Daisuke Yamana

Sustained repression and translocation of Ntcp and expression of Mrp4 for cholestasis after rat 90% partial hepatectomy

BACKGROUND & AIMS To clarify the mechanism of persistent cholestasis after massive hepatectomy, the relationship between such cholestasis and the expression and localization of organic anion transporters for bile acids was examined in a rat model. METHODS Male Sprague-Dawley rats were subjected to 90% hepatectomy, and tissues were harvested at 0, 1, 3, and 7 days for microarray analysis, quantitative real-time polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry to examine the expression of multidrug resistance protein 4 (Mrp4), bile salt export pump (Bsep), and sodium-dependent taurocholate cotransporting polypeptide (Ntcp). RESULTS Persistently elevated levels of serum bile acids were observed at days 3 and 7. RT-PCR and Western blotting indicated that the expression of Mrp4, a bile acid export pump located in the basolateral membrane, was increased at day 3. The expression of Ntcp, a transporter used to uptake bile acids from the sinusoids, was significantly decreased throughout the period. The levels of Bsep, an export pump localized to the canalicular membrane, were unchanged. Immunohistochemistry revealed the localization of Mrp4 and Bsep in the basolateral and canalicular membranes, respectively. On the other hand, at days 3 and 7, Ntcp was localized in the cytoplasm and was hardly detected in the basolateral membrane. CONCLUSIONS These results suggested that the sustained repression and translocation of Ntcp and the expression of Mrp4 at the basolateral membrane seem to be responsible for the high blood bile acids levels after massive hepatectomy.

Lectin-like Receptor Ly49s3 on Dendritic Cells Contributes to the Differentiation of Regulatory T Cells in the Rat Thymus

Naturally occurring regulatory T cells (nTregs), important for immune regulation and the maintenance of self-tolerance, develop in the thymus. The Hirosaki hairless rat (HHR), derived from the Sprague–Dawley rat (SDR), was shown to have decreased peripheral lymphocyte number, small thymus, and leukocyte infiltration in its dermis. In the HHR thymus, the medulla was underdeveloped and nTreg number was decreased. Array comparative genome hybridization revealed the deletion of an NK cell lectin-like receptor gene, Ly49s3, detecting MHC class I molecules on target cells, in the chromosome 4q42 region in HHRs. The gene was expressed in thymic conventional dendritic cells (cDCs) in SDRs, but not in HHRs. When CD4–single-positive or CD4+CD8−CD25− thymocytes were cultured with thymic cDCs, the expression of nTreg marker genes was lower when these cells were from HHRs than from SDRs, suggesting that HHR cDCs are deficient in the ability to induce and maintain nTreg differentiation. Expression of the genes was recovered when Ly49s3 was expressed on HHR thymic cDCs. Expression levels of MHC class II genes, presumably from cDCs, were parallel to those of nTreg marker genes in mixed-cell cultures. However, in the presence of an anti-MHC class I Ab, blocking interaction between Ly49s3 and MHC class I molecules, the expression of the former genes was upregulated, whereas the latter was downregulated. These results suggest that Ly49s3 contributes to nTreg regulation along with MHC class II molecules, whose effects alone are insufficient, and loss of Ly49s3 from thymic cDCs is the reason for the nTreg deficiency in HHRs.

Glutathione S-transferase A4 is a positive marker for rat hepatic foci induced by clofibrate and genotoxic carcinogens

Peroxisome proliferators (PP), including clofibrate (CF), are non‐genotoxic rodent carcinogens, and oxidative DNA damages are suggested as a causative event for carcinogenesis. Gene expression profiles differ between hepatic lesions induced by PP and genotoxic carcinogens. Our previous study revealed that expression of L‐bifunctional enzyme (enoyl‐CoA hydratase/3‐hydroxyacyl‐CoA dehydrogenase, BE) was repressed in preneoplastic lesions induced by PP, whereas it was enhanced in the surrounding tissues. In the present study, we immunohistochemically examined expression of the specific glutathione S‐transferase (GST) form, GST‐A4, which detoxifies 4‐hydroxy‐alkenal, the end‐product of lipid peroxides, and nuclear factor‐erythroid 2‐related factor 2 (Nrf2), a transcription factor for many genes encoding drug‐metabolizing enzymes and defending enzymes against oxidative stress, during rat hepatocarcinogenesis induced by CF and genotoxic carcinogens. GST‐A4 and Nrf2 were not expressed in BE‐negative foci at 8 weeks of CF administration, but were expressed in the foci at 60 weeks. GST‐A4‐positive foci appeared at later stages than BE‐negative foci, but its localization was coincidental with that of the latter foci. The areas of GST‐A4‐positive foci were larger than those of BE‐negative foci without GST‐A4 expression. Most GST‐A4‐positive foci were also positive for Nrf2. In rat livers induced by genotoxic carcinogens, GST‐P‐negative foci as well as GST‐P‐positive foci were demonstrated. GST‐A4 and Nrf2 were expressed in GST‐P‐negative foci, whereas they were not expressed in most GST‐P‐positive foci. Thus, GST‐A4‐positive foci developed in rat livers by CF and genotoxic carcinogen administration, indicating that the enzyme is a positive marker for hepatic foci induced by these different carcinogens.

Histone acetylation and steroid receptor coactivator expression during clofibrate‐induced rat hepatocarcinogenesis

Peroxisome proliferators (PPs), non‐genotoxic rodent carcinogens, cause the induction of the peroxisomal fatty acid β‐oxidation system, including bifunctional enzyme (BE) and 3‐ketoacyl‐CoA thiolase (TH), in the liver. GST M1 gene is polymorphic in Sprague–Dawley rats, NC‐ and KS‐type. The KS‐type rats showed enhanced susceptibility to ethyl‐α‐chlorophenoxyisobutyrate (clofibrate, CF), one of the PPs. The degree of BE induction was higher in the KS‐type and preneoplastic foci developed after 6–8 weeks of treatment, whereas no foci developed in the NC‐type. In the preset study, factors involved in different BE inducibility were investigated. There were no differences in hepatic peroxisome proliferator‐activated receptor (PPAR) α levels between them. Among various coactivators for PPARα, only steroid receptor coactivator (SRC)‐3 level was higher in the KS‐type. To investigate the association between PPARα and SRC‐3 or other proteins, nuclear extracts from CF‐treated livers were applied to a PPARα column. In the KS‐type, 110, 72, and 42 kDa proteins were bound and these were identified as SRC‐3, BE, and TH, respectively. EMSA supported the binding of these proteins to PPARα associated to the BE enhancer in CF‐treated KS‐type, but not in the NC‐type. Histone H3 acetylation was increased 11‐fold in the KS‐type by CF treatment but not in the NC‐type. As BE and TH are responsible for acetyl‐CoA production and SRC‐3 possesses a histone acetyltransferase activity, these results suggest that enhanced BE induction in the KS‐type livers is due to acetylation‐mediated transcriptional activation and epigenetic mechanisms might be involved in CF‐induced rat hepatocarcinogenesis.

Decrease of hepatic stellate cells in rats with enhanced sensitivity to clofibrate‐induced hepatocarcinogenesis

To examine the possible involvement of nonparenchymal cells in the development of preneoplastic hepatic lesions induced by clofibrate (CF), alterations of these cells were investigated immunohistochemically in glutathione S‐transferase M1 gene polymorphic rats (KS and NC types) with different cancer susceptibilities. After CF administration for 8 weeks, α‐smooth muscle actin (α‐SMA)‐positive hepatic stellate cells (HSC) were markedly decreased in sensitive KS‐type rats, but not in the NC‐type rats. Kupffer cells were decreased with similar extents between them. The sinusoidal endothelial cells were not changed in either type. The other markers for HSC, vimentin and CRBP1, also confirmed the decrease of HSC in the KS type. The decrease of HSC was not observed at 4 weeks of CF administration. Preneoplastic peroxisomal bifunctional enzyme‐negative foci were detected in the KS‐type rats at 8 weeks of CF administration, but not at 4 weeks. Human HSC were cultured in the presence of clofibric acid and expression of most HSC marker genes, such as vimentin and α‐SMA (ACTA2), evaluated by a microarray, was not altered by the treatment, suggesting that HSC loss in the KS‐type rats was not due to the direct toxic effect of CF. The expression levels of most HSC marker genes were low in both control and CF‐treated rat livers. A possible link between HSC loss and the development of preneoplastic hepatic foci is discussed. (Cancer Sci 2011; 102: 735–741)