Yoshitaka Tamai

Identification of membrane-type receptor for bile acids (M-BAR)

Bile acids play an essential role in the solubilization and absorption of dietary fat and lipid-soluble vitamins. Bile acids also modulate the transcription of various genes for enzymes and transport proteins for their own and cholesterol homeostasis through binding to nuclear receptors. Here we report a novel category of bile acid receptor, a membrane-type G protein-coupled receptor (GPCR), BG37. Bile acids induced rapid and dose-dependent elevation of intracellular cAMP levels in BG37-expressing cells, but not in mock-transfected cells, independently of nuclear receptor expression. The rank order of potency of various bile acids for BG37-expressing cells was different from that for the nuclear receptor-mediated response. These observations demonstrate the presence of two independent signaling pathways for bile acids; membrane-type GPCR for rapid signaling and nuclear receptors for delayed signaling. Expression of BG37 was detected in various specific tissues, suggesting its physiological role, although it remains to be further characterized.

Formation of Multiple Hearts in Mice following Deletion of β-catenin in the Embryonic Endoderm

Using Cre/loxP, we conditionally inactivated the beta-catenin gene in cells of structures that exhibit important embryonic organizer functions: the visceral endoderm, the node, the notochord, and the definitive endoderm. Mesoderm formation was not affected in the mutant embryos, but the node was missing, patterning of the head and trunk was affected, and no notochord or somites were formed. Surprisingly, deletion of beta-catenin in the definitive endoderm led to the formation of multiple hearts all along the anterior-posterior (A/P) axis of the embryo. Ectopic hearts developed in parallel with the normal heart in regions of ectopic Bmp2 expression. We provide evidence that ablation of beta-catenin in embryonic endoderm changes cell fate from endoderm to precardiac mesoderm, consistent with the existence of bipotential mesendodermal progenitors in mouse embryos.

Colonic polyposis caused by mTOR-mediated chromosomal instability in Apc+/Δ716 Cdx2+/- compound mutant mice

The mammalian homeobox transcription factor CDX2 has key roles in intestinal development and differentiation. Heterozygous Cdx2 mice develop one or two benign hamartomas in the proximal colon, whereas heterozygous ApcΔ716 mice develop numerous adenomatous polyps, mostly in the small intestine. Here we show that the colonic polyp number is about six times higher in Apc+/Δ716 Cdx2+/− compound mutant mice. Levels of both APC and CDX2 were significantly lower in the distal colon, which caused high anaphase bridge index (ABI) associated with a higher frequency of loss of heterozygosity (LOH) at Apc. In cultured rat intestinal epithelial and human colon cancer cell lines, suppression of CDX2 by antisense RNA caused marked increases in ABI and chromosomal aberrations. This was mediated by stimulation of the mTOR pathway, causing translational deregulation and G1-S acceleration, associated with low levels of p27 and activation of cyclin E–Cdk2. We obtained similar results in the colonic mucosa of Apc+/Δ716 Cdx2+/− compound mutant mice. Forced activation of mTOR through upstream regulator Akt also increased ABI in colon cancer cells. High ABI in all cell lines was suppressed by mTOR inhibitors LY294002 and rapamycin. These results suggest that reduced expression of CDX2 is important in colon tumorigenesis through mTOR-mediated chromosomal instability.

Targeted deletion of keratins 18 and 19 leads to trophoblast fragility and early embryonic lethality

It has been reported previously that keratin 8 (K8)‐deficient mice of one strain die from a liver defect at around E12.5, while those of another strain suffer from colorectal hyperplasia. These findings have generated considerable confusion about the function of K8, K18 and K19 that are co‐expressed in the mouse blastocyst and internal epithelia. To resolve this issue, we produced mice doubly deficient for K18 and K19 leading to complete loss of keratin filaments in early mouse development. These embryos died at around day E9.5 with 100% penetrance. The absence of keratins caused cytolysis restricted to trophoblast giant cells, followed by haematomas in the trophoblast layer. Up to that stage, embryonic development proceeded unaffected in the absence of keratin filaments. K18/19‐deficient mouse embryos die earlier than any other intermediate filament knockouts reported so far, suggesting that keratins, in analogy to their well established role in epidermis, are essential for the integrity of a specialized embryonic epithelium. Our data also offer a rationale to explore the involvement of keratin mutations in early abortions during human pregnancies.

Hepatic De Novo Lipogenesis Is Present in Liver-Specific ACC1-Deficient Mice

ABSTRACT Acetyl coenzyme A (acetyl-CoA) carboxylase (ACC) catalyzes carboxylation of acetyl-CoA to form malonyl-CoA. In mammals, two isozymes exist with distinct physiological roles: cytosolic ACC1 participates in de novo lipogenesis (DNL), and mitochondrial ACC2 is involved in negative regulation of mitochondrial β-oxidation. Since systemic ACC1 null mice were embryonic lethal, to clarify the physiological role of ACC1 in hepatic DNL, we generated the liver-specific ACC1 null mouse by crossbreeding of an Acc1lox(ex46) mouse, in which exon 46 of Acc1 was flanked by two loxP sequences and the liver-specific Cre transgenic mouse. In liver-specific ACC1 null mice, neither hepatic Acc1 mRNA nor protein was detected. However, to compensate for ACC1 function, hepatic ACC2 protein and activity were induced 1.4 and 2.2 times, respectively. Surprisingly, hepatic DNL and malonyl-CoA were maintained at the same physiological levels as in wild-type mice. Furthermore, hepatic DNL was completely inhibited by an ACC1/2 dual inhibitor, 5-tetradecyloxyl-2-furancarboxylic acid. These results strongly demonstrate that malonyl-CoA from ACC2 can access fatty acid synthase and become the substrate for the DNL pathway under the unphysiological circumstances that result with ACC1 disruption. Therefore, there does not appear to be strict compartmentalization of malonyl-CoA from either of the ACC isozymes in the liver.

Requirement for tumor suppressor Apc in the morphogenesis of anterior and ventral mouse embryo.

Tumor suppressor Apc (adenomatous polyposis coli) is implicated in the Wnt signaling pathway that is involved in the early embryonic development and tumorigenesis in vertebrates. While the heterozygous null mutant mice develop intestinal polyps, the homozygous embryos die before gastrulation. To investigate the role of Apc in later embryonic development, we constructed a novel hypomorphic Apc allele whose expression was attenuated by approximately 80%. In the hypomorphic Apc homozygous ES cells, reduction in Apc expression caused beta-catenin accumulation and Wnt signaling activation. The homozygous mutant mouse embryos survived 3 days longer than the null mutant embryos. Interestingly, they showed anterior truncation, partial axis duplication, and defective ventral morphogenesis. To determine the tissues where Apc functions for anterior and ventral morphogenesis, we constructed chimeric embryos whose epiblast was derived predominantly from the Apc hypomorphic homozygous cells but the visceral endoderm was from the wild type. Although these chimeric embryos still showed some anterior defects, their ventral morphogenesis was rescued. In addition, marker studies indicated that the axial mesendoderm was also defective in the homozygous embryos. Our results provide genetic evidence that expression of Apc at the normal level is essential for both anterior and ventral development, in the epiblast derivatives and visceral endoderm.

Role of MGAT2 and DGAT1 in the release of gut peptides after triglyceride ingestion

Triglyceride ingestion releases gut peptides from enteroendocrine cells located in the intestinal epithelia and provides feedback regulations of gastrointestinal function. The precise mechanisms sensing lipids in the intestinal wall, however, are not well characterized. In the current study, we investigated the release of gut peptides following oral triglyceride loading in mice deficient for monoacylglycerol acyltransferase 2 (MGAT2KO) and diacylglycerol acyltransferase 1 (DGAT1KO), enzymes that sequentially re-synthesize triglyceride to secrete as chylomicron at the small intestine. In wild-type (Wt) mice, oral triglyceride loading resulted in hypertriglycemia. In addition, plasma glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) were significantly increased 30 min after triglyceride loading, before decaying in 2h. In MGAT2KO and DGAT1KO mice, oral triglyceride loading did not result in hypertriglycemia and the increase in GIP was significantly suppressed in both KO mouse strains. In contrast, the increases in plasma GLP-1 and PYY in both KO mouse strains were comparable to Wt mice 30 min after triglyceride loading, however, they remained elevated in DGAT1KO mice even 2h after triglyceride loading. In parallel to the changes in GLP-1 and PYY, gastric emptying was delayed after oral triglyceride loading in MGAT2KO mice comparably to Wt type mice and was further delayed in DGAT1KO mice. STC-1 and GLUTag, GLP-1-producing intestinal endocrine L-cell lines, displayed a significant level of DGAT1 activity but not MGAT activity. These findings suggest that synthesis and/or secretion of triglyceride-rich lipoproteins play an important role in the release of GIP. Moreover, DGAT1 may directly regulate the release of GLP-1 and PYY in L-cells.

Cloning and expression of the mouse Pse gene encoding a novel Ets family member.

Human prostate-specific Ets (hPSE) is a novel Ets transcription factor and is exclusively expressed in human prostate glandular epithelium. To explore the role of PSE, we cloned the mouse Pse (mPse) and examined its pattern of expression. A sequence analysis indicated that mPse contains a conserved carboxy-terminal ETS DNA-binding domain and central Pointed domain, and the overall amino acid sequence shares 86% identity with that of hPSE. The ETS DNA-binding domain is highly conserved between human and mouse (98.8% sequence identity) and is similar to Drosophila dets4 (76.7% identity), but not similar to other Ets factors. A Northern blotting analysis revealed that mPse shows organ-specific expression. An in situ hybridization analysis of the prostate and intestine showed that mPse transcripts were present in their epithelial cells. mPse transactivates the promoter of the MASPIN gene in transient transfection assay. These results suggest that mPse encodes a novel Ets family member and is expressed in epithelial cells of restricted organs.

Keratin-8 null mice have different gallbladder and liver susceptibility to lithogenic diet-induced injury

Keratin transgenic mouse models and the association of human keratin mutations with liver disease highlight the importance of keratins in protecting the liver from environmental insults, but little is known regarding keratins and their function in the gallbladder. We characterized keratin expression pattern and filament organization in normal and keratin polypeptide-8 (K8)-null, K18-null and K19-null gallbladders, and examined susceptibility to liver and gallbladder injury induced by a high-fat lithogenic diet (LD) in K8-null mice. The major keratins of normal mouse gallbladder are K8>K19>K18 which become markedly depleted in K8-null mice with minor K18/K19 remnants and limited K7 over-expression. Compensatory K18/K20 protein and RNA overexpression occur in K19-null but not in K18-null gallbladders, probably because of the higher levels of K19 than K18 in normal gallbladder. LD challenge causes more severe liver injury in K8-null than wild-type mice without altering keratin protein levels. In contrast, wild-type and K8-null gallbladders are equally susceptible to LD-induced injury and stone formation, but wild-type gallbladders do overexpress keratins upon LD challenge. LD-induced injury triggers keratin hyperphosphorylation in wild-type livers and gallbladders. Hence, mouse gallbladder K8/K18/K19 expression is induced in response to cholelithiasis injury. A high-fat LD increases the susceptibility of K8-null mice to liver but not gallbladder injury, which suggests that keratin mutations may increase the risk of liver damage in patients with steatohepatitis. Differences between K8-null mouse gallbladder and hepatocyte susceptibility to injury may be related to their minimal versus absent keratin expression, respectively.

Serum components and activated Ha‐ras antagonize expression of perivenous marker genes stimulated by β‐catenin signaling in mouse hepatocytes

Hepatocytes of the periportal and perivenous zones of the liver lobule show marked differences in the contents and activities of many enzymes and other proteins. Previous studies from our and other groups have pointed towards an important role of β‐catenin‐dependent signaling in the regulation of expression of genes encoding proteins with preferential perivenous localization, whereas, in contrast, signaling through Ras‐dependent pathway(s) may induce a ‘periportal’ phenotype. We have now conducted a series of experiments to further investigate this hypothesis. In transgenic mice with scattered expression of an activated Ha‐ras (Ha‐rasG12V) mutant in liver, expression of the perivenous markers glutamine synthetase and two cytochrome P450 isoforms was completely abolished in those hepatocytes demonstrating constitutively activated extracellular signal‐regulated kinase activity, even though they were located directly adjacent to central veins. Similarly, incubation of primary hepatocytes or hepatoma cells with increasing amounts of serum caused a concentration‐dependent attenuation of expression of perivenous marker mRNAs, whereas the expression of periportal markers was increased. The inhibitory effect of high amounts of serum on the expression of perivenous markers was also observed if their expression was stimulated by activation of β‐catenin signaling, and comparable inhibitory effects were seen in cells stably transfected with a T‐cell factor/lymphoid‐enhancing factor‐driven luciferase reporter. Epidermal growth factor could partly mimic serum effects in hepatoma cells, and its effect could be blocked by an inhibitor of extracellular signal‐regulated kinase activity. These data suggest that activation of the Ras/mitogen‐activated protein kinase (extracellular signal‐regulated kinase) pathway favors periportal gene expression while simultaneously antagonizing a perivenous phenotype of hepatocytes.