Takaya Gotoh

Developmentally regulated role for Ras‐GRFs in coupling NMDA glutamate receptors to Ras, Erk and CREB

p140 Ras‐GRF1 and p130 Ras‐GRF2 constitute a family of calcium/calmodulin‐regulated guanine–nucleotide exchange factors that activate the Ras GTPases. Studies on mice lacking these exchange factors revealed that both p140 Ras‐GRF1 and p130 Ras‐GRF2 couple NMDA glutamate receptors (NMDARs) to the activation of the Ras/Erk signaling cascade and to the maintenance of CREB transcription factor activity in cortical neurons of adult mice. Consistent with this function for Ras‐GRFs and the known neuroprotective effect of CREB activity, ischemia‐induced CREB activation is reduced in the brains of adult Ras‐GRF knockout mice and neuronal damage is enhanced. Interestingly, in cortical neurons of neonatal animals NMDARs signal through Sos rather than Ras‐GRF exchange factors, implying that Ras‐GRFs endow NMDARs with functions unique to mature neurons.

Large scale replication analysis of loci associated with lipid concentrations in a Japanese population

Background: Recent genome wide association studies discovered seven novel loci that influence plasma concentrations of triglycerides, high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol in Europeans. To date, large scale replication studies using populations with known differences in genome-wide linkage disequilibrium (LD) pattern have not been undertaken. Methods: To address this issue, we tested associations between single nucleotide polymorphisms (SNPs) within the seven novel loci and plasma lipid profiles in 21 010 Japanese individuals. Results: Multiple linear regression analyses showed that the rs3812316 in MLXIPL was strongly associated with triglyceride concentrations (p∼3.0×10−11, 7.1 mg/dl decrease per minor C allele) and that rs599839 in CELSR2/PSRC1/SORT1 was strongly associated with LDL cholesterol concentrations (p∼3.1×10−11, 4.7 mg/dl decrease per minor G allele) in the Japanese population. SNPs near ANGPTL3, TRIB1 and GALNT2 showed evidence for associations with triglyceride concentrations (3.6×10−6<p<5.1×10−5). SNP near TRIB1 showed association with LDL cholesterol concentrations (p∼1.2×10−5). On the other hand, SNPs in NCAN/CILP2/PBX4 and MVK/MMAB were not associated with any plasma lipid profiles in the Japanese population. Ethnic differences in LD pattern would explain the lack of association between these two loci and plasma lipid concentrations in the Japanese population. Conclusion: Associations between the novel loci and plasma lipid concentrations were generally conserved in the Japanese population, with the exception of NCAN/CILP2/PBX4 and MVK/MMAB.

Regulation of Ras Signaling Specificity by Protein Kinase C

ABSTRACT Ras proteins have the capacity to bind to and activate at least three families of downstream target proteins: Raf kinases, phosphatidylinositol 3 (PI 3)-kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). We have previously shown that the Ras/Ral-GEF and Ras/Raf pathways oppose each other upon nerve growth factor stimulation, with the former promoting proliferation and the latter promoting cell cycle arrest. Moreover, the pathways are not activated equally. While the Ras/Raf/Erk signaling pathway is induced for hours, the Ras/Ral-GEF/Ral signaling pathway is induced for only minutes. Here we show that this preferential down-regulation of Ral signaling is mediated, at least in part, by protein kinase C (PKC). In particular, we show that PKC activation by phorbol ester treatment of cells blocks growth factor-induced Ral activation while it enhances Erk activation. Moreover, suppression of growth factor-induced PKC activation enhances and prolongs Ral activation. PKC does not influence the basal activity of the Ral-GEF designated Ral-GDS but suppresses its activation by Ras. Interestingly, Ras binding to the C-terminal Ras binding domain of Ral-GDS is not affected by PKC activity. Instead, suppression of Ral-GDS activation occurs through the region N terminal to the catalytic domain, which becomes phosphorylated in response to phorbol ester treatment of cells. These findings identify a role for PKC in determining the specificity of Ras signaling by its ability to differentially modulate Ras effector protein activation.

The High-Risk Human Papillomavirus Type 16 E6 Counters the GAP Function of E6TP1 toward Small Rap G Proteins

ABSTRACT We have recently identified E6TP1 (E6-targeted protein 1) as a novel high-risk human papillomavirus type 16 (HPV16) E6-binding protein. Importantly, mutational analysis of E6 revealed a strong correlation between the transforming activity and its abilities to bind and target E6TP1 for ubiquitin-mediated degradation. As a region within E6TP1 has high homology with GAP domains of known and putative Rap GTPase-activating proteins (GAPs), these results raised the possibility that HPV E6 may alter the Rap small-G-protein signaling pathway. Using two different approaches, we now demonstrate that human E6TP1 exhibits GAP activity for Rap1 and Rap2, confirming recent findings that a closely related rat homologue exhibits Rap-specific GAP activity. Using mutational analysis, we localize the GAP activity to residues 240 to 945 of E6TP1. Significantly, we demonstrate that coexpression of HPV16 E6, by promoting the degradation of E6TP1, enhances the GTP loading of Rap. These results support a role of Rap small-G-protein pathway in E6-mediated oncogenesis.

An intronic variable number of tandem repeat polymorphisms of the cold-induced autoinflammatory syndrome 1 ( CIAS1) gene modifies gene expression and is associated with essential hypertension

Cold-induced autoinflammatory syndrome 1 (CIAS1) gene is a member of the NALP subfamily of the CATERPILLER protein family that is expressed predominantly in peripheral blood leukocytes, which is to regulate apoptosis or inflammation through the activation of NF-κB and caspase. Recent genetic analyses suggested an association between inflammation and oxidative stress-related genes in the development of hypertension. This is the first genetic study indicating an association between the CIAS1 gene and susceptibility to essential hypertension (EH). The frequency of subject with the homozygote of 12 repeat allele was significantly higher in patients with hypertension compared with control subjects (987 cases, 924 controls) (P=0.030; odds ratio=1.24) at a novel VNTR polymorphism of CIAS1 intron 4 loci. We also found that the mean of systolic blood pressure of homozygotes of 12 repeat allele was 6.4 mmHg higher than those of homozygotes of non-12 repeat allele in male random population (P=0.009). The frequency of six SNPs spanning of the CIAS1 gene was not significantly between patients and controls. The real-time PCR analysis showed that among healthy young adults, 12-12 subjects expressed CIAS1 mRNA in peripheral leukocytes significantly more abundantly than homozygote of non-12 repeat alleles subjects (P<0.05). Reporter gene assay of the CIAS1-VNTR in HL60 stimulated by lipopolysaccharides showed that the intronic sequence involving 12 repeat increased the expression of luciferase compared with 9, 7, and 6 repeats. Thus, we propose here the CIAS1 is associated with EH through the dominant expression of transcripts, which may depend on the CIAS1-VNTR genotype.

Regulatory SNP in the RBP4 Gene Modified the Expression in Adipocytes and Associated With BMI

Retinol‐binding protein 4 (RBP4) is a recently identified adipokine that was involved in insulin resistance. RBP4 is predominantly expressed from the liver in normal metabolic state to transport retinoids throughout the body, but the exact physiological function and the regulatory mechanisms of adipocyte‐derived RBP4 have not been revealed. We conducted the genetic analysis about metabolic parameters in Japanese and Mongolian; the minor allele carriers of regulatory single‐nucleotide polymorphism (SNP −803G>A) showed significantly higher BMI in Japanese men (P = 0.009) and women (P = 0.017), and in Mongolian women (P = 0.009). Relative quantification of RBP4 transcripts in −803GA heterozygotes showed that the minor allele–linked haplotype‐derived mRNA was significantly more abundant than the transcript from major allele. RBP4 promoter assay in 3T3L1 adipocytes revealed that the minor allele increased the promoter activity double to triple and the administration of 9‐cis‐retinoic acid (RA) and 8‐bromo‐cyclic adenosine monophosphate (8‐Br‐cAMP) enhanced the activity. Multiple alignment analysis of human, mouse, rat, and cattle RBP4 promoter suggested conserved seven transcription factor binding motifs. Electrophoretic mobility shift assay showed the −803G>A SNP modulate the affinity against unidentified DNA‐binding factor, which was assumed to be a suppressive factor. These results collectively suggested that the minor allele of RBP4 regulatory SNP enhanced the expression in adipocytes, which may be associated with the adipogenesis.

Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation.

Exosomes mediate intercellular communication, and mesenchymal stem cells (MSC) or their secreted exosomes affect a number of pathophysiologic states. Clinical applications of MSC and exosomes are increasingly anticipated. Radiation therapy is the main therapeutic tool for a number of various conditions. The cellular uptake mechanisms of exosomes and the effects of radiation on exosome-cell interactions are crucial, but they are not well understood. Here we examined the basic mechanisms and effects of radiation on exosome uptake processes in MSC. Radiation increased the cellular uptake of exosomes. Radiation markedly enhanced the initial cellular attachment to exosomes and induced the colocalization of integrin CD29 and tetraspanin CD81 on the cell surface without affecting their expression levels. Exosomes dominantly bound to the CD29/CD81 complex. Knockdown of CD29 completely inhibited the radiation-induced uptake, and additional or single knockdown of CD81 inhibited basal uptake as well as the increase in radiation-induced uptake. We also examined possible exosome uptake processes affected by radiation. Radiation-induced changes did not involve dynamin2, reactive oxygen species, or their evoked p38 mitogen-activated protein kinase-dependent endocytic or pinocytic pathways. Radiation increased the cellular uptake of exosomes through CD29/CD81 complex formation. These findings provide essential basic insights for potential therapeutic applications of exosomes or MSC in combination with radiation.

Aly/REF, a factor for mRNA transport, activates RH gene promoter function

The rhesus (Rh) blood group antigens are of considerable importance in transfusion medicine as well as in newborn or autoimmune hemolytic diseases due to their high antigenicity. We identified a major DNaseI hypersensitive site at the 5′ flanking regions of both RHD and RHCE exon 1. A 34 bp fragment located at −191 to −158 from a translation start position, and containing the TCCCCTCCC sequence, was involved in enhancing promoter activity, which was assessed by luciferase reporter gene assay. A biotin‐labelled 34 bp probe isolated an mRNA transporter protein, Aly/REF. The specific binding of Aly/REF to RH promoter in erythroid was confirmed by chromatin immunoprecipitation assay. The silencing of Aly/REF by siRNA reduced not only the RH promoter activity of the reporter gene but also transcription from the native genome. These facts provide second proof of Aly/REF as a transcription coactivator, initially identified as a coactivator for the TCRα enhancer function. Aly/REF might be a novel transcription cofactor for erythroid‐specific genes.

Assessing the applicability of FISH-based prematurely condensed dicentric chromosome assay in triage biodosimetry

AbstractThe dicentric chromosome assay (DCA) has been regarded as the gold standard of radiation biodosimetry. The assay, however, requires a 2-d peripheral blood lymphocyte culture before starting metaphase chromosome analyses to estimate biological doses. Other biological assays also have drawbacks with respect to the time needed to obtain dose estimates for rapid decision on the correct line of medical treatment. Therefore, alternative technologies that suit requirements for triage biodosimetry are needed. Radiation-induced DNA double strand breaks in G0 lymphocytes can be detected as interphase chromosome aberrations by the cell fusion-mediated premature chromosome condensation (PCC) method. The method, in combination with fluorescence in situ hybridization (FISH) techniques, has been proposed in early studies as a powerful tool for obtaining biological dose estimates without 2-d lymphocyte culture procedures. The present work assesses the applicability of FISH-based PCC techniques using pan-centromeric and telomeric peptide nucleic acid (PNA) probes in triage mode biodosimetry and demonstrates that an improved rapid procedure of the prematurely condensed dicentric chromosome (PCDC) assay has the potential for evaluating exposed radiation doses in as short as 6 h after the collection of peripheral blood specimens.

Phenotype-dependent production of des-γ-carboxy prothrombin in hepatocellular carcinoma

BackgroundDes-γ-carboxy prothrombin (DCP) is an established tumor marker for hepatocellular carcinoma (HCC), but the precise mechanism of its production remains unknown. We have recently demonstrated that cytoskeletal rearrangement during the phenotypic changes involved in epithelial mesenchymal transition (EMT) plays a crucial role in DCP production through the impairment of vitamin K uptake. However, DCP production in long-lasting severe hypoxic conditions with nutrient deprivation–such as transarterial embolization–remains unknown.MethodsWe examined the effects of long-lasting hypoxia with nutrient deprivation, as well as the constitutive expression of hypoxia-inducible factor (HIF)-1α, on EMT status, DCP production, and protein synthesis in human hepatoma cell lines by enzyme-linked immunosorbent assay, immunofluorescent studies, and western blotting. Immunohistochemistry findings for DCP, anti-hepatocyte paraffin 1 (Hep Par 1), and vimentin were examined using human resected HCC samples.ResultsBoth severe hypoxia with nutrient deprivation and HIF-1α transfection led to the cessation of DCP production, by attenuating protein synthesis through the hypophosphorylation of mammalian target of rapamycin and its effector proteins, indicative of a further phenotypic shift involving impaired liver-specific protein synthesis. In immunohistochemistry, the distribution of DCP- and Hep Par 1-positive HCC cells was mostly similar and vimentin-positive HCC cells were frequently observed in the areas that were negative for Hep Par 1 and/or DCP.ConclusionsHCC cells produce DCP when they undergo mild phenotypic changes. However, when HCC cells adopt mesenchymal properties they lose their capacity for protein synthesis, and the production of DCP is attenuated. Building upon our previous works, it appears that DCP could be a unique tumor marker that reflects the stepwise phenotypic changes of HCC.