Sigma receptor 1 (SigR1) is an endoplasmic reticulum resident integral membrane protein whose functions remain unclear. Although the liver shows the highest expression of SigR1, its role in this organ is unknown. SigR1 is overexpressed in many cancers and its expression is correlated to hormonal status in hormone-dependent cancers. To better understand the role of SigR1 in hepatocytes we focused our work on the regulation of its expression in tumoral liver. In this context, hepatocellular adenomas, benign hepatic tumors associated with estrogen intake are of particular interest. The expression of SigR1 mRNA was assessed in hepatocellular adenoma (HCA) patients using qPCR. The impact of estrogen on the expression of SigR1 was studied in vivo (mice) and in vitro (HepG2 and Huh7 cells). The effect of HNF1α on the expression of SigR1 was studied in vivo by comparing wild type mice to HNF1 knockout mice. Estrogen enhanced SigR1 expression through its nuclear receptor ERα. HNF1α mutated HCA (H-HCA) significantly overexpressed SigR1 compared to all other HCA subtypes. HNF1 knockout mice showed an increase in SigR1 expression. Overexpressing SigR1 in cellular models increases proliferation rate and storage of lipid droplets, which phenocopies the H-HCA phenotype. SigR1 is involved in hepatocyte proliferation and steatosis and may play an important role in the control of the H-HCA phenotype.

Tridax procumbens, a traditional medicinal plant, was analyzed in the present work for its antioxidant power through its free radicals scavenging capacity using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) test and its potential anti ROS (Reactiv Oxygen Species). The phytochemical screening revealed in the leaves of Tridax procumbens the presence of various metabolites such as flavonoids, tannins, phenols and mucilage and the absence of alkaloids, reducing sugars, leucoanthocyanins, anthocyans, anthraquinons and saponins. The antioxidant potential determination shows that T. procumbens has a very significant antioxidant power: IC50 = 0.17 mg/mL for DPPH test and C = 1mg / ml inhibit completely the production of ROS. Its high content of total polyphenols (11.49 mg/g EAG) and flavonoids (24.01 gm/g CE) could explain this activity. The toxicity test shows that the semi-ethanolic extract of T. procumbens is not toxic at any dose up of 10 mg / ml. This extract can be used at reasonable doses to fight against hyperlipidemia and non-communicable diseases such as hypertension, arteriosclerosis and neurodegenerative diseases.

Shigella, the agent of bacillary dysentery, invades epithelial cells by locally inducing actin reorganization. Upon cell invasion, Shigella induces calcium (Ca2+) signalling, but its role in invasion has remained unclear. Here we show that components involved in inositol 1, 4, 5- trisphosphate (InsP(3)) signalling are implicated in Shigella invasion. Although global Ca2+ responses are dispensable for bacterial invasion, local Ca2+ responses of unprecedented long duration are associated with invasion sites. Fluorescence recovery after photobleaching experiments indicate that diffusion of small solutes is hindered at Shigella-invasion sites and that diffusion hindrance is dependent on bacterially induced actin reorganization. Computational simulations and experimental challenge of the model support the notion that local accumulation of InsP(3) permitted by restricted diffusion and enrichment of InsP3 receptors account for sustained local Ca2+ increases at entry sites. Thus, cytoskeletal reorganization through diffusion hindrance shapes the duration of local Ca2+ signals.

Hint2, one of the five members of the superfamily of the histidine triad AMP-lysine hydrolase proteins, is expressed in mitochondria of various cell types. In human adrenocarcinoma cells, Hint2 modulates Ca2+ handling by mitochondria. As Hint2 is highly expressed in hepatocytes, we investigated if this protein affects Ca2+ dynamics in this cell type. We found that in hepatocytes isolated from Hint2−/− mice, the frequency of Ca2+ oscillations induced by 1 μM noradrenaline was 150% higher than in the wild-type. Using spectrophotometry, we analyzed the rates of Ca2+ pumping in suspensions of mitochondria prepared from hepatocytes of either wild-type or Hint2−/− mice; we found that Hint2 accelerates Ca2+ pumping into mitochondria. We then resorted to computational modeling to elucidate the possible molecular target of Hint2 that could explain both observations. On the basis of a detailed model for mitochondrial metabolism proposed in another study, we identified the respiratory chain as the most probable target of Hint2. We then used the model to predict that the absence of Hint2 leads to a premature opening of the mitochondrial permeability transition pore in response to repetitive additions of Ca2+ in suspensions of mitochondria. This prediction was then confirmed experimentally.

Extracellular ATP regulates many hepatic functions by stimulating purinergic receptors. Only the G protein‐coupled P2Y receptors have been studied in hepatocytes. We investigated the functional expression of P2X receptors, the ATP‐gated channels in rat hepatocytes. P2X4 and P2X7 transcripts and proteins were detected by RT‐PCR and by both Western blotting and immunocytochemistry. High concentrations of ATP, and 2′‐and 3′‐O‐(4‐benzoylbenzoyl)‐ATP the preferring agonist of P2X7, induced membrane blebbing and significant uptake of 4‐[(3‐methyl‐2(3H)‐benzoxazolylidene)methyl]‐1‐[3‐(triethylammonio)propyl]diiodide, both of which were inhibited by oxidised ATP, a blocker of P2X receptors. These results provide evidence that P2X4 and P2X7 receptors are expressed and functional on rat hepatocytes, possibly playing an important role in the purinergic signaling complex in these cells.

Background/Aims: During liver regeneration, a network of cytokines and growth factors interact with hepatocytes, helping to restore the liver mass and functions after partial tissue loss. Agonists that trigger Ca2+ signals in the liver contribute to this process, although little is known about calcium signalling during liver regeneration.Results: We observed two phases in which the hepatocyte response to calcium-mobilising agonists was greatly reduced versus control cells at 24 h and five days after partial hepatectomy. We found that both phases of hepatocyte desensitisation involved the down-regulation of cell surface receptors and the type II InsP(3) receptor. Single cell studies with flash photolysis of caged InsP3 revealed that InsP(3)-mediated Ca2+ release was slower in regenerating hepatocytes at 24, 48 h and 5 days than in control cells. Also, the temporal pattern of vasopressin-elicited intracellular calcium oscillations studied on fura2-loaded cells was altered, with the duration of each Ca2+ peak being longer. Finally, we showed an association between hepatocyte desensitisation and progression through the cell cycle towards the S phase at 24 h after hepatectomy.Conclusions: Our study supports the remodelling of hepatocyte calcium signalling during liver regeneration, and that this change is partly linked with cell cycle progression. (c) 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Calcium entry is a component of the processes regulating the proliferative phenotype of some types of cancer. In non-excitable cells, capacitative calcium entry (CCE) and non-capacitative calcium entry (NCCE) are thought to be the main pathways of Ca2+ influx into cells. Thus, blocking calcium entry may prevent normal and pathological cell proliferation and there is evidence to suggest that molecules blocking calcium entry also have antiproliferative properties. Carboxyamidotriazole (CAI), a novel inhibitor of the non-voltage-dependent calcium entry has been shown to have such properties in model systems in vitro and in vivo. We used Hep G2 and Huh-7 human hepatoma cells to investigate the effects of calcium entry blockers on cell proliferation. CAI (10 μM) and 2-APB (20 μM) completely blocked CCE in thapsigargin-treated Huh-7, and CAI and 2-APB inhibited cell proliferation with IC50 of 4.5 and 43 μM, respectively. The plateau phase of the [Ca2+]i increases triggered by 10% FCS were abolished in the absence of external Ca2+ and in the presence of CAI or 2-APB. We, therefore, suggest that CCE is the main pathway involved in regulation of the processes leading to cell proliferation.

Liver regeneration after partial hepatectomy is a plastic process during which the mechanisms that coordinate liver mass restoration compensate one another through a complex regulatory network of cytokines, growth factors, and hormones. Vasopressin, an agonist that triggers highly organized Ca2+ signals in the liver, may be one of these factors, although little in vivo evidence is available in support of this hypothesis. We provide evidence that hypothalamic vasopressin secretion is stimulated early after partial hepatectomy. Although hepatocytes were fully responsive to vasopressin during the first hours of regeneration, they became desensitized and exhibited slow oscillating Ca2+ responses to vasopressin on the following days. On the first day, hepatocyte V1a receptor density decreased and its lobular gradient increased in hepatectomized rats. By antagonizing the V1a receptor in vivo, we demonstrated that vasopressin contributes to NF-κB and cyclin (D1 and A) activation, to hepatocyte progression in the cell cycle, and to liver mass restoration. Finally, vasopressin exerted a choleretic effect shortly after hepatectomy, both in the isolated perfused liver and in the intact rat. In conclusion, we provide compelling in vivo evidence that vasopressin contributes significantly to growth initiation and bile flow stimulation in the early stages of liver regeneration.

Beta-catenin is a key mediator of the Wnt pathway, which plays a critical role in embryogenesis and oncogenesis. As a transcriptional activator, beta-catenin binds the transcription factors, T-cell factor and lymphoid enhancer factor, and regulates gene expression in response to Wnt signaling. Abnormal activation of beta-catenin has been linked to various types of cancer. In a yeast two-hybrid screen, we identified the four and a half of LIM-only protein 2 (FHL2) as a novel beta-catenin-interacting protein. Here we show specific interaction of FHL2 with beta-catenin, which requires the intact structure of FHL2 and armadillo repeats 1-9 of beta-catenin. FHL2 cooperated with beta-catenin to activate T-cell factor/lymphoid enhancer factor-dependent transcription from a synthetic reporter and the cyclin D1 and interleukin-8 promoters in kidney and colon cell lines. In contrast, coexpression of beta-catenin and FHL2 had no synergistic effect on androgen receptor-mediated transcription, whereas each of these two coactivators independently stimulated AR transcriptional activity. Thus, the ability of FHL2 to stimulate the trans-activating function of beta-catenin might be dependent on the promoter context. The detection of increased FHL2 expression in hepatoblastoma, a liver tumor harboring frequent beta-catenin mutations, suggests that FHL2 might enforce beta-catenin transactivation activity in cancer cells. These findings reveal a new function of the LIM coactivator FHL2 in transcriptional activation of Wnt-responsive genes.

The role of hepatitis B virus HBx protein in the carcinogenesis associated with chronic viral infection remains ill-defined. Indeed, pleiotropic effects have been ascribed to HBx: in addition to its well-documented ability to indirectly stimulate transcription, the protein has been reported to affect cell growth, signal transduction, DNA repair and apoptosis. In this work, we generated Chang (CCL-13)-derived cell lines constitutively expressing wild type or mutant HBx, as a model of HBx-host cell interaction closer to the chronic infection setting, than the classically used transient expression systems. We document the potentiation by HBx of the apoptotic cell death pathway in the recipient cells. This effect is unlikely to rely on p53 activity since the protein is functionally inactivated in CCL-13. In addition, antioxidants and cyclosporin A failed to reduce the apoptotic response back to the normal level, suggesting that production of reactive oxygen species and calcineurin activation are not directly involved in the proapoptotic effect of HBx. In contrast, our data show that transactivation and stimulation of apoptosis are tightly linked HBx activities. Finally, expression of transactivation-active protein did not result in detectable change in the pattern of MAP kinases phosphorylation nor did it affect the ability of the host cell to repair in vitro irradiated plasmid DNA.

The chromosome 8p is associated with a large number of allelic imbalances in epithelial tumors including hepatocellular carcinoma (HCC), However, no tumor suppressor gene has been identified so far in this particular region of the genome. To further clarify the pattern of allelic deletions on chromosome 8p in HCC, we have undertaken high-density polymorphic marker analysis of 109 paired normal and primary tumor samples using 40 microsatellites positioned every 2 cm in average throughout 8p, We found that 60% of the tumors exhibited loss of heterozygosity (LOH) at one or more loci at 8p with three distinct minimal deleted areas : a 13 cm region in the distal part of 8p21, a 9 cm area in the more proximal portion of 8p22 and a 5 cm area in 8p23, These data strongly suggest the presence of at least three novel tumor suppressor loci on 8p in hepatocellular carcinoma.

With a view to obtaining human monoclonal antibodies against HIV-1 antigens we used the Epstein-Barr virus immortalization technique to induce lymphoblastoid cell lines from peripheral blood lymphocytes of 10 people who were seropositive for HIV-1 and had no clinical symptoms. A number of polyclonal lines were obtained which synthesized antibodies against most of the major proteins and glycoproteins of HIV-1. Three stable clones were characterized for class, secretion characteristics and specificity. Two of these clones produce antibodies which react with gp41, and the third reacts with p25. One of the anti-gp41 antibodies was found to have neutralizing activity.