy was suppressed by overexpression of a dominant-negative MEK5 mutant, and not by overexpression of a dominant-negative MEK1 mutant. These findings indicate that the major pathway responsible for the hypertrophic responses to CT-1 is not JAK-STAT3 pathway nor MEK1-ERK1/2 pathway, but MEK5-ERK5 pathway. Show less

A novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif. The role of RGPR-p117 in cell function is unknown. The nuclear localization of RGPR-p117 was investigated using cloned normal rat kidney proximal tubular epithelial NRK52E cells in vitro. RGPR-p117 mRNA was expressed in NRK52E cells, and its expression was stimulated by culture with parathyroid hormone (10(-7) M) or phorbol 12-myristate (10(-6) M). RGPR-p117 was found to localize in the cytoplasm and nucleus with immunocytochemical and Western blot analysis using HA-RGPR-p117/phCMV2-transfected NRK52E cells. Overexpression of HA-RGPR-p117 was found to have a significant stimulatory effect on regucalcin mRNA expression in NRK52E cells. This study demonstrates that RGPR-p117 is localized in the nucleus of kidney cells, and may be involved in gene expression. Show less

Combinatorial actions of transcription factors in multiprotein complexes dictate gene expression profiles in cardiac development and disease. The Hairy-related transcription factor (HRT) family of basic helix-loop-helix proteins is composed of transcriptional repressors highly expressed in the cardiovascular system. However, it has remained unclear whether HRT proteins modulate gene expression driven by cardiac transcriptional activators. Here, we have shown that HRT proteins inhibit cardiac gene transcription by interfering with GATA transcription factors that are implicated in cardiac development and hypertrophy. HRT proteins inhibited GATA-dependent transcriptional activation of cardiac gene promoters such as the atrial natriuretic factor (ANF) promoter. Adenovirus-mediated expression of Hrt2 suppressed mRNA expression of ANF and other cardiac-specific genes in cultured cardiomyocytes. Among various signaling molecules implicated in cardiomyocyte growth, constitutively active Akt1/protein kinase B alpha relieved Hrt2-mediated inhibition of GATA-dependent transcription. HRT proteins physically interacted with GATA proteins, and the basic domain of HRT was critical for physical association as well as transcriptional inhibition. These results suggest that HRT proteins may regulate specific sets of cardiac genes by modulating the function of GATA proteins and other cardiac transcriptional activators in a signal-dependent manner. Show less