Motoyuki Otsuka

Impaired microRNA processing causes corpus luteum insufficiency and infertility in mice

The microRNA (miRNA) processing enzyme Dicer1 is required for zygotic and embryonic development, but the early embryonic lethality of Dicer1 null alleles in mice has limited our ability to address the role of Dicer1 in normal mouse growth and development. To address this question, we used a mouse mutant with a hypomorphic Dicer1 allele (Dicer(d/d)) and found that Dicer1 deficiency resulted in female infertility. This defect in female Dicer(d/d) mice was caused by corpus luteum (CL) insufficiency and resulted, at least in part, from the impaired growth of new capillary vessels in the ovary. We found that the impaired CL angiogenesis in Dicer(d/d) mice was associated with a lack of miR17-5p and let7b, 2 miRNAs that participate in angiogenesis by regulating the expression of the antiangiogenic factor tissue inhibitor of metalloproteinase 1. Furthermore, injection of miR17-5p and let7b into the ovaries of Dicer(d/d) mice partially normalized tissue inhibitor of metalloproteinase 1 expression and CL angiogenesis. Our data indicate that the development and function of the ovarian CL is a physiological process that appears to be regulated by miRNAs and requires Dicer1 function.

Genome-wide association study identifies a susceptibility locus for HCV-induced hepatocellular carcinoma

To identify the genetic susceptibility factor(s) for hepatitis C virus–induced hepatocellular carcinoma (HCV-induced HCC), we conducted a genome-wide association study using 432,703 autosomal SNPs in 721 individuals with HCV-induced HCC (cases) and 2,890 HCV-negative controls of Japanese origin. Eight SNPs that showed possible association (P < 1 × 10−5) in the genome-wide association study were further genotyped in 673 cases and 2,596 controls. We found a previously unidentified locus in the 5′ flanking region of MICA on 6p21.33 (rs2596542, Pcombined = 4.21 × 10−13, odds ratio = 1.39) to be strongly associated with HCV-induced HCC. Subsequent analyses using individuals with chronic hepatitis C (CHC) indicated that this SNP is not associated with CHC susceptibility (P = 0.61) but is significantly associated with progression from CHC to HCC (P = 3.13 × 10−8). We also found that the risk allele of rs2596542 was associated with lower soluble MICA protein levels in individuals with HCV-induced HCC (P = 1.38 × 10−13).

Hepatitis C Virus Core Protein Enhances p53 Function through Augmentation of DNA Binding Affinity and Transcriptional Ability

Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis, and hepatocellular carcinoma. Since there are several reports indicating that some viruses influence the tumor suppressor p53 function, we determined the effects of HCV proteins on p53 function and its mechanism determined by use of a reporter assay. Among seven HCV proteins investigated (core, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), only core protein augmented the transcriptional activity of p53 and increased the expression of p21 waf1 protein, which is a major target of p53. Core protein increased both DNA-binding affinity of p53 in electrophoretic morbidity shift assay and transcriptional ability of p53 itself in a reporter assay. The direct interaction between core protein and C terminus of p53 was also shown by glutathioneS-transferase fusion protein binding assay. In addition, core protein interacted with hTAFII28, a component of the transcriptional factor complex in vivo and in vitro. These results suggest that HCV core protein interacts with p53 and modulates p53-dependent promoter activities during HCV infection.

Serum metabolomics reveals γ-glutamyl dipeptides as biomarkers for discrimination among different forms of liver disease

BACKGROUND & AIMS We applied a metabolome profiling approach to serum samples obtained from patients with different liver diseases, to discover noninvasive and reliable biomarkers for rapid-screening diagnosis of liver diseases. METHODS Using capillary electrophoresis and liquid chromatography mass spectrometry, we analyzed low molecular weight metabolites in a total of 248 serum samples obtained from patients with nine types of liver disease and healthy controls. RESULTS We found that γ-glutamyl dipeptides, which were biosynthesized through a reaction with γ-glutamylcysteine synthetase, were indicative of the production of reduced glutathione, and that measurement of their levels could distinguish among different liver diseases. Multiple logistic regression models facilitated the discrimination between specific and other liver diseases and yielded high areas under receiver-operating characteristic curves. The area under the curve values in training and independent validation data were 0.952 and 0.967 in healthy controls, 0.817 and 0.849 in drug-induced liver injury, 0.754 and 0.763 in asymptomatic hepatitis B virus infection, 0.820 and 0.762 in chronic hepatitis B, 0.972 and 0.895 in hepatitis C with persistently normal alanine transaminase, 0.917 and 0.707 in chronic hepatitis C, 0.803 and 0.993 in cirrhosis type C, and 0.762 and 0.803 in hepatocellular carcinoma, respectively. Several γ-glutamyl dipeptides also manifested potential for differentiating between nonalcoholic steatohepatitis and simple steatosis. CONCLUSIONS γ-Glutamyl dipeptides are novel biomarkers for liver diseases, and varying levels of individual or groups of these peptides have the power to discriminate among different forms of hepatic disease.

Macrophage Deletion of p38α Partially Impairs Lipopolysaccharide-Induced Cellular Activation

The activation of p38α, a MAPK family member, is associated with macrophage activation by microbial pattern molecules, such as LPS. The requirement of p38α in inflammatory responses has been shown in a number of studies using chemical inhibitors, though the inhibitors also inhibit p38β and perhaps some other enzymes. In this study, we used conditional knockout of p38α in macrophages to address the role of p38α in macrophage activation. We found that p38α deficiency causes a significant inhibition in the production of LPS-induced TNF-α, IL-12, and IL-18, but it has little or no effect on IL-6 or IFN-β production. Knockout of p38α in macrophages did not affect LPS-induced activation of the other major signaling pathways (NF-κB, Jnk, and Erk), nor did it affect the transcriptional activity of NF-κB. It had little inhibitory effect on LPS-induced AP-1 activity, but it significantly inhibited LPS-induced C/EBP-β and CREB activation, indicating that the role of p38α in cytokine production in macrophages is at least in part through its regulation of C/EBP-β and CREB activation. In addition, we also confirmed that p38α is important for phagocytosis of bacteria by macrophages. Our in vivo studies with two murine models showed that p38α is involved in sepsis. Collectively, our data demonstrate that p38α is an important player in inflammatory responses.

Interaction between the HCV NS3 protein and the host TBK1 protein leads to inhibition of cellular antiviral responses

The persistent nature of hepatitis C virus (HCV) infection suggests that HCV encodes proteins that enable it to overcome host antiviral responses. Toll‐like receptor 3 (TLR3)‐mediated signaling, which recognizes the double‐stranded RNA that is produced during viral replication and induces type I interferons, including interferon β (IFN‐β), is crucial to the host defense against viruses. Recent studies suggest that a TIR domain–containing adaptor protein, TRIF, and two protein kinases, TANK‐binding kinase‐1 (TBK1) and IκB kinase‐ϵ (IKKϵ), play essential roles in TLR3‐mediated IFN‐β production through the activation of the transcriptional factor interferon regulatory factor 3 (IRF‐3). We report that the HCV NS3 protein interacts directly with TBK1, and that this binding results in the inhibition of the association between TBK1 and IRF‐3, which leads to the inhibition of IRF‐3 activation. In conclusion, these results suggest the mechanisms of the inhibition of the innate immune responses of HCV infection by NS3 protein. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;41:1004–1012.)

Vitamin K2 inhibits the growth and invasiveness of hepatocellular carcinoma cells via protein kinase A activation.

Heatocellular carcinoma (HCC) is a common human malignancy. Its high mortality rate is mainly a result of high intrahepatic recurrence and portal venous invasion (PVI). We previously reported that the development of PVI is related to levels of des‐gamma‐carboxy prothrombin (DCP), a serum protein that increases at a notably higher rate in patients with HCC. Because DCP is produced by a vitamin K shortage, we examined the biological effects of extrinsic supplementation of vitamin K2 in HCC cells in vitro and in vivo. Consequently, vitamin K2 inhibits the growth and invasion of HCC cells through the activation of protein kinase A, which modulates the activities of several transcriptional factors and inhibits the small GTPase Rho, independent of suppression of DCP. In addition, administration of vitamin K2 to nude mice inoculated with liver tumor cells reduced both tumor growth and body weight loss. In conclusion, similar to an acyclic retinoid—which was previously reported to prevent the recurrence of HCC—vitamin K2, another lipid‐soluble vitamin, may be a promising therapeutic means for the management of HCC. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:243–251.)

Cell surface 4-1BBL mediates sequential signaling pathways 'downstream' of TLR and is required for sustained TNF production in macrophages

The stimulation of Toll-like receptors (TLRs) on macrophages triggers production of the cytokine tumor necrosis factor (TNF). TNF production occurs within 1 h of TLR stimulation and is sustained for 1 d. Here we document a function for the TNF family member 4-1BB ligand (4-1BBL) in sustaining TLR-induced TNF production. TLR signaling induced 4-1BBL, and 4-1BBL interacted with TLRs on the macrophage surface. The influence of 4-1BBL on TNF production was independent of its receptor (4-1BB) and did not require the adaptors MyD88 or TRIF. It did not influence TLR4-induced activation of transcription factor NF-κB (an early response) but was required for TLR4-induced activation of transcription factors CREB and C/EBP (a late event). Transient TLR4-MyD88 complexes appeared during the first hour after lipopolysaccharide stimulation, and TLR4–4-1BBL interactions were detected between 2 h and 8 h after lipopolysaccharide stimulation. Our results indicate that two different TLR4 complexes sequentially form and selectively control early and late TNF production.

MicroRNA122 is a key regulator of α-fetoprotein expression and influences the aggressiveness of hepatocellular carcinoma

α-fetoprotein (AFP) is not only a widely used biomarker in hepatocellular carcinoma (HCC) surveillance, but is also clinically recognized as linked with aggressive tumour behaviour. Here we show that deregulation of microRNA122, a liver-specific microRNA, is a cause of both AFP elevation and a more biologically aggressive phenotype in HCC. We identify CUX1, a direct target of microRNA122, as a common central mediator of these two effects. Using liver tissues from transgenic mice in which microRNA122 is functionally silenced, an orthotopic xenograft tumour model, and human clinical samples, we further demonstrate that a microRNA122/CUX1/microRNA214/ZBTB20 pathway regulates AFP expression. We also show that the microRNA122/CUX1/RhoA pathway regulates the aggressive characteristics of tumours. We conclude that microRNA122 and associated signalling proteins may represent viable therapeutic targets, and that serum AFP levels in HCC patients may be a surrogate marker for deregulated intracellular microRNA122 signalling pathways in HCC tissues.

Calcineurin Negatively Regulates TLR-Mediated Activation Pathways

In innate immunity, microbial components stimulate macrophages to produce antimicrobial substances, cytokines, other proinflammatory mediators, and IFNs via TLRs, which trigger signaling pathways activating NF-κB, MAPKs, and IFN response factors. We show in this study that, in contrast to its activating role in T cells, in macrophages the protein phosphatase calcineurin negatively regulates NF-κB, MAPKs, and IFN response factor activation by inhibiting the TLR-mediated signaling pathways. Evidence for this novel role for calcineurin was provided by the findings that these signaling pathways are activated when calcineurin is inhibited either by the inhibitors cyclosporin A or FK506 or by small interfering RNA-targeting calcineurin, and that activation of these pathways by TLR ligands is inhibited by the overexpression of a constitutively active form of calcineurin. We further found that IκB-α degradation, MAPK activation, and TNF-α production by FK506 were reduced in macrophages from mice deficient in MyD88, Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF), TLR2, or TLR4, whereas macrophages from TLR3-deficient or TLR9 mutant mice showed the same responses to FK506 as those of wild-type cells. Biochemical studies indicate that calcineurin interacts with MyD88, TRIF, TLR2, and TLR4, but not with TLR3 or TLR9. Collectively, these results suggest that calcineurin negatively regulates TLR-mediated activation pathways in macrophages by inhibiting the adaptor proteins MyD88 and TRIF, and a subset of TLRs.