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Kazuaki Yoneno,
Tadakazu Hisamatsu, Katsuyoshi Shimamura,
Nobuhiko Kamada,
Riko Ichikawa,
Mina T Kitazume,
Maiko Mori,
Michihide Uo,
Yuka Namikawa,
Katsuyoshi Matsuoka,
Toshiro Sato,
Kazutaka Koganei,
Akira Sugita,
Takanori Kanai,
Toshifumi Hibi
[show abstract]
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ABSTRACT: Bile acids (BAs) play important roles not only in lipid metabolism, but also in signal transduction. TGR5, a transmembrane receptor of BAs, is an immunomodulative factor, but its detailed mechanism remains unclear. Here, we aimed to delineate how BAs operate in immunological responses via the TGR5 pathway in human mononuclear cell lineages. We examined TGR5 expression in human peripheral blood monocytes, several types of in vitro differentiated macrophages (Mϕs) and dendritic cells. Mϕs differentiated with macrophage colony-stimulating factor and interferon-γ (Mγ-Mϕs), which are similar to the human intestinal lamina propria CD14(+) Mϕs that contribute to Crohn's disease (CD) pathogenesis by production of pro-inflammatory cytokines, highly expressed TGR5 compared with any other type of differentiated Mϕ and dendritic cells. We also showed that a TGR5 agonist and two types of BAs, deoxycholic acid and lithocholic acid, could inhibit tumour necrosis factor-α production in Mγ-Mϕs stimulated by commensal bacterial antigen or lipopolysaccharide. This inhibitory effect was mediated by the TGR5-cAMP pathway to induce phosphorylation of c-Fos that regulated nuclear factor-κB p65 activation. Next, we analysed TGR5 levels in lamina propria mononuclear cells (LPMCs) obtained from the intestinal mucosa of patients with CD. Compared with non-inflammatory bowel disease, inflamed CD LPMCs contained more TGR5 transcripts. Among LPMCs, isolated CD14(+) intestinal Mϕs from patients with CD expressed TGR5. In isolated intestinal CD14(+) Mϕs, a TGR5 agonist could inhibit tumour necrosis factor-α production. These results indicate that TGR5 signalling may have the potential to modulate immune responses in inflammatory bowel disease.
Immunology 05/2013; 139(1):19-29. · 3.32 Impact Factor
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Jonathan Chang,
Tadakazu Hisamatsu, Katsuyoshi Shimamura,
Kazuaki Yoneno,
Masayuki Adachi,
Hiroshi Naruse,
Toru Igarashi,
Hajime Higuchi,
Katsuyoshi Matsuoka,
Mina T Kitazume,
Setsu Ando,
Nobuhiko Kamada,
Takanori Kanai,
Toshifumi Hibi
[show abstract]
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ABSTRACT: AIM: Liver macrophages play integral roles in both the progression and resolution of hepatic inflammation and fibrosis, comprising opposing functions that largely coincide with the activation state of nearby hepatic stellate cells (HSC). While cross-talk between HSC and macrophages may be essential at various stages of inflammation and fibrogenesis, many facets of this interaction have yet to be thoroughly explored. Here, we examine the potential roles of HSC-derived signaling molecules as mediators of liver macrophage differentiation. METHODS: Human peripheral blood mononuclear cells (PBMC) were differentiated to macrophages in the presence or absence of cultured HSC-derived conditioned media. The phenotype of resulting macrophages was characterized by examination of cell surface marker expression, antigen-presenting capabilities and cytokine secretion. RESULTS: Conditioned media from activated human HSC promoted the differentiation of a unique set of macrophages that differed in morphology and function from both classical (M1) and alternative (M2) macrophages, expressing increased levels of CD14 and CD16, as well as a distinct interleukin (IL)-6(high) /IL-10(low) /transforming growth factor (TGF)-β(high) expression profile. These macrophages expressed high levels of CD206, CD209, CD80 and human leukocyte antigen DR, though no significant increases in antigen presentation were apparent. HSC-derived macrophages exhibited specific activation of p38 mitogen-activated protein kinase, and inhibition of this activation by p38 inhibitors during differentiation effectively reversed increases in IL-6 and TGF-β. CONCLUSION: The present results suggest that HSC-derived signaling molecules promote differentiation of liver macrophages with both pro-inflammatory and profibrotic functions. Furthermore, these effects appear to be mediated, at least partially, in a p38-dependent manner.
Hepatology Research 09/2012; · 2.20 Impact Factor
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Kengo Tomita,
Toshiaki Teratani,
Takahiro Suzuki,
Tetsuya Oshikawa,
Hirokazu Yokoyama, Katsuyoshi Shimamura,
Kiyoshi Nishiyama,
Norikazu Mataki,
Rie Irie,
Tohru Minamino, [......],
Hidetsugu Saito,
Ippei Shimizu,
Yohko Yoshida,
Ryota Hokari,
Kazuo Sugiyama,
Kazuo Hatsuse,
Junji Yamamoto,
Takanori Kanai,
Soichiro Miura,
Toshifumi Hibi
[show abstract]
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ABSTRACT: The tumor suppressor p53 is a primary sensor of stressful stimuli, controlling a number of biologic processes. The aim of our study was to examine the roles of p53 in non-alcoholic steatohepatitis (NASH).
Male wild type and p53-deficient mice were fed a methionine- and choline-deficient diet for 8weeks to induce nutritional steatohepatitis. mRNA expression profiles in normal liver samples and liver samples from patients with non-alcoholic liver disease (NAFLD) were also evaluated.
Hepatic p53 and p66Shc signaling was enhanced in the mouse NASH model. p53 deficiency suppressed the enhanced p66Shc signaling, decreased hepatic lipid peroxidation and the number of apoptotic hepatocytes, and ameliorated progression of nutritional steatohepatitis. In primary cultured hepatocytes, transforming growth factor (TGF)-β treatment increased p53 and p66Shc signaling, leading to exaggerated reactive oxygen species (ROS) accumulation and apoptosis. Deficient p53 signaling inhibited TGF-β-induced p66Shc signaling, ROS accumulation, and hepatocyte apoptosis. Furthermore, expression levels of p53, p21, and p66Shc were significantly elevated in human NAFLD liver samples, compared with results obtained with normal liver samples. Among NAFLD patients, those with NASH had significantly higher hepatic expression levels of p53, p21, and p66Shc compared with the group with simple steatosis. A significant correlation between expression levels of p53 and p66Shc was observed.
p53 in hepatocytes regulates steatohepatitis progression by controlling p66Shc signaling, ROS levels, and apoptosis, all of which may be regulated by TGF-β. Moreover, p53/p66Shc signaling in the liver appears to be a promising target for the treatment of NASH.
Journal of Hepatology 05/2012; 57(4):837-43. · 9.26 Impact Factor
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Jun Miyoshi,
Tomoharu Yajima,
Susumu Okamoto,
Katsuyoshi Matsuoka,
Nagamu Inoue,
Tadakazu Hisamatsu, Katsuyoshi Shimamura,
Atsushi Nakazawa,
Takanori Kanai,
Haruhiko Ogata,
Yasushi Iwao,
Makio Mukai,
Toshifumi Hibi
[show abstract]
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ABSTRACT: BackgroundHost–intestinal microbial interaction plays an important role in the pathogenesis of inflammatory bowel diseases (IBDs). The
surface molecules of the intestinal epithelium act as receptors for bacterial adhesion and regulate the intestinal bacteria.
Some known receptors are the mucosal blood type antigens, which are regulated by the fucosyltransferase2 (FUT2) gene, and individuals who express these antigens in the gastrointestinal tract are called secretors. Recent research has
revealed that the FUT2 gene is associated with Crohn’s disease (CD) in western populations.
MethodsTo clarify the contribution of mucosal blood type antigens in IBD, we determined the incidence of five previously reported
single-nucleotide polymorphisms of the FUT2 gene in Japanese patients. We also used immunohistochemistry to investigate the antigen expression in mucosal specimens from
IBD patients and animal models.
ResultsGenetic analysis revealed that all of the patients with colonic CD were secretors, whereas the incidence of secretors was
80, 80, 67, and 80%, respectively, for the control, ileocolonic CD, ileal CD, and ulcerative colitis groups (P=0.036). Abnormal expression of blood type antigens was observed only in colonic CD. Interleukin-10−/− mice, but not dextran sulfate sodium colitis mice, had enhanced colonic expression of blood type antigens, and the expression
of these antigens preceded the development of colitis in the interleukin-10−/− mice.
Conclusions
FUT2 secretor status was associated with colonic-type CD. This finding, taken together with the immunohistochemistry data, suggests
that the abnormal expression of blood type antigens in the colon may be a unique and essential factor for colonic CD.
KeywordsColonic Crohn’s disease–
FUT2
–Blood type antigen
Journal of Gastroenterology 04/2012; 46(9):1056-1063. · 4.16 Impact Factor
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ABSTRACT: Cyclooxygenase-2 (COX-2) is a key enzyme that produces prostaglandin E2 (PGE2) and plays an important role in colorectal tumor growth. In addition, recent researches focused on 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which degrades PGE2. Here we determined the effect of 5-aminosalicylic acid (5-ASA) on COX-2 and 15-PGDH expression and investigated its preventive effect for colorectal cancer (CRC).
HT-29 cells were used in the in vitro experiments. c-Ha-ras transgenic mice were employed in order to explore the chemopreventive effects. Western blotting analysis was performed and the protein expression of COX-2 and 15-PGDH was quantified.
5-ASA significantly suppressed COX-2 expression and induced 15-PGDH expression in HT-29 cells. In the transgenic mice, oral 5-ASA intake reduced the incidence of colorectal tumor formation and the tumor size. Furthermore, we observed a down-regulation of COX-2 and an up-regulation of 15-PGDH in the tissue from colons of these mice.
5-ASA exerts a preventive effect against colorectal tumor development through mediation of COX-2 and 15-PGDH expression.
Anticancer research 04/2012; 32(4):1193-202. · 1.73 Impact Factor
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Toshiaki Teratani,
Kengo Tomita,
Takahiro Suzuki,
Tetsuya Oshikawa,
Hirokazu Yokoyama, Katsuyoshi Shimamura,
Susumu Tominaga,
Sadayuki Hiroi,
Rie Irie,
Yoshikiyo Okada,
Chie Kurihara,
Hirotoshi Ebinuma,
Hidetsugu Saito,
Ryota Hokari,
Kazuo Sugiyama,
Takanori Kanai,
Soichiro Miura,
Toshifumi Hibi
[show abstract]
[hide abstract]
ABSTRACT: Some studies have indicated that dietary cholesterol has a role in the progression of liver fibrosis. We investigated the mechanisms by which dietary cholesterol might contribute to hepatic fibrogenesis.
C57BL/6 mice were fed a high-cholesterol diet or a control diet for 4 weeks; liver fibrosis then was induced by bile-duct ligation or carbon tetrachloride administration. Hepatic stellate cells (HSCs) were isolated from mice fed high-cholesterol diets or from Niemann-Pick type C1-deficient mice, which accumulate intracellular free cholesterol.
After bile-duct ligation or carbon tetrachloride administration, mice fed high-cholesterol diets had significant increases in liver fibrosis and activation of HSCs compared with mice fed control diets. There were no significant differences in the degree of hepatocellular injury or liver inflammation, including hepatocyte apoptosis or Kupffer cell activation, between mice fed high-cholesterol or control diets. Levels of free cholesterol were much higher in HSCs from mice fed high-cholesterol diets than those fed control diets. In cultured HSCs, accumulation of free cholesterol in HSCs increased levels of Toll-like receptor 4 (TLR4), leading to down-regulation of bone morphogenetic protein and activin membrane-bound inhibitor (a pseudoreceptor for transforming growth factor [TGF]β); the HSCs became sensitized to TGFβ-induced activation. Liver fibrosis was not aggravated by the high-cholesterol diet in C3H/HeJ mice, which express a mutant form of TLR4; HSCs that express mutant TLR4 were not activated by accumulation of free cholesterol.
Dietary cholesterol aggravates liver fibrosis because free cholesterol accumulates in HSCs, leading to increased TLR4 signaling, down-regulation of bone morphogenetic protein and activin membrane-bound inhibitor, and sensitization of HSC to TGFβ. This pathway might be targeted by antifibrotic therapies.
Gastroenterology 01/2012; 142(1):152-164.e10. · 11.68 Impact Factor
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ABSTRACT: A Petunia inflata isolate with a novel phenotype of a purple corolla limb with green corolla segments (GCS) was characterized. The GCS have stomata and trichomes on the adaxial side, and resemble calyx segments in epidermal morphology. The GCS phenotype was inherited in a recessive manner. In the GCS plant, a novel inhibitor/defective spm-like transposable element (dPifTp1) was inserted in the second intron of the Floral Binding Protein 2 (FBP2) gene. The sequence of the resulting transcript contained five silent mutations as compared the corresponding open reading frame of P. x hybrida FBP2 mRNA. The GCS phenotype co-segregated with an FBP2 fragment containing a dPifTp1 insertion. The transcript level of the FBP2 gene in GCS flowers was markedly lower than that in wild-type (WT) flowers, suggesting that partially inhibited FBP2 gene expression caused the morphogenesis of calyx-like tissue in the corolla segments of GCS flowers. Gene expression pattern analysis using a full-length Petunia floral cDNA microarray indicated that some photosynthesis-related genes were expressed at significantly higher levels in the GCS of GCS flowers, but the mRNA levels of most other genes in the GCS were similar to those in the WT corolla. Taken together, these data suggest that the partial loss of FBP2 expression does not shift global gene expression in the corolla segments of the GCS flower toward that of calyx, even though calyx-like morphogenesis was established in the corolla segments.
Planta 09/2008; 228(3):401-9. · 3.00 Impact Factor
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ABSTRACT: Small interfering RNA (siRNA) species with 21-25 nucleotides in length guide mRNA cleavage, translational arrest, and heterochromatin formation in RNA interference (RNAi). To delineate the target region of RNAi, a construct harboring a transcriptional fusion between parts of the target mRNA and the beta-glucuronidase gene was biolistically delivered into tobacco leaves showing an RNAi phenotype and the assay sequence was transiently expressed. The RNAi effect was monitored by amplification of this chimeric transcript. By using this assay method, we addressed the transitive RNA silencing of a tobacco endoplasmic reticulum omega-3 fatty acid desaturase gene (NtFAD3). In the NtFAD3 RNAi plants, the target region of RNAi was restricted in the inducer region corresponding to a stem sequence of the hairpin double-stranded RNA, indicating that endogenous NtFAD3 mRNA was not a template for an RNA-dependent RNA polymerase. The secondary NtFAD3 siRNAs were produced in the crossbred plants between the NtFAD3 overexpressed plant and the NtFAD3 RNAi plant. Similarly, the secondary siRNAs were generated in the systemically silenced scion. Although these secondary siRNAs originated preferentially from the 3' region downstream of the inducer region, the secondary siRNAs produced in the silenced scion (non-cell autonomous secondary siRNAs) resulted in the strong degradation of the target mRNA, but the secondary siRNAs in the crossbred plants (cell-autonomous secondary siRNAs) showed limited RNA degradation activity. These results showed that this in vivo assay for determination of RNAi efficiency is a useful tool to delineate RNAi mechanisms.
Plant Molecular Biology 05/2007; 63(6):803-13. · 4.15 Impact Factor
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[show abstract]
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ABSTRACT: Anthocyanin synthesis and chlorophyll degradation in regenerated torenia (Torenia fournieri Linden ex Fourn.) shoots induced by osmotic stress with 7% sucrose were examined to identify the genes regulating the underlying molecular mechanism. To achieve this, suppression subtractive hybridization was performed to enrich the cDNAs of genes induced in anthocyanin-synthesizing and chlorophyll-degrading regenerated shoots. The nucleotide sequences of 1,388 random cDNAs were determined, and these were used in the preparation of cDNA microarrays for high-throughput screening. From 1,056 cDNAs analyzed in the microarrays, 116 nonredundant genes were identified, which were up regulated by 7% sucrose to induce anthocyanin synthesis and chlorophyll degradation in regenerated shoots. Of these, eight genes were selected and RNAi transformants prepared, six of which exhibited anthocyanin synthesis inhibition and/or chlorophyll degradation in their leaf discs. Notably, the RNAi transformants of the glucose 6-phosphate/phosphate translocator gene displayed inhibition both of anthocyanin synthesis and chlorophyll degradation in both leaf discs and regenerated shoots. There was also less accumulation of anthocyanin in the petals, and flowering time was shortened. The genes we identified as being up-regulated in the regenerated torenia shoots may help further elucidate the molecular mechanism underlying the induction of anthocyanin synthesis and chlorophyll degradation.
Journal of Plant Research 06/2006; 119(3):217-30. · 1.75 Impact Factor
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Analytical Biochemistry 10/2005; 344(2):284-6. · 3.00 Impact Factor
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ABSTRACT: Petunia cDNA libraries were prepared from whole flower parts (including buds), pollen, and pollen tubes to generate expressed sequence tags (ESTs). A total of 7001 random clones were subjected to unidirectional sequencing, resulting in identification of 611 groups of related sequences and 2410 singletons. Highly conserved 1098 ESTs were functionally assigned. ESTs encoding proteins involved in the calcium-dependent signal pathway and in cell wall metabolism such as pectin degradation and modification were frequently found in the pollen and pollen tube libraries. The 2976 cDNA clones from the bud/flower cDNA libraries were used for the construction of microarrays. The 112 functionally annotated genes were up-regulated in the buds just before opening, including the genes for anthocyanin pigmentation and protein degradation. These ESTs and microarrays will serve the analysis of floral traits of petunias.
Plant Science.
