IHH Cells Research Articles

Hepatitis C Core Protein Induces a Genotype-Specific Susceptibility of Hepatocytes to TNF-Induced Death In Vitro and In Vivo.

Hepatitis C virus (HCV) core protein is a multifunctional protein that is involved in the proliferation, inflammation, and apoptosis mechanism of hepatocytes. HCV core protein genetic variability has been implicated in various outcomes of HCV pathology and treatment. In the present study, we aimed to analyze the role of the HCV core protein in tumor necrosis factor α (TNFα)-induced death under the viewpoint of HCV genetic variability. Immortalized hepatocytes (IHH), and not the Huh 7.5 hepatoma cell line, stably expressing HCV subtype 4a and HCV subtype 4f core proteins showed that only the HCV 4a core protein could increase sensitivity to TNFα-induced death. Development of two transgenic mice expressing the two different core proteins under the liver-specific promoter of transthyretin (TTR) allowed for the in vivo assessment of the role of the core in TNFα-induced death. Using the TNFα-dependent model of lipopolysaccharide/D-galactosamine (LPS/Dgal), we were able to recapitulate the in vitro results in IHH cells in vivo. Transgenic mice expressing the HCV 4a core protein were more susceptible to the LPS/Dgal model, while mice expressing the HCV 4f core protein had the same susceptibility as their littermate controls. Transcriptome analysis in liver biopsies from these transgenic mice gave insights into HCV core molecular pathogenesis while linking HCV core protein genetic variability to differential pathology in vivo.

Generation of an Ihh-mKate2-Dre knock-in mouse line.

Indian hedgehog (Ihh), a member of the Hh family, plays important roles in vertebrate development and homeostasis. To improve our understanding of the function of Ihh-expressing cells and their progeny as well, we generate an Ihh-mKate2tomm20 -Dre knock-in mouse line that can label Ihh-positive cells with a fluorescence protein mKate2 and trace Ihh-positive cells and their progeny via Dre-mediated recombination. Consistent with previous reports, we verified Ihh expression in hypertrophic chondrocytes of growth plate and granulosa cells of ovarian follicles by mKate2 immunostaining, and meanwhile confirmed Dre activity in these cells via a Dre reporter mouse line Rosa26-confetti2. We also found, for the first time, that Ihh can mark some cell types, including retinal ganglion cells, Purkinje cells, and gallbladder epithelial cells. Taken together, the Ihh-mKate2tomm20 -Dre mouse is a genetic tool for examining the precise expression profile of Ihh and tracing Ihh-expressing cells and their progeny.

Acetyl-CoA metabolism drives epigenome change and contributes to carcinogenesis risk in fatty liver disease

BackgroundThe incidence of non-alcoholic fatty liver disease (NAFLD)-associated hepatocellular carcinoma (HCC) is increasing worldwide, but the steps in precancerous hepatocytes which lead to HCC driver mutations are not well understood. Here we provide evidence that metabolically driven histone hyperacetylation in steatotic hepatocytes can increase DNA damage to initiate carcinogenesis.MethodsGlobal epigenetic state was assessed in liver samples from high-fat diet or high-fructose diet rodent models, as well as in cultured immortalized human hepatocytes (IHH cells). The mechanisms linking steatosis, histone acetylation and DNA damage were investigated by computational metabolic modelling as well as through manipulation of IHH cells with metabolic and epigenetic inhibitors. Chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) and transcriptome (RNA-seq) analyses were performed on IHH cells. Mutation locations and patterns were compared between the IHH cell model and genome sequence data from preneoplastic fatty liver samples from patients with alcohol-related liver disease and NAFLD.ResultsGenome-wide histone acetylation was increased in steatotic livers of rodents fed high-fructose or high-fat diet. In vitro, steatosis relaxed chromatin and increased DNA damage marker γH2AX, which was reversed by inhibiting acetyl-CoA production. Steatosis-associated acetylation and γH2AX were enriched at gene clusters in telomere-proximal regions which contained HCC tumour suppressors in hepatocytes and human fatty livers. Regions of metabolically driven epigenetic change also had increased levels of DNA mutation in non-cancerous tissue from NAFLD and alcohol-related liver disease patients. Finally, genome-scale network modelling indicated that redox balance could be a key contributor to this mechanism.ConclusionsAbnormal histone hyperacetylation facilitates DNA damage in steatotic hepatocytes and is a potential initiating event in hepatocellular carcinogenesis.

Evaluation of the expression of fatty acid synthase and O-GlcNAc transferase in patients with liver cancer by exploration of transcriptome databases and experimental approaches.

Tumor occurrence and development are closely related to metabolism abnormalities. One of the metabolic networks that is dysregulated during carcinogenesis is the fatty acid synthesis pathway, which is mainly controlled by fatty acid synthase (FASN). We previously demonstrated in proliferating HepG2 liver cancer cells that FASN expression depends on the catalytic activity of O-GlcNAc transferase (OGT) and the activation of the mechanistic/mammalian target of rapamycin (mTOR) pathway. The aim of the present study was to go further in these investigations by analyzing datasets and tissues of patients with liver cancer. To that purpose, transcriptome databases were explored, and reverse transcription-quantitative PCR, western blotting and immunohistochemistry were used. Database analyses revealed that FASN and OGT gene expression was higher in certain cancer tissues, including liver hepatocellular carcinoma, compared with that in non-cancerous tissues. At the protein level, FASN expression was higher in the liver cancer-derived cell lines HepG2 and Hep3B compared with the immortalized human hepatocytes IHH cell line. However, neither the expression of OGT nor of its product O-GlcNAcylation showed any significant difference among the three hepatic cell lines. Subsequently, the expression of FASN and OGT at the protein and mRNA levels was evaluated in human liver cancer and non-tumoral tissues from the same patients with different liver lesions. The results from western blotting demonstrated a significant increase in OGT ands O-GlcNAcylation expression in liver cancer tissues independently of the type of lesion characterizing the non-tumoral counterpart. As previously reported for HepG2 proliferating cells, the protein level of FASN was positively correlated with the activation of mTOR and, although a rather upward trend, a high variability in its expression was monitored between patients. However, the results from immunohistochemistry showed no particular modification for OGT and O-GlcNAcylation expression and a significant increase in FASN expression in cancer tissues compared with that in adjacent non-tumoral tissues. Non-significant changes were observed for FASN and OGT mRNA levels between tumoral and non-tumoral samples, with a high variability between patients. Taken together, these results demonstrated that FASN expression was higher in hepatic cancer tissues in comparison with non-tumoral tissues. Furthermore, OGT expression and activity were shown to vary greatly between cell or cancer type, making any generalization difficult.

The Relevance of SOCS1 Methylation and Epigenetic Therapy in Diverse Cell Populations of Hepatocellular Carcinoma.

The suppressor of cytokine signaling 1 (SOCS1) is a tumor suppressor gene found to be hypermethylated in cancers. It is involved in the oncogenic transformation of cirrhotic liver tissues. Here, we investigated the clinical relevance of SOCS1 methylation and modulation upon epigenetic therapy in diverse cellular populations of hepatocellular carcinoma (HCC). HCC clinical specimens were evaluated for SOCS1 methylation and mRNA expression. The effect of 5-Azacytidine (5-AZA), a demethylation agent, was assessed in different subtypes of HCC cells. We demonstrated that the presence of SOCS1 methylation was significantly higher in HCC compared to peri-HCC and non-tumoral tissues (52% vs. 13% vs. 14%, respectively, p < 0.001). In vitro treatment with a non-toxic concentration of 5-AZA significantly reduced DNMT1 protein expression for stromal subtype lines (83%, 73%, and 79%, for HLE, HLF, and JHH6, respectively, p < 0.01) compared to cancer stem cell (CSC) lines (17% and 10%, for HepG2 and Huh7, respectively), with the strongest reduction in non-tumoral IHH cells (93%, p < 0.001). 5-AZA modulated the SOCS1 expression in different extents among the cells. It was restored in CSC HCC HepG2 and Huh7 more efficiently than sorafenib. This study indicated the relevance of SOCS1 methylation in HCC and how cellular heterogeneity influences the response to epigenetic therapy.

Azaspiracids Increase Mitochondrial Dehydrogenases Activity in Hepatocytes: Involvement of Potassium and Chloride Ions

Background: Azaspiracids (AZAs) are marine toxins that are produced by Azadinium and Amphidoma dinoflagellates that can contaminate edible shellfish inducing a foodborne poisoning in humans, which is characterized by gastrointestinal symptoms. Among these, AZA1, -2, and -3 are regulated in the European Union, being the most important in terms of occurrence and toxicity. In vivo studies in mice showed that, in addition to gastrointestinal effects, AZA1 induces liver alterations that are visible as a swollen organ, with the presence of hepatocellular fat droplets and vacuoles. Hence, an in vitro study was carried out to investigate the effects of AZA1, -2, and -3 on liver cells, using human non-tumor IHH hepatocytes. Results: The exposure of IHH cells to AZA1, -2, or -3 (5 × 10−12–1 × 10−7 M) for 24 h did not affect the cell viability and proliferation (Sulforhodamine B assay and 3H-Thymidine incorporation assay), but they induced a significant concentration-dependent increase of mitochondrial dehydrogenases activity (MTT reduction assay). This effect depends on the activity of mitochondrial electron transport chain complex I and II, being counteracted by rotenone and tenoyl trifluoroacetone, respectively. Furthermore, AZAs-increased mitochondrial dehydrogenase activity was almost totally suppressed in the K+-, Cl−-, and Na+-free media and sensitive to the specific inhibitors of KATP and hERG potassium channels, Na+/K+, ATPase, and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. Conclusions: These results suggest that AZA mitochondrial effects in hepatocytes derive from an imbalance of intracellular levels of K+ and, in particular, Cl− ions, as demonstrated by the selective reduction of toxin effects by CFTR chloride channel inhibition.

Ipriflavone attenuates the degeneration of cartilage by blocking the Indian hedgehog pathway

BackgroundTo determine if ipriflavone, a novel and safe inhibitor of Indian hedgehog (Ihh) signaling, can attenuate cartilage degeneration by blocking the Ihh pathway.MethodsHuman chondrocytes were used to evaluate Ihh signaling, cell proliferation, apoptosis, gene, and protein expression of chondrocytes by cell proliferation and apoptosis assays, real-time qPCR, and Western blotting at 48 h after ipriflavone treatment. Human cartilage explants were further used to validate the cell culture results. The effects of ipriflavone on cartilage degeneration in vivo were assessed using the rat ACLT OA model. Two-month-old male SD rats were randomized into 3 groups (n = 75): (1) sham, (2) ACLT alone, and (3) ACLT+ ipriflavone. Ipriflavone was administered intragastrically at 24 h after ACLT for 6 weeks. The extent of OA progression was evaluated by the OARSI score and immunohistochemistry at 12 weeks after surgery. The Ihh signaling pathway and OA-related genes were quantified by real-time PCR.ResultsCell proliferation in the cells treated with ipriflavone was increased to 36.40% ± 1.32% (5 μM) and 28.54% ± 0.74% (10 μM) from 11.99% ± 0.35% (DMSO) (P < 0.001), and apoptosis was decreased to 12.64% ± 3.7% (5 μM) and 15.18% ± 3.13% (10 μM) from 25.76% ± 5.1% (DMSO) (P < 0.05). Ipriflavone blocked Runx-2 mainly through the Smo-Gli2 pathway. A similar result was found in the cartilage explant culture. Ihh signaling in vivo was inhibited in animals treated with ipriflavone. Safranin-O staining revealed a less cartilage damage with lower OARSI scores (P < 0.05) in the ipriflavone-treated animals compared with untreated animals. The gene expression of Smo and Gli2 was inhibited significantly by ipriflavone (P < 0.05). The OA-related gene and protein type X, MMP-13, and type II collagen-C fragment were reduced, while type II collagen and Agg were increased in the ipriflavone-treated animals (P < 0.05).ConclusionsCatabolic genes were disrupted by blocking the Ihh pathway. This finding suggests that disruption of Ihh signaling with ipriflavone provides chondral protection in rat posttraumatic OA.

Lipid and glucose metabolism in hepatocyte cell lines and primary mouse hepatocytes: a comprehensive resource for in vitro studies of hepatic metabolism.

The liver is a critical tissue for maintaining glucose, fatty acid, and cholesterol homeostasis. Primary hepatocytes represent the gold standard for studying the mechanisms controlling hepatic glucose, lipid, and cholesterol metabolism in vitro. However, access to primary hepatocytes can be limiting, and therefore, other immortalized hepatocyte models are commonly used. Here, we describe substrate metabolism of cultured AML12, IHH, and PH5CH8 cells, hepatocellular carcinoma-derived HepG2s, and primary mouse hepatocytes (PMH) to identify which of these cell lines most accurately phenocopy PMH basal and insulin-stimulated metabolism. Insulin-stimulated glucose metabolism in PH5CH8 cells, and to a lesser extent AML12 cells, responded most similarly to PMH. Notably, glucose incorporation in HepG2 cells were 14-fold greater than PMH. The differences in glucose metabolic activity were not explained by differential protein expression of key regulators of these pathways, for example glycogen synthase and glycogen content. In contrast, fatty acid metabolism in IHH cells was the closest to PMHs, yet insulin-responsive fatty acid metabolism in AML12 and HepG2 cells was most similar to PMH. Finally, incorporation of acetate into intracellular-free cholesterol was comparable for all cells to PMH; however, insulin-stimulated glucose conversion into lipids and the incorporation of acetate into intracellular cholesterol esters were strikingly different between PMHs and all tested cell lines. In general, AML12 cells most closely phenocopied PMH in vitro energy metabolism. However, the cell line most representative of PMHs differed depending on the mode of metabolism being investigated, and so careful consideration is needed in model selection.

PO-094 Persistent Myc activity prevents resolution of liver injury

IntroductionFas/CD95 is a member of the death receptor superfamily that can induce apoptosis and it functions as the key component of the extrinsic death pathway. This study reports on a relevant function of Fas death receptor that can regulate transcription factors p53, MYC, NF-κ B and pro-apoptotic gene PUMA, that are important in intrinsic death pathway in hepatocellular carcinoma (HCC).Material and methodsFor the in vivo study, genes Fas, and FasL from 111 samples (39 HCC, 30 peri-HCC, 31 cirrhosis, and 11 normal) from patients undergoing liver resection without any prior treatments were analysed. For the in vitro study, hepatocytes cell line IHH and human HCC cell lines HepG2, HUH7, JHH6 were used. Apoptosis-induction was performed by using anti-Fas (DX2) at a concentration of 250 ng/ml and 500 ng/ml for 24 hours. Flow cytometry and quantitative real-time PCR were performed to analyse the data.Results and discussionThe expressions of Fas and FasL mRNA were significantly noticed in HCC as compared to normal tissues. FasL mRNA was high expressed in viral-related compared to metabolic-related HCC while Fas was high in HCC tissues without vascular invasion compared to tissues with vascular invasion. For in vitro study, after treatment of anti-CD95, TNF-α was significantly up regulated in HEPG2 compare to IHH cells. Transcription factors p53, MYC and NF-κ B were down regulated in HEPG2 cells while up regulated or no difference change in IHH cells. Pro-apoptotic gene Puma was only up regulated in IHH cell line. Telomerase was up regulated in IHH and significantly down regulated in HEPG2.ConclusionsWe observed a modulation of transcription factors p53, MYC, NF-κ B and pro-apoptotic genes Puma, TNF-α, and Telomerase after induction by CD95 antibody in acute phase treatment for 24 hours. The expression of PUMA and MYC were regulated by the transcription factors p53, thus suggesting the involvement of Fas in p53-dependent apoptosis pathway.

Pharmacokinetics and pharmacodynamics of thiopurines in an in vitro model of human hepatocytes: Insights from an innovative mass spectrometry assay

AimTo apply an innovative LC-MS/MS method to quantify thiopurine metabolites in human hepatocytes and to associate them to cytotoxicity. MethodsImmortalized human hepatocytes (IHH cells) were treated for 48 and 96 h, with 1.4 × 10−4 M azathioprine and 1.1 × 10−3 M mercaptopurine, concentrations corresponding to the IC50 values calculated after 96 h exposure in previous cytotoxicity analysis. After treatments, cells were collected for LC-MS/MS analysis to quantify 11 thiopurine metabolites with different level of phosphorylation and viable cells were counted by trypan blue exclusion assay to determine thiopurines in vitro effect on cell growth and survival. Statistical significance was determined by analysis of variance (ANOVA). ResultsAzathioprine and mercaptopurine had a significant time-dependent cytotoxic effect (p-value ANOVA = 0.012), with a viable cell count compared to controls of 55.5% and 67.5% respectively after 48 h and 23.7% and 36.1% after 96 h; no significant difference could be observed between the two drugs. Quantification of thiopurine metabolites evidenced that the most abundant metabolite was TIMP, representing 57.1% and 40.3% of total metabolites after 48 and 96 h. Total thiopurine metabolites absolute concentrations decreased over time: total mean content decreased from 469.9 pmol/million cells to 83.6 pmol/million cells (p-value ANOVA = 0.0070). However, considering the relative amount of thiopurine metabolites, TGMP content significantly increased from 11.4% cells to 26.4% (p-value ANOVA = 0.017). A significant association between thiopurine effects and viable cell counts could be detected only for MeTIMP: lower MeTIMP concentrations were associated with lower cell survival (p-value ANOVA = 0.011). Moreover, the ratio between MeTIMP and TGMP metabolites directly correlated with cell survival (p-value ANOVA = 0.037). ConclusionDetailed quantification of thiopurine metabolites in a human hepatocytes model provided useful insights on the association between thioguanine and methyl-thioinosine nucleotides with cell viability.

Influence of components of tumour microenvironment on the response of HCT-116 colorectal cancer to the ruthenium-based drug NAMI-A

Solid tumours are constituted of tumour cells, healthy cells recruited from the host tissues and soluble factors released by both these cell types. The present investigation examines the capacity of co-cultures between the HCEC colon epithelial cells and the HCT-116 colorectal cancer cells (mimicking the primary site of tumour growth) and between IHH hepatocytes and the HCT-116 colorectal cancer cells (metastatic site) to influence the effects of NAMI-A (imidazolium trans-imidazoledimethylsulphoxidetetrachloro ruthenate) on the tumour cells themselves. The growth of HCT-116 cells is significantly influenced when the cancer cells are sown on a monolayer of HCEC. The release of soluble factors by the healthy cells promotes, in HCT-116 colorectal cancer cells, the transcription of genes involved in growth, invasion and migration. NAMI-A is not cytotoxic to HCT-116 cells grown on plastics or co-cultured with HCEC or IHH cells, and maintains its ability to control the cell pseudo-metastatic ability, mimicked by the migration in the scratch test. The effects of NAMI-A on HCT-116 migration are supported by its inhibition of the transcription of the ABL-2, ATF-3 and RND-1 genes. In conclusion the study highlights the need of test systems more complex than a single cancer cell culture to study an anticancer drug in vitro and reinforces the hypothesis that NAMI-A targets the ability of the cancer cell to interact with the tumour microenvironment and with the signals that support its metastatic ability.

Role of oxidative stress mediated by glutathione-s-transferase in thiopurines' toxic effects.

Azathioprine (AZA), 6-mercaptopurine (6-MP), and 6-thioguanine (6-TG) are antimetabolite drugs, widely used as immunosuppressants and anticancer agents. Despite their proven efficacy, a high incidence of toxic effects in patients during standard-dose therapy is recorded. The aim of this study is to explain, from a mechanistic point of view, the clinical evidence showing a significant role of glutathione-S-transferase (GST)-M1 genotype on AZA toxicity in inflammatory bowel disease patients. To this aim, the human nontumor IHH and HCEC cell lines were chosen as predictive models of the hepatic and intestinal tissues, respectively. AZA, but not 6-MP and 6-TG, induced a concentration-dependent superoxide anion production that seemed dependent on GSH depletion. N-Acetylcysteine reduced the AZA antiproliferative effect in both cell lines, and GST-M1 overexpression increased both superoxide anion production and cytotoxicity, especially in transfected HCEC cells. In this study, an in vitro model to study thiopurines' metabolism has been set up and helped us to demonstrate, for the first time, a clear role of GST-M1 in modulating AZA cytotoxicity, with a close dependency on superoxide anion production. These results provide the molecular basis to shed light on the clinical evidence suggesting a role of GST-M1 genotype in influencing the toxic effects of AZA treatment.

Molecular insights into the anti-cancer properties of traditional Tibetan medicine Yukyung Karne.

BackgroundYukyung karne (YK) is a traditional Tibetan formulation used for many centuries for the treatment of ovarian cancer. However, the pharmacological basis of its anticancer property is not well understood. In the present study, the anticancer property of YK was investigated in cell culture.MethodsThe growth inhibitory property of YK was evaluated in SKOV6, IHH, HepG2 and HEK293 cell lines using MTT assay. The pro-apoptotic activity of drug was analyzed by terminal deoxynuleotidyl transferase dUTP nick end labeling (TUNEL) and DNA fragmentation assays. Confocal microscopy was used to show the release of cytochrome c and its co-localization with mitochondria with the help of dsRed mitotracker in SKOV6 cells. The inhibition in cell proliferation was also visualized by confocal microscopy after BrDU incorporation. The activation of tumor suppressor p53 was evaluated by Western blotting while VEGF levels in culture supernatant were measured by a colorimetric method.ResultsYK specifically and efficiently induced apoptotic killing of the human ovarian cancer SKOV6 cells as indicated by increased DNA fragmentation and nick end DNA labeling. Confocal microscopy suggested inhibition of cell proliferation and increase in cytochrome c release via perturbation in mitochondrial membrane potential (Δψm). Further, YK up-regulated the expression of tumor suppressor p53 and key cyclin-dependent kinase inhibitor p21, and inhibited VEGF secretion by cells. Interestingly, YK also exhibited a synergy with paclitaxel which is a well-known anti-cancer therapeutic drug.ConclusionsThe pharmacological properties of YK to impose growth arrest and trigger pro-apoptotic death in cells amply justify its usage in primary as well as adjunct therapy for ovarian cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1472-6882-14-380) contains supplementary material, which is available to authorized users.

698P - Cox-2 Status Modulates the Activation of Hedgehog Pathway and Consequently the Expression of Stem Cell Markers in Patients with Pancreatic Ductal Adenocarcinoma

ABSTRACT Aim: Inflammation is a key driver in the development of pancreatic ductal adenocarcinoma (PDAC). Cyclooxygenase-2 (COX-2) and Interleukin (IL)-6 are key modulators of inflammation and act as a potential link between chronic pancreatitis and subsequent carcinogenesis. In this study, we analyzed the association between COX-2 and IL-6 status and the expression of Hedgehog signaling pathway and stem cell markers and their relationship with the clinico-pathological features and prognosis of PDAC patients. Methods: The expressions of COX-2 and IL-6 were examined using immunohistochemistry in 79 resection specimens of PDAC. The expression of the Hedgehog components SHH, IHH, DHH, SMO, PTCH1, PTCH2, and stem cell markers CD24, CD44, OCT3/4 and PROM1 were analyzed by qRT-PCR. The correlation among gene expression levels was performed by the Pearson product moment correlation method. Survival analysis was conducted via Kaplan-Meier product-limit method. Results: 49 patients (62%) had COX-2+ tumors, while 37 patients (47%) had IL-6+ tumors. SHH (p = 0.001), IHH (p = 0.011) and SMO (p = 0.040) were significantly more expressed in patients COX-2+. In addition, IHH was significantly more expressed in patients with IL-6+ tumors (p = 0.040). CD44 expression was associated with DHH (rho = 0.742), IHH (rho = 0.826) and SMO (rho = 0.899). PROM1 expression was associated with DHH (rho = 0.772), IHH (rho = 0.847) and SMO (rho = 0.915). T stage was higher in patients with COX-2+ (p = 0.020), whereas no associations were found between COX-2 and node involvement (p = 0.071) or grading (p = 0.273) as well as between IL-6 expression and the clinico-pathological characteristics of these patients. No significant differences in terms of survival were found based on COX-2 or IL-6 status. Conclusions: Taken together, our data suggest for a model in which COX-2 expression modulates the activation of the Hedgehog pathway and, subsequently, the expression of stemness markers. These data may lead to a better characterization of patients to include in clinical trials in order to develop future therapeutic strategies. Disclosure: All authors have declared no conflicts of interest.

Macrophage‐derived hedgehog ligands promotes fibrogenic and angiogenic responses in human schistosomiasis mansoni

Schistosomiasis mansoni is a major cause of portal fibrosis and portal hypertension. The Hedgehog pathway regulates fibrogenic repair in some types of liver injury. Determine if Hedgehog pathway activation occurs during fibrosis progression in schistosomiasis and to determine if macrophage-related mechanisms are involved. Immunohistochemistry was used to characterize the cells that generate and respond to Hedgehog ligands in 28 liver biopsies from patients with different grades of schistosomiasis fibrosis staged by ultrasound. Cultured macrophages (RAW264.7 and primary rat Kupffer cells) and primary rat liver sinusoidal endothelial cells (LSEC) were treated with schistosome egg antigen (SEA) and evaluated using qRT-PCR. Inhibition of the Hedgehog pathway was used to investigate its role in alternative activation of macrophages (M2) and vascular tube formation. Patients with schistosomiasis expressed more ligands (Shh and Ihh) and target genes (Patched and Gli2) than healthy individuals. Activated LSEC and myofibroblasts were Hedgehog responsive [Gli2(+)] and accumulated in parallel with fibrosis stage (P < 0.05). Double IHC for Ihh/CD68 showed that Ihh(+) cells were macrophages. In vitro studies demonstrated that SEA-stimulated macrophages to express Ihh and Shh mRNA (P < 0.05). Conditioned media from such macrophages induced luciferase production by Shh-LightII cells (P < 0.001) and Hedgehog inhibitors blocked this effect (P < 0.001). SEA-treated macrophages also up-regulated their own expression of M2 markers, and Hh pathway inhibitors abrogated this response (P < 0.01). Inhibition of the Hedgehog pathway in LSEC blocked SEA-induced migration and tube formation. SEA stimulates liver macrophages to produce Hh ligands, which promote alternative activation of macrophages, fibrogenesis and vascular remodelling in schistosomiasis.

The human hepatocyte cell lines IHH and HepaRG: models to study glucose, lipid and lipoprotein metabolism

Metabolic diseases reach epidemic proportions. A better knowledge of the associated alterations in the metabolic pathways in the liver is necessary. These studies need in vitro human cell models. Several human hepatoma models are used, but the response of many metabolic pathways to physiological stimuli is often lost. Here, we characterize two human hepatocyte cell lines, IHH and HepaRG, by analysing the expression and regulation of genes involved in glucose and lipid metabolism. Our results show that the glycolysis pathway is activated by glucose and insulin in both lines. Gluconeogenesis gene expression is induced by forskolin in IHH cells and inhibited by insulin in both cell lines. The lipogenic pathway is regulated by insulin in IHH cells. Finally, both cell lines secrete apolipoprotein B-containing lipoproteins, an effect promoted by increasing glucose concentrations. These two human cell lines are thus interesting models to study the regulation of glucose and lipid metabolism.

Expression and significance of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 in hepatocyte steatosis model

To investigate the expression of IGF-1 and IGFBP-3 in nonalcoholic fatty steatosis hepatocyte models induced by oleic acid. Nonalcoholic fatty steatosis hepatocyte models induced by oleic acid on immortalized human hepatocyte, Oil red O staining and intracellular triglycerides were detected for observing the situation of IHH cells fatty degeneration. IHH cells were divided into control group, NAFLD group, which the control group cultured in DMEM/F12 medium, NAFLD group were treated with oleic acid, 0.5 mmol/L treatment for 72 h. The expression of mRNA and protein of IGF-1 and IGFBP3 were measured by immunofluorescent staining, Western blot and RT-PCR methods. Between the two groups were compared using the t- test. The steatosis models of the hepatocytes were established successfully with 0.5 mmol/L oleic acid. Lipid droplets were observed through Oil red O staining. The level of hepatocyte TG was increased (275.7+/-27.2) mug/mg from (150.2+/-15.6) mug/mg (t = 21.67, P less than 0.01). Compared with the control group, the mRNA of IGF-1 (0.76+/-0.04 vs 4.82+/-1.51, t = 17.915, P less than 0.01), IGFBP-3 (1.58+/-0.93 vs 5.41+/-1.37, t = 12.893, P less than 0.01) and protein expression of IGF-1 (1.00+/-0.29 vs 2.56+/-0.71, t = 29.17, P less than 0.01), IGFBP-3 (0.65+/-0.36 vs 1.23+/-0.91, t = 32. 12, P less than 0.01) significantly decreased in oleic acid-treated group. The results of immunofluorescence staining also confirm the significantly decreased protein expression of IGF-1 and IGFBP-3 in NAFLD group compared with control group. The expression of IGF-1 and IGFBP-3 decreased in nonalcoholic fatty steatosis hepatocyte models, which will provide the experimental basis for the further study of the mechanism of the limited height of some children with non-alcoholic fatty liver disease in clinical.

Indian hedgehog signaling is required for proper formation, maintenance and migration of Xenopus neural crest

Neural crest induction is the result of the combined action at the neural plate border of FGF, BMP, and Wnt signals from the neural plate, mesoderm and nonneural ectoderm. In this work we show that the expression of Indian hedgehog (Ihh, formerly named Banded hedgehog) and members of the Hedgehog pathway occurs at the prospective neural fold, in the premigratory and migratory neural crest. We performed a functional analysis that revealed the requirement of Ihh signaling in neural crest development. During the early steps of neural crest induction loss of function experiments with antisense morpholino or locally grafted cyclopamine-loaded beads suppressed the expression of early neural crest markers concomitant with the increase in neural and epidermal markers. We showed that changes in Ihh activity produced no alterations in either cell proliferation or apoptosis, suggesting that this signal involves cell fate decisions. A temporal analysis showed that Hedgehog is continuously required not only in the early and late specification but also during the migration of the neural crest. We also established that the mesodermal source of Ihh is important to maintain specification and also to support the migratory process. By a combination of embryological and molecular approaches our results demonstrated that Ihh signaling drives in the migration of neural crest cells by autocrine or paracrine mechanisms. Finally, the abrogation of Ihh signaling strongly affected only the formation of cartilages derived from the neural crest, while no effects were observed on melanocytes. Taken together, our results provide insights into the role of the Ihh cell signaling pathway during the early steps of neural crest development.

The SV2 variant of KLF6 is down-regulated in hepatocellular carcinoma and displays anti-proliferative and pro-apoptotic functions

KLF6 protein is a transcription factor that plays important functions in hepatocellular carcinoma (HCC), which is one of the leading causes of death by cancer worldwide. Previous studies showed the existence of three splice variants of KLF6, termed SV1, SV2, and SV3. An increased SV1/KLF6 mRNA ratio in HCC was already described. In this study, we aimed to investigate the expression of the SV2 variant in HCC samples and its role in hepatic cells. We measured the expression of the SV2 variant in HCC and adjacent tissue samples by q-RT-PCR. We established IHH and HepG2 stable cell lines over-expressing the SV2 variant and measured cell growth and apoptotic rate. We observed a reduced expression of the SV2 variant in HCC samples versus surrounding tissues and normal liver. Interestingly, our findings demonstrate that the over-expression of the SV2 variant in IHH and HepG2 cells leads to a significant reduction of proliferation associated with cell death by apoptosis. We further demonstrate that the SV2 expression leads to an induction of the cell-cycle-controlling p21(CIP/WAF1) and the pro-apoptotic Bax genes, mediated by the p53 protein. We show further that the SV2 expression in IHH and HepG2 cells induces their sensitivity to the anti-cancer drug, gemcitabine. We reveal a reduced expression of the SV2 variant of KLF6 in HCC samples and describe anti-proliferative and pro-apoptotic functions for this variant in hepatic cells.