[Show abstract][Hide abstract] ABSTRACT: Previously, we proposed a rare autosomal recessive inherited enteropathy characterized by persistent blood and protein loss from the small intestine as chronic nonspecific multiple ulcers of the small intestine (CNSU). By whole-exome sequencing in five Japanese patients with CNSU and one unaffected individual, we found four candidate mutations in the SLCO2A1 gene, encoding a prostaglandin transporter. The pathogenicity of the mutations was supported by segregation analysis and genotyping data in controls. By Sanger sequencing of the coding regions, 11 of 12 other CNSU patients and 2 of 603 patients with a diagnosis of Crohn's disease were found to have homozygous or compound heterozygous SLCO2A1 mutations. In total, we identified recessive SLCO2A1 mutations located at seven sites. Using RT-PCR, we demonstrated that the identified splice-site mutations altered the RNA splicing, and introduced a premature stop codon. Tracer prostaglandin E2 uptake analysis showed that the mutant SLCO2A1 protein for each mutation exhibited impaired prostaglandin transport. Immunohistochemistry and immunofluorescence analyses revealed that SLCO2A1 protein was expressed on the cellular membrane of vascular endothelial cells in the small intestinal mucosa in control subjects, but was not detected in affected individuals. These findings indicate that loss-of-function mutations in the SLCO2A1 gene encoding a prostaglandin transporter cause the hereditary enteropathy CNSU. We suggest a more appropriate nomenclature of "chronic enteropathy associated with SLCO2A1 gene" (CEAS).
[Show abstract][Hide abstract] ABSTRACT: Acyl-coenzyme A: cholesterol acyltransferase (ACAT) catalyzes the conversion of free cholesterol (FC) to cholesterol ester, which prevents excess accumulation of FC. We recently found that FC accumulation in hepatic stellate cells (HSCs) plays a role in progression of liver fibrosis, but the effect of ACAT1 on liver fibrosis has not been clarified. In this study, we aimed to define the role of ACAT1 in the pathogenesis of liver fibrosis.
ACAT1-deficient and wild-type mice, or Toll-like receptor 4 (TLR4)(-/-)ACAT1(+/+) and TLR4(-/-)ACAT1(-/-) mice were subjected to bile duct ligation (BDL) for 3 weeks or were given carbon tetrachloride (CCl4) for 4 weeks to induce liver fibrosis.
ACAT1 was the major isozyme in mice and human primary HSCs, and ACAT2 was the major isozyme in mouse primary hepatocytes and Kupffer cells. ACAT1 deficiency significantly exaggerated liver fibrosis in the mouse models of liver fibrosis, without affecting the degree of hepatocellular injury or liver inflammation, including hepatocyte apoptosis or Kupffer cell activation. ACAT1 deficiency significantly increased FC levels in HSCs, augmenting TLR4 protein and downregulating expression of transforming growth factor-β (TGFβ) pseudoreceptor Bambi (bone morphogenetic protein and activin membrane-bound inhibitor), leading to sensitization of HSCs to TGFβ activation. Exacerbation of liver fibrosis by ACAT1 deficiency was dependent on FC accumulation-induced enhancement of TLR4 signaling.
ACAT1 deficiency exaggerates liver fibrosis mainly through enhanced FC accumulation in HSCs. Regulation of ACAT1 activities in HSCs could be a target for treatment of liver fibrosis.
Journal of Hepatology 03/2014; 61(1). DOI:10.1016/j.jhep.2014.03.018 · 11.34 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Although nonalcoholic steatohepatitis (NASH) is associated with hypercholesterolemia, the underlying mechanisms of this association have not been clarified. We aimed to elucidate the precise role of cholesterol in the pathophysiology of NASH. C57BL/6 mice were fed a control, high-cholesterol (HC), methionine-choline-deficient (MCD), or MCD+HC diet for 12 weeks or a control, HC, high-fat (HF), or HF+HC diet for 24 weeks. Increased cholesterol intake accelerated liver fibrosis in both the mouse models without affecting the degree of hepatocellular injury or Kupffer cell activation. The major causes of the accelerated liver fibrosis involved free cholesterol (FC) accumulation in hepatic stellate cells (HSCs), which increased Toll-like receptor 4 protein (TLR4) levels through suppression of the endosomal-lysosomal degradation pathway of TLR4, and thereby sensitized the cells to transforming growth factor (TGF)β-induced activation by down-regulating the expression of bone morphogenetic protein and activin membrane-bound inhibitor. Mammalian-cell cholesterol levels are regulated by way of a feedback mechanism mediated by sterol regulatory element-binding protein 2 (SREBP2), maintaining cellular cholesterol homeostasis. Nevertheless, HSCs were sensitive to FC accumulation because the high intracellular expression ratio of SREBP cleavage-activating protein (Scap) to insulin-induced gene (Insig) disrupted the SREBP2-mediated feedback regulation of cholesterol homeostasis in these cells. HSC activation subsequently enhanced the disruption of the feedback system by Insig-1 down-regulation. In addition, the suppression of peroxisome proliferator-activated receptor γ signaling accompanying HSC activation enhanced both SREBP2 and microRNA-33a signaling. Consequently, FC accumulation in HSCs increased and further sensitized these cells to TGFβ-induced activation in a vicious cycle, leading to exaggerated liver fibrosis in NASH.
These characteristic mechanisms of FC accumulation in HSCs are potential targets to treat liver fibrosis in liver diseases including NASH.
[Show abstract][Hide abstract] 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.
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.
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.
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; 43(6). DOI:10.1111/j.1872-034X.2012.01111.x · 2.74 Impact Factor
[Show abstract][Hide abstract] 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. DOI:10.1016/j.jhep.2012.05.013 · 11.34 Impact Factor
[Show abstract][Hide abstract] 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. DOI:10.1016/S0016-5085(12)60577-0 · 1.83 Impact Factor
[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.
[Show abstract][Hide abstract] 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.
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–
–Blood type antigen
Journal of Gastroenterology 09/2011; 46(9):1056-1063. DOI:10.1007/s00535-011-0425-7 · 4.52 Impact Factor