Masatoshi Muraoka

Disruption of CXC Motif Chemokine Ligand-14 in Mice Ameliorates Obesity-induced Insulin Resistance

In obese individuals, white adipose tissue (WAT) is infiltrated by large numbers of macrophages, resulting in enhanced inflammatory responses that contribute to insulin resistance. Here we show that expression of the CXC motif chemokine ligand-14 (CXCL14), which targets tissue macrophages, is elevated in WAT of obese mice fed a high fat diet (HFD) compared with lean mice fed a regular diet. We found that HFD-fed CXCL14-deficient mice have impaired WAT macrophage mobilization and improved insulin responsiveness. Insulin-stimulated phosphorylation of Akt kinase in skeletal muscle was severely attenuated in HFD-fed CXCL14+/- mice but not in HFD-fed CXCL14-/- mice. The insulin-sensitive phenotype of CXCL14-/- mice after HFD feeding was prominent in female mice but not in male mice. HFD-fed CXCL14-/- mice were protected from hyperglycemia, hyperinsulinemia, and hypoadiponectinemia and did not exhibit increased levels of circulating retinol-binding protein-4 and increased expression of interleukin-6 in WAT. Transgenic overexpression of CXCL14 in skeletal muscle restored obesity-induced insulin resistance in CXCL14-/- mice. CXCL14 attenuated insulin-stimulated glucose uptake in cultured myocytes and to a lesser extent in cultured adipocytes. These results demonstrate that CXCL14 is a critical chemoattractant of WAT macrophages and a novel regulator of glucose metabolism that functions mainly in skeletal muscle.

p57Kip2 Is Degraded through the Proteasome in Osteoblasts Stimulated to Proliferation by Transforming Growth Factor β1

Cyclin-dependent kinase inhibitory proteins are negative regulators of the cell cycle. Although all the cyclin-dependent kinase inhibitory proteins may be involved in cell cycle control during a differentiation process, only p57Kip2 is shown to be essential for embryonic development. However, the role of p57 in the control of the cell cycle is poorly understood. Using osteoblasts derived from the calvaria of rat fetus, we show that p57 is accumulated in cells starved by low serum. Cyclin-dependent kinase 2 activity was suppressed in these cells with a significant amount bound to p57. Treatment of the cells with transforming growth factor β1 dramatically reduced the amount of p57, resulting in an activation of cyclin-dependent kinase 2 activity and the stimulation of cell proliferation. The decrease in p57 was inhibited by treating the cells with proteasome inhibitors, Z-Leu-Leu-Leu-aldehyde or lactacystin, but not with Z-Leu-Leu-aldehyde, which is an inhibitor of calpain, indicating that p57 is degraded through the proteasome pathway. p57 was also shown to be ubiquitinated in vitro. Because transforming growth factor β1 not only stimulates the growth but also inhibits the differentiation of the cells in this system, our results may suggest a possible involvement of p57 in the control of osteoblastic cell proliferation and differentiation.

Drastic genetic instability of tumors and normal tissues in Turcot syndrome

Turcot syndrome is characterized by an association of malignant brain tumors and colon cancer developing in the patients teens. Since the mechanism of carcinogenesis in Turcot syndrome is still unclear, we analysed genetic changes in tumors from a Turcot patient with no family history of the condition. All tumors, including one astrocytoma, three colon carcinomas, and two colon adenomas, exhibited severe replication error (RER), and all colon tumors showed somatic muations at repeated regions of TGFβRII, E2F-4, hMSH3, and/or hMSH6 genes. Somatic APC mutations were detected in three of three colon carcinomas, and somatic p53 mutations were detected in the astrocytoma and two of three colon carcinomas, both of which showed two mutations without allele loss. We also found that normal colon mucosa, normal skin fibroblasts and normal brain tissue from this patient showed respective high frequencies of RER, in contrast to usual HNPCC patients in which RER was very rare in normal tissues. These results suggest that extreme DNA instability in normal tissues causes the early development of multiple cancer in Turcot syndrome. A missense mutation (GAG to AAG) at codon 705 of hPMS2 gene was detected in one allele of this patient, which was inherited from his mother without tumors. Additional unknown germline mutation may contribute to the genetic instability in normal tissues.

Familial polyposis: recent advances☆

Familial adenomatous polyposis (FAP) is a dominantly inherited disease characterized by the development of numerous adenomatous polyps and a high risk of colorectal cancer. Affected individuals have multiple adenomas at various intermediate stages between benign and malignant, so FAP is a supportive model for the adenoma-carcinoma sequence of colorectal carcinogenesis. The APC (adenomatous polyposis coli) gene, which is a tumor suppressor gene, was discovered in 1991, and mutation of this gene was found to be the cause of FAP. To date, more than 150 germ-line mutations have been determined in FAP patients, and more than 300 somatic mutations of the APC gene have been detected in colorectal adenomas and carcinomas from both FAP and non-FAP patients - almost all of these resulting in truncated APC protein. The nature of germline and somatic mutations is described in detail herein, including the finding of several codons in which no germ-line, but only somatic mutation, frequently occurs. Existence of somatic APC mutations in adenomas from both FAP and non-FAP patients suggests that inactivation of the APC gene by two mutations is generally involved in the development of adenoma. Further development of adenoma to advanced carcinoma is associated with loss of heterozygosity (LOH) of the APC gene and additional inactivation of multiple tumor suppressor genes (possibly through mutation and LOH) that include the p53 gene, DCC gene and tumor suppresor genes on chromosomes 1p, 8p and 22q in both FAP and non-FAP patients. Recent findings on the correlation of these genetic changes with the adenomacarcinoma sequence are also described. In vitro models for carcinogenesis, and experiments of suppression of tumorigenicity in advanced colon carcinoma cells by introduction of a normal single chromosome or a normal suppressor gene are also referred to. Furthermore, evidence of inactivation of the APC gene in extracolonic tumors, in FAP patients, including desmoid and adrenocortical tumors, is demonstrated. This confirms the pleiotropic effect of the mutant APC gene on both colonic and extracolonic manifestations in FAP patients, and that the normal APC gene acts as a growth control gene throughout the body

Expression of CD44 variants in gastric carcinoma with or without Epstein-Barr virus.

The significance of CD44 variants in gastric carcinoma has not been fully investigated in terms of the pathological features of the carcinoma, including its association with Epstein‐Barr virus (EBV). In this study, a total of 104 primary gastric carcinoma tissues (EBV‐associated gastric carcinomas, EBVaGC, and EBV‐negative carcinomas) were evaluated by immunohistochemistry. When the immunoreactivity of formalin‐fixed, paraffin‐embedded sections was graded on a scale of 0–3, the frequencies of grades 0–1, 2 and 3 were, respectively, 77%, 16% and 7% using monoclonal antibody (MAb) 3G5, which recognizes V3–5, and 70%, 14% and 15% with MAb 2F10, which recognizes V6. The expression of CD44 variants is independently correlated with lymph node metastasis and EBV‐association in gastric carcinoma. Significant correlations were observed between V3–5 expression and lymph vessel invasion or lymph node metastasis, and between V6 expression and lymph node metastasis. The expression of both variants was significantly correlated with EBV‐association. EBV‐association and lymph node metastasis contributed independently to CD44 variant expression by multivariate analysis. Thus, the mechanism and significance of CD44 variant‐expression are different in gastric carcinomas with or without EBV. EBVaGC is a distinct type of gastric carcinoma which should be considered separately from EBV‐negative carcinoma. Int. J. Cancer 74:450–454, 1997.

Malignant transformation and EGFR activation of immortalized mouse liver epithelial cells caused by HBV enhancer-X from a human hepatocellular carcinoma.

We have previously observed that all human hepatocellular carcinomas (HCCs) from HBV carriers examined had the integrated X region. In this study, HBV DNA was isolated from an integration site in one HCC that had a single, very small integrated viral DNA including the X region, but it had no expression of X gene as poly(A)RNA. It was found that HBV DNA was present between alphoid repetitive sequences, and it included Enhancer and X regions, encompassing the adr sequence from 910 to 1811. Nucleotides for 8 amino acids at the 3’ end, a stop codon of X gene and a poly(A) signal downstream of X gene were lost by integration, and nucleotides for 7 amino acids and a stop codon were substituted by a connected alphoid sequence. When this cloned HBV DNA was transfected with an expression vector to an immortalized mouse liver epithelial cell line, MLE‐10, malignant transformation occurred. Transformants having expressed poly(A)RNA of the X gene showed anchorage‐independent growth in soft agar and tumor formation in the subcutis of nude mice. The mRNA level of EGFR was found to be remarkably enhanced in X‐transformed cells, in contrast with the absence of this mRNA in parental and ras‐transformed MLE‐10. Our data provide evidence that the Enhancer‐X region alone is the key contributor to the malignant change of pre‐malignant liver cells in HBV carriers through activation of some specific genes, such as EGFR. Int. J. Cancer 85:518–522, 2000.

Variety of Nucleotide Sugar Transporters with Respect to the Interaction with Nucleoside Mono- and Diphosphates

Nucleotide sugar transporters have long been assumed to be antiporters that exclusively use nucleoside monophosphates as antiport substrates. Here we present evidence indicating that two other types of nucleotide sugar transporters exist that differ in their antiport substrate specificity. Biochemical studies using microsomes derived from Saccharomyces cerevisiae cells expressing either human (h) UGTrel7 or the Drosophila (d) FRC (Fringe connection) transporter revealed that (i) efflux of preloaded UDP-glucuronic acid from the yeast microsomes expressing hUGTrel7 was strongly enhanced by UDP-GlcNAc added in the external medium, but not by UMP or UDP, suggesting that hUGTrel7 may be described as a UDP-sugar/UDP-sugar antiporter, and (ii) addition of UDP-sugars, UDP, or UMP in the external medium stimulated the efflux of preloaded UDP-GlcNAc from the yeast microsomes expressing dFRC to a comparable extent, suggesting that UDP, as well as UMP, may serve as an antiport substrate of dFRC. Antiport of UDP-sugars with these specific substrates was reproduced and definitively confirmed using proteoliposomes reconstituted from solubilized and purified transporters. Possible physiological implications of these observations are discussed.

Tumor-suppressive Function of Protein-tyrosine Phosphatase Non-receptor Type 23 in Testicular Germ Cell Tumors Is Lost upon Overexpression of miR142–3p microRNA

Background: The PTPN23 gene is a candidate tumor suppressor involved in the tumorigenesis of various organs. Results: Expression of PTPN23 in testicular germ cell tumor cells is negatively regulated by miR-142-3p. Conclusion: A lack of PTPN23 protein expression in human TGCTs is inversely correlated with miR-142-3p expression. Significance: Loss of PTPN23 expression mediated by miR-142--3p may be a key event in the pathogenesis of TGCTs. Protein-tyrosine phosphatase non-receptor type 23 (PTPN23) is a candidate tumor suppressor involved in the tumorigenesis of various organs. However, its physiological role(s) and detailed expression profile(s) have not yet been elucidated. We investigated the function and regulation of PTPN23 in the formation of testicular germ cell tumors (TGCTs). Expression of PTPN23 in human TGCT cell lines was significantly lower than that in spermatogonial stem cells in mice. Overexpression of PTPN23 in NEC8, a human TGCT cell line, suppressed soft agar colony formation in vitro and tumor formation in nude mice in vivo. These data indicate that PTPN23 functions as a tumor suppressor in TGCTs. Multiple computational algorithms predicted that the 3′ UTR of human PTPN23 is a target for miR-142-3p. A luciferase reporter assay confirmed that miR-142-3p bound directly to the 3′ UTR of PTPN23. Introduction of pre-miR-142 in the PTPN23 transfectant of NEC8 led to suppressed expression of PTPN23 and increased soft agar colony formation. Quantitative RT-PCR data revealed a significantly higher expression of miR-142-3p in human seminomas compared with normal testes. No difference in mRNA expression between seminoma and non-seminoma samples was detected by in situ hybridization. Both quantitative RT-PCR and immunohistochemical analyses revealed that PTPN23 expression was significantly lower in TGCTs than in normal testicular tissues. Finally, a lack of PTPN23 protein expression in human TGCTs correlated with a relatively higher miR-142-3p expression. These data suggest that PTPN23 is a tumor suppressor and that repression of PTPN23 expression by miR-142-3p plays an important role in the pathogenesis of TGCTs.