Daisuke Okuzaki

Lats2 is an essential mitotic regulator required for the coordination of cell division

Tumor suppressor Lats2 is a member of the conserved Dbf2 kinase family. It localizes to the centrosome and has been implicated in regulation of the cell cycle and apoptosis. However, the in vivo function of this kinase remains unclear. Here, we show that complete disruption of the gene encoding Lats2 in mice causes developmental defects in the nervous system and embryonic lethality. Furthermore, mutant cells derived from total LATS2-knock-out embryos exhibit mitotic defects including centrosome fragmentation and cytokinesis defects, followed by nuclear enlargement and multinucleation. We show that the Mob1 family, a regulator of mitotic exit, associates with Lats2 to induce its activation. We also show that the complete LATS2-knock-out cells exhibit an acceleration of exit from mitosis and marked down-regulation of critical mitotic regulators. These results suggest that Lats2 plays an essential mitotic role in coordinating accurate cytokinesis completion, governing the stabilization of other mitotic regulators.

Expression of MicroRNA miR-122 Facilitates an Efficient Replication in Nonhepatic Cells upon Infection with Hepatitis C Virus

ABSTRACT Hepatitis C virus (HCV) is one of the most common etiologic agents of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. In addition, HCV infection is often associated with extrahepatic manifestations (EHM), including mixed cryoglobulinemia and non-Hodgkins lymphoma. However, the mechanisms of cell tropism of HCV and HCV-induced EHM remain elusive, because in vitro propagation of HCV has been limited in the combination of cell culture-adapted HCV (HCVcc) and several hepatic cell lines. Recently, a liver-specific microRNA called miR-122 was shown to facilitate the efficient propagation of HCVcc in several hepatic cell lines. In this study, we evaluated the importance of miR-122 on the replication of HCV in nonhepatic cells. Among the nonhepatic cell lines expressing functional HCV entry receptors, Hec1B cells derived from human uterus exhibited a low level of replication of the HCV genome upon infection with HCVcc. Exogenous expression of miR-122 in several cells facilitates efficient viral replication but not production of infectious particles, probably due to the lack of hepatocytic lipid metabolism. Furthermore, expression of mutant miR-122 carrying a substitution in a seed domain was required for efficient replication of mutant HCVcc carrying complementary substitutions in miR-122-binding sites, suggesting that specific interaction between miR-122 and HCV RNA is essential for the enhancement of viral replication. In conclusion, although miR-122 facilitates efficient viral replication in nonhepatic cells, factors other than miR-122, which are most likely specific to hepatocytes, are required for HCV assembly.

MicroRNA-mediated downregulation of mTOR/FGFR3 controls tumor growth induced by Src-related oncogenic pathways.

The tyrosine kinase c-Src is upregulated in various human cancers, but the molecular mechanisms underlying c-Src-mediated tumor growth remain unclear. Here we examined the involvement of microRNAs in the c-Src-mediated tumor growth. Microarray profiling revealed that c-Src activation downregulates a limited set of microRNAs, including miR-99a, which targets oncogenic mammalian target of rapamycin (mTOR) and fibroblast growth factor receptor 3 (FGFR3). Re-expression of miR-99a suppressed tumor growth of c-Src-transformed cells, and this effect was restored by the overexpression of mTOR. The downregulation of miR-99a was also observed in epidermal growth factor- and Ras-transformed cells, and it was suppressed by inhibiting the mitogen-activated protein kinase (MAPK) pathway. Furthermore, miR-99a downregulation is associated with mTOR/FGFR3 upregulation in various human lung cancer cells/tissues. The tumorigenicity of these cells was suppressed by the introduction of miR-99a. These findings suggest that the miR-99a-mTOR/FGFR3 pathway is crucial for controlling tumor growth in a wide range of human cancers that harbor upregulation of the Src-related oncogenic pathways.

Increased Th17-Inducing Activity of CD14+ CD163low Myeloid Cells in Intestinal Lamina Propria of Patients With Crohn’s Disease

BACKGROUND & AIMS Abnormal activity of innate immune cells and T-helper (Th) 17 cells has been implicated in the pathogenesis of autoimmune and inflammatory diseases, including Crohns disease (CD). Intestinal innate immune (myeloid) cells have been found to induce development of Th17 cells in mice, but it is not clear if this occurs in humans or in patients with CD. We investigated whether human intestinal lamina propria cells (LPCs) induce development of Th17 cells and whether these have a role in the pathogenesis of CD. METHODS Normal intestinal mucosa samples were collected from patients with colorectal cancer and noninflamed and inflamed regions of mucosa were collected from patients with CD. LPCs were isolated by enzymatic digestion and analyzed for expression of HLA-DR, lineage markers CD14 and CD163 using flow cytometry. RESULTS Among HLA-DR(high) Lin(-) cells, we identified a subset of CD14(+) CD163(low) cells in intestinal LPCs; this subset expressed Toll-like receptor (TLR) 2, TLR4, and TLR5 mRNAs and produced interleukin (IL)-6, IL-1β, and tumor necrosis factor in response to lipopolysaccharide. In vitro co-culture with naïve T cells revealed that CD14(+) CD163(low) cells induced development of Th17 cells. CD14(+) CD163(low) cells from inflamed regions of mucosa of patients with CD expressed high levels of IL-6, IL-23p19, and tumor necrosis factor mRNAs, and strongly induced Th17 cells. CD14(+) CD163(low) cells from the noninflamed mucosa of patients with CD also had increased abilities to induce Th17 cells compared with those from normal intestinal mucosa. CONCLUSIONS CD14(+) CD163(low) cells in intestinal LPCs from normal intestinal mucosa induce differentiation of naive T cells into Th17 cells; this activity is increased in mucosal samples from patients with CD. These findings show how intestinal myeloid cell types could contribute to pathogenesis of CD and possibly other Th17-associated diseases.

MicroRNA-mediated upregulation of integrin-linked kinase promotes Src-induced tumor progression

The tyrosine kinase c-Src is upregulated in various human cancers; however, the molecular mechanisms underlying c-Src-mediated tumor progression remain unclear. Here we show that downregulation of microRNA (miR)-542-3p is tightly associated with tumor progression via c-Src-related oncogenic pathways. In c-Src-transformed fibroblasts and human cancer cells that overexpress c-Src, miR-542-3p is substantially downregulated, and the ectopic expression of miR-542-3p suppresses tumor growth. We identified the integrin-linked kinase (ILK) as a conserved target of miR-542-3p. ILK upregulation promotes cell adhesion and invasion by activating the integrin–focal adhesion kinase (FAK)/c-Src pathway, and can also contribute to tumor growth via the AKT and glycogen synthase kinase 3β pathways. MiR-542-3p expression is downregulated by the activation of c-Src-related signaling molecules, including epidermal growth factor receptor, K-Ras and Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase. In human colon cancer tissues, downregulation of miR-542-3p is significantly correlated with the upregulation of c-Src and ILK. Our results suggest that the novel c-Src–miR-542-3p–ILK–FAK circuit plays a crucial role in controlling tumor progression.

Nucleoredoxin sustains Wnt/β-catenin signaling by retaining a pool of inactive dishevelled protein.

Overexpression of Dishevelled (Dvl), an essential component of the Wnt signaling pathway, is frequently associated with tumors, and thus the Dvl protein level must be tightly controlled to sustain Wnt signaling without causing tumors. Kelch-like 12 (KLHL12) targets Dvl for ubiquitination and degradation, suggesting its potential importance in avoiding aberrant Dvl overexpression. However, the regulatory mechanism of the KLHL12 activity remained elusive. We show here that nucleoredoxin (NRX) determines the Dvl protein level, which is revealed by analyses on NRX(-/-) mice showing skeletal and cardiovascular defects. Consistent with the previously reported Dvl-inhibiting function of NRX, Wnt/β-catenin signaling is hyperactivated in NRX(-/-) osteoblasts. However, the signal activity is suppressed in cardiac cells, where KLHL12 is highly expressed. Biochemical analyses reveal that Dvl is rapidly degraded by accelerated ubiquitination in NRX(-/-) mouse embryonic fibroblasts, and they fail to activate Wnt/β-catenin signaling in response to Wnt ligands. Moreover, experiments utilizing purified proteins show that NRX expels KLHL12 from Dvl and inhibits ubiquitination. These findings reveal an unexpected function of NRX, retaining a pool of inactive Dvl for robust activation of Wnt/β-catenin signaling upon Wnt stimulation.

Fission yeast meu14+ is required for proper nuclear division and accurate forespore membrane formation during meiosis II.

Using a meiosis-specific subtracted cDNA library of Schizosaccharomyces pombe, we identified meu14+ as a gene whose expression is upregulated during meiosis. Transcription of meu14+ is induced abruptly after the cell enters meiosis. Its transcription is dependent on the meiosis-specific transcription factor Mei4. In meu14Δ cells, the segregation and modification of the SPBs (spindle pole bodies) and microtubule elongation during meiosis II were aberrant. Meiotic meu14Δ cells consequently produced a high frequency of abnormal tetranucleate cells harboring aberrant forespore membranes and failed to produce asci. In wild-type cells harboring the integrated meu14+-gfp fusion gene, Meu14-GFP first appeared inside the nuclear region at prophase II, after which it accumulated beside the two SPBs at metaphase II. Thereafter, it formed two ring-shaped structures that surrounded the nucleus at early anaphase II. At post-anaphase II, it disappeared. Meu14-GFP appears to localize at the border of the forespore membrane that later develops into spore walls at the end of sporulation. This was confirmed by coexpressing Spo3-HA, a component of the forespore membrane, with Meu14-GFP. Taken together, we conclude that meu14+ is crucial in meiosis in that it participates in both the nuclear division during meiosis II and the accurate formation of the forespore membrane.

Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles

Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly.

MiR-424/503-Mediated Rictor Upregulation Promotes Tumor Progression

mTOR complex 2 (mTORC2) signaling is upregulated in multiple types of human cancer, but the molecular mechanisms underlying its activation and regulation remain elusive. Here, we show that microRNA-mediated upregulation of Rictor, an mTORC2-specific component, contributes to tumor progression. Rictor is upregulated via the repression of the miR-424/503 cluster in human prostate and colon cancer cell lines that harbor c-Src upregulation and in Src-transformed cells. The tumorigenicity and invasive activity of these cells were suppressed by re-expression of miR-424/503. Rictor upregulation promotes formation of mTORC2 and induces activation of mTORC2, resulting in promotion of tumor growth and invasion. Furthermore, downregulation of miR-424/503 is associated with Rictor upregulation in colon cancer tissues. These findings suggest that the miR-424/503–Rictor pathway plays a crucial role in tumor progression.

Gin4 of S. cerevisiae is a bud neck protein that interacts with the Cdc28 complex

A number of proteins make up the Cdc28 complex in Saccharomyces cerevisiae, and regulate CDK activity. The cell cycle regulator Nik1 (Hsl1) is a protein kinase that interacts with the Cdc28 complex. The growth inhibitor Gin4 is structurally similar to Nik1 and may play a redundant role in the regulation of the cell cycle. We investigated the functions of Gin4 with respect to those of Nik1.