Role Of Hepatocytes Research Articles

Hepatocyte-specific mitogen-activated protein kinase phosphatase 1 in sexual dimorphism and susceptibility to alcohol induced liver injury.

It is well established that females are more susceptible to the toxic effects of alcohol, although the exact mechanisms are still poorly understood. Previous studies noted that alcohol reduces the expression of mitogen-activated protein kinase phosphatase 1 (MKP1), a negative regulator of mitogen-activated protein kinases (MAPK) in the liver. However, the role of hepatocyte- specific MKP1 in the pathogenesis of alcohol-associated liver disease (ALD) remains uncharacterized. This study aimed to evaluate the role of hepatocyte-specific MKP1 in the susceptibility and sexual dimorphism in alcohol-induced liver injury. C57Bl/6 mice were used in an intragastric ethanol feeding model of alcohol-associated steatohepatitis (ASH). Hepatocyte-specific Mkp1-/- knockout and (Mkp1+/+ "f/f" male and female mice were subjected to the NIAAA chronic plus binge model. Primary mouse hepatocytes were used for in vitro studies. Liver RNA sequencing was performed on an Illumina NextSeq 500. Liver injury was evaluated by plasma alanine transaminase (ALT), hepatic ER stress and inflammation markers. Statistical analysis was carried out using ANOVA and the unpaired Student's t-test. ASH was associated with the severe injury accompanied by increased endoplasmic reticulum (ER) stress and significant downregulation of Dusp1 mRNA expression. In vitro, ethanol treatment resulted in a time-dependent decrease in Dusp1 mRNA and protein expression in primary hepatocytes in both males and females; however, this effect was significantly more pronounced in hepatocytes from females. In vivo, female mice developed more liver injury in a chronic plus binge model which was accompanied by a significant decrease in liver Dusp1 mRNA expression. In comparison, liver Dusp1 was not changed in male mice, while they developed milder injury to alcohol. Mkp1 deletion in hepatocytes led to increased alcohol induced liver injury, ER stress and inflammation in both sexes. Hepatocyte Mkp1 plays a significant role in alcohol induced liver injury. Alcohol downregulates Mkp1 expression in hepatocytes in a sex dependent manner and could play a role in sexual dimorphism in increased female susceptibility to alcohol.

Hepatocyte-specific regulation of autophagy and inflammasome activation via MyD88 during lethal Ehrlichia infection.

Hepatocytes play a crucial role in host response to infection. Ehrlichia is an obligate intracellular bacterium that causes potentially life-threatening human monocytic ehrlichiosis (HME) characterized by an initial liver injury followed by sepsis and multi-organ failure. We previously showed that infection with highly virulent Ehrlichia japonica (E. japonica) induces liver damage and fatal ehrlichiosis in mice via deleterious MyD88-dependent activation of CASP11 and inhibition of autophagy in macrophage. While macrophages are major target cells for Ehrlichia, the role of hepatocytes (HCs) in ehrlichiosis remains unclear. We investigated here the role of MyD88 signaling in HCs during infection with E. japonica using primary cells from wild-type (WT) and MyD88-/- mice, along with pharmacologic inhibitors of MyD88 in a murine HC cell line. Similar to macrophages, MyD88 signaling in infected HCs led to deleterious CASP11 activation, cleavage of Gasdermin D, secretion of high mobility group box 1, IL-6 production, and inflammatory cell death, while controlling bacterial replication. Unlike macrophages, MyD88 signaling in Ehrlichia-infected HCs attenuated CASP1 activation but activated CASP3. Mechanistically, active CASP1/canonical inflammasome pathway negatively regulated the activation of CASP3 in infected MyD88-/- HCs. Further, MyD88 promoted autophagy induction in HCs, which was surprisingly associated with the activation of the mammalian target of rapamycin complex 1 (mTORC1), a known negative regulator of autophagy. Pharmacologic blocking mTORC1 activation in E. japonica-infected WT, but not infected MyD88-/- HCs, resulted in significant induction of autophagy, suggesting that MyD88 promotes autophagy during Ehrlichia infection not only in an mTORC1-indpenedent manner, but also abrogates mTORC1-mediated inhibition of autophagy in HCs. In conclusion, this study demonstrates that hepatocyte-specific regulation of autophagy and inflammasome pathway via MyD88 is distinct than MyD88 signaling in macrophages during fatal ehrlichiosis. Understanding hepatocyte-specific signaling is critical for the development of new therapeutics against liver-targeting pathogens such as Ehrlichia.

The role of Sirtuin 2 in sustaining functional integrity of the liver

AimSirtuin 2 (SIRT2) is a NAD+-dependent deacetylase involved in various biological functions via deacetylation of proteins, including histone protein. Hepatic fat accumulation from aging and excess caloric intake contribute to development of non-alcoholic fatty liver disease. The study aim was to elucidate the role of SIRT2 in lipid metabolism homeostasis. Materials and methodsSIRT2+/+ (C57BL/6) and SIRT2−/− were randomly assigned to normal diet or high-fat diet (HFD) groups and fed for 6 weeks. Histological features of the livers were evaluated by hematoxylin and eosin and Masson's trichrome staining, and the levels of selected factors were determined by quantitative reverse transcription-polymerase chain reaction and western blot analysis. Key findingsAlthough the SIRT2−/− mice were viable, their livers exhibited higher glycogen accumulation, and skeletal muscle showed features of increased metabolic demand. The SIRT2−/− mice attenuated HFD-induced weight gain, visceral adipose tissue formation, and fat accumulation in the liver in which the expressions of genes involved in metabolic substrate transport were modified. Additionally, the hepatocellular senescence and upregulated cell-cycle factors upon HFD intake in SIRT2−/− livers suggested a role of SIRT2 in gene expression during abnormal metabolism. Moreover, the fibrotic phenotype of liver tissue without fat accumulation and the increased expression of genes involved in liver fibrosis in the HFD-fed SIRT2−/− mice indicated that SIRT2 had a role in hepatocyte and hepatic stellate cell activation. SignificanceOur results indicated that SIRT2 has a critical role in regulating lipid metabolic homeostasis and in sustaining liver integrity by modulating related gene expression.

Calcium-dependent insulin resistance in hepatocytes: mathematical model

Hepatocyte insulin resistance is one of the early factors of developing type II diabetes. If insulin resistance is treated early, type II diabetes could be prevented. In recent years, scientists have been conducting extensive research on the underlying issues on a cellular and molecular level. It was found that the modulation of IP3-receptors, the mitochondrial ability to form the mitochondria-associated membranes (MAMs) and the endoplasmic reticulum stress during Ca2+ signaling play a key role in hepatocyte being able to maintain euglycemia and provide metabolic flexibility. However, researchers cannot agree on what factor is the key one in resulting in insulin resistance. In this work, we propose a mathematical model of Ca2+ signaling. We included in the model all the major contributors of a proper Ca2+ signaling during both the fasting and the postprandial state. Our modeling results are in good agreement with available experimental data. The analysis of modeling results suggests that MAMs dysfunction alone cannot result in abnormal Ca2+ signaling and the wrong modulation of IP3-receptors is a more definite reason. However, both the MAMs dysfunction and the IP3 signaling dysregulation combined can lead to a robust Ca2+ signal and improper glucose release. In addition, our model results suggest a strong dependence of Ca2+ oscillations pattern on morphological characteristics of the ER and the mitochondria.

Inositol 1,4,5-trisphosphate receptor type 3 plays a protective role in hepatocytes during hepatic ischemia-reperfusion injury

Hepatic ischemia-reperfusion injury is seen in a variety of clinical conditions, including hepatic thrombosis, systemic hypotension, and liver transplantation. Calcium (Ca2+) signaling mediates several pathophysiological processes in the liver, but it is not known whether and how intracellular Ca2+ channels are involved in the hepatocellular events secondary to ischemia-reperfusion. Using an animal model of hepatic ischemia-reperfusion injury, we observed a progressive increase in expression of the type 3 isoform of the inositol trisphosphate receptor (ITPR3), an intracellular Ca2+ channel that is not normally expressed in healthy hepatocytes. ITPR3 expression was upregulated, at least in part, by a combination of demethylation of the ITPR3 promoter region and the increased transcriptional activity of the nuclear factor of activated T-cells (NFAT). Additionally, expression of pro-inflammatory interleukins and necrotic surface area were less pronounced in livers of control animals compared to liver-specific ITPR3 KO mice subjected to hepatic damage. Corroborating these findings, ITPR3 expression and activation of NFAT were observed in hepatocytes of liver biopsies from patients who underwent liver ischemia caused by thrombosis after organ transplant. Together, these results are consistent with the idea that ITPR3 expression in hepatocytes plays a protective role during hepatic injury induced by ischemia-reperfusion.

Hepatic FNDC5 is a potential local protective factor against Non-Alcoholic Fatty Liver

The proteolytic cleavage of Fibronectin type III domain-containing 5 (FNDC5) generates soluble irisin. Initially described as being mainly produced in muscle during physical exercise, irisin mediates adipose tissue thermogenesis and also regulates carbohydrate and lipid metabolism. The aim of this study was to evaluate the hepatic expression of FNDC5 and its role in hepatocytes in Non-Alcoholic Fatty Liver (NAFL). Here we report that hepatic expression of FNDC5 increased with hepatic steatosis and liver injury without impacting the systemic level of irisin in mouse models of NAFLD (HFD and MCDD) and in obese patients. The increased Fndc5 expression in fatty liver resulted from its upregulation in hepatocytes and non-parenchymal cells in mice. The local production of Fndc5 in hepatocytes was influenced by genotoxic stress and p53-dependent pathways. The down-regulation of FNDC5 in human HepG2 cells and in primary mouse hepatocytes increased the expression of PEPCK, a key enzyme involved in gluconeogenesis associated with a decrease in the expression of master genes involved in the VLDL synthesis (CIDEB and APOB). These alterations in FNDC5-silenced cells resulted to increased steatosis and insulin resistance in response to oleic acid and N-acetyl glucosamine, respectively. The downregulation of Fndc5 also sensitized primary hepatocytes to apoptosis in response to TNFα, which has been associated with decreased hepatoprotective autophagic flux. In conclusion, our human and experimental data strongly suggest that the hepatic expression of FNDC5 increased with hepatic steatosis and its upregulation in hepatocytes could dampen the development of NAFLD by negatively regulating steatogenesis and hepatocyte death.

Sensing of Liver Iron Content Requires Cell-Cell Communication between Hepatocytes and Liver Sinusoidal Endothelial Cells

The liver stores iron and senses systemic and tissue iron availability. Hepatocytes control iron homeostasis by producing the peptide hormone hepcidin that controls dietary iron absorption and iron release from intracellular stores. Recent data challenged the exclusive role of hepatocytes in controlling iron levels. Indeed, liver sinusoidal endothelial cells (LSECs) increase BMP2 and BMP6 levels in response to iron, which control hepcidin expression in a paracrine manner. However the molecular mechanism(s) of how BMPs respond to iron levels remain unknown. We established primary murine LSEC cultures and exposed these to iron sources. Unexpectedly, BMP2 mRNA expression is strongly reduced by iron treatment, while BMP6 levels are only mildly increased. This finding suggests that intracellular iron content cannot directly activate BMP2 transcription and only slightly contribute to BMP6 upregulation in LSEC cultures. However, if LSECs are co-cultured with iron-loaded primary hepatocytes the expression of BMP2 and BMP6 is increased and the fold induction of BMP6 is greater compared to LSECs cultured alone, suggesting that the iron status of hepatocytes instructs the LSEC BMP response. These data are supported by findings in a genetic mouse model of iron overload (Slc40a1C326S/C326S). Hepatocytes isolated from Slc40a1C326S/C326S mice display an iron-loaded molecular signature and the expected low mRNA expression of Transferrin Receptor 1 (Tfr1). By contrast, LSECs show high expression of Tfr1, indicating intracellular iron deficiency. Despite this, hepatic BMP levels are increased, suggesting that BMP2 and BMP6 expression are directly related to the intracellular iron content of hepatocytes but not LSECs. RNA-sequencing of isolated hepatic cell populations is ongoing to identify putative hepatocyte regulators involved in the iron-mediated BMP2 and BMP6 regulation. Disclosures Muckenthaler: Silence Therapeutics: Consultancy; Novartis: Research Funding.

Endoplasmic Reticulum Stress in Metabolic Liver Diseases and Hepatic Fibrosis.

Endoplasmic reticulum (ER) stress is a major contributor to liver disease and hepatic fibrosis, but the role it plays varies depending on the cause and progression of the disease. Furthermore, ER stress plays a distinct role in hepatocytes versus hepatic stellate cells (HSCs), which adds to the complexity of understanding ER stress and its downstream signaling through the unfolded protein response (UPR) in liver disease. Here, the authors focus on the current literature of ER stress in nonalcoholic and alcoholic fatty liver diseases, how ER stress impacts hepatocyte injury, and the role of ER stress in HSC activation and hepatic fibrosis. This review provides insight into the complex signaling and regulation of the UPR, parallels and distinctions between different liver diseases, and how ER stress may be targeted as an antisteatotic or antifibrotic therapy to limit the progression of liver disease.

Integrative proteomics and immunochemistry analysis of the factors in the necrosis and repair in acetaminophen-induced acute liver injury in mice.

Acetaminophen (APAP) overdose-induced acute liver injury (AILI) is a significant clinical problem worldwide, the hepatotoxicity mechanisms are well elucidated, but the factors involved in the necrosis and repair still remain to be investigated. APAP was injected intraperitoneally in male Institute of Cancer Research (ICR) mice. Quantitative proteome analysis of liver tissues was performed by 2-nitrobenzenesulfenyl tagging, two-dimensional-nano high-performance liquid chromatography separation, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. Diffrenetial proteins were verified by the immunochemistry method. 36 and 44 differentially expressed proteins were identified, respectively, at 24 hr after APAP (200 or 300 mg·kg -1 ) administration. The decrease in the mitochondrial protective proteins Prdx6, Prdx3, and Aldh2 accounted for the accumulation of excessive reactive oxygen species (ROS) and aldehydes, impairing mitochondria structure and function. The Gzmf combined with Bax and Apaf-1 jointly contributed to the necrosis. The blockage of Stat3 activation led to the overexpression of unphosphorylated Stat3 and the overproduction of Bax. The overexpression of unphosphorylated Stat3 represented necrosis; the alternation from Stat3 to p-Stat3 in necrotic regions represented hepatocytes from death to renewal. The high expressions of P4hα1, Ncam, α-SMA, and Cygb were involved in the liver repair, they were not only the markers of activated HSC but also represented an intermediate stage of hepatocytes from damage or necrosis to renewal. Our data provided a comprehensive report on the profile and dynamic changes of the liver proteins in AILI; the involvement of Gzmf and the role of Stat3 in necrosis were revealed; and the role of hepatocyte in liver self-repair was well clarified.

Sinusoidal endothelial cells and vascular endothelial growth factor are key players in an in-vitro model of oxaliplatin-induced sinusoidal obstruction syndrome

Introduction: Oxaliplatin as part of systemic treatment for colorectal liver metastases (CLM) has been associated with the development of sinusoidal obstruction syndrome (SOS). While the role of hepatocytes and sinusoidal endothelial cells in the pathogenesis of SOS remains unclear, bevacizumab, an inhibitor of vascular endothelial growth factor (VEGF), is considered to prevent SOS. The aim of this study was to analyse cytotoxicity of oxaliplatin-based treatment regimens and expression of VEGF in an in-vitro model of SOS. Methods: Human hepatic sinusoidal endothelial cells (HHSEC) and HepG2 cells (human liver cancer cell line) were incubated with either oxaliplatin, bevacizumab or both for 12 to 60 hours. Cytotoxicity was analysed by CellTox green assay. Protein levels of VEGF and matrix metallopeptidase 9 (MMP9) were measured by ELISA. Cytoskeletal changes were identified by F-Actin immunofluorescence. Cell proliferation and migration were analysed using a scratch assay. Liver transaminases were measured by photometric determination. Results: Supernatant of HHSEC had increased VEGF levels after 48h hours of treatment with oxaliplatin compared to supernatant of HepG2 cells (382.2 vs. 23.73 pg/mL). Additional administration of bevacizumab on HHSEC facilitated a significantly decreased cytotoxicity compared to oxaliplatin alone (4.13 vs. 5.52 RFU, P=0.004). Conclusion: Our findings suggest that oxaliplatin has a major impact on HHSEC resulting in increased expression of VEGF as a key player of oxaliplatin-induced SOS. Additionally, anti-VEGF seems to protect the HHSEC from the cytotoxic effect of oxaliplatin. These results may improve our understanding of the pathogenesis of SOS and optimize multimodal treatment of patients with CLM.

Abstract 1102: IL-6/STAT3 activation in hepatocytes drives pro-metastatic niche formation in the liver

Abstract The liver is the most common site of metastasis in pancreatic ductal adenocarcinoma (PDAC). This metastatic tropism is dependent, at least in part, on the formation of a “pro-metastatic” niche that supports tumor cell seeding and colonization in the liver. However, mechanisms that direct the formation of this niche remain poorly understood. We show using the LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) model of PDAC that pancreatic tumor development enhances the susceptibility of the liver to metastatic seeding by inducing recruitment of F4/80+ and Ly6G+ myeloid cells and fibrosis within the liver. 3' mRNA sequencing (QuantSeq) on RNA isolated from the liver of KPC mice versus control PC mice revealed that the liver produces a specific set of myeloid chemoattractants, particularly serum amyloid A1 and A2 (SAA1/2), early during PDAC development. In addition, gene set enrichment analysis (GSEA) on genes upregulated in the liver of KPC mice demonstrated a significant enrichment of the interleukin 6 (IL-6)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway. Consistent with this finding, phosphorylation of STAT3 was detected in 20-30% of F4/80+ myeloid cells and 80-90% of hepatocytes. A requirement for IL-6/STAT3 signaling in the formation of a pro-metastatic niche was determined by comparing the metastatic potential of wild type mice, Il-6 knockout (Il-6-/-) mice, and mice treated with anti-IL-6 receptor (IL-6R) antibody after orthotopic implantation of KPC-derived PDAC cells. Compared to wild type mice, the liver of Il-6-/- mice and mice treated with anti-IL-6R antibody was less susceptible to metastatic seeding and showed significantly less accumulation of myeloid cells, fibrosis, and production of SAA1/2 in the liver. We obtained similar results with mice that lack Stat3 specifically in hepatocytes (Stat3flox/flox Alb-Cre), demonstrating that IL-6/STAT3 signaling in hepatocytes is necessary for the formation of a pro-metastatic niche in the liver. Further, using Saa1/2 double knockout (Saa-/-) mice, we found that SAA1/2 production by hepatocytes was required for formation of the pro-metastatic niche in the liver and increased susceptibility to metastatic seeding. Patients with a history of liver metastasis also showed higher levels of SAA1/2 in the plasma compared to normal donors, and SAA overexpression was detected in hepatocytes in liver biopsy samples collected from PDAC patients. Collectively, our study reveals a novel role for hepatocytes in directing the formation of a pro-metastatic niche in the liver during PDAC development and identifies IL-6/STAT3/SAA1/2 signaling as a promising therapeutic target for prevention of metastasis in PDAC. Citation Format: Jae W. Lee, Stacy K. Thomas, Chad A. Komar, Whitney L. Gladney, Xia Hua, Dong Xin, Abraham Shaked, Mitesh J. Borad, Ramesh K. Ramanathan, Ailing Ji, Nancy R. Webb, Maria C. de Beer, Frederick C. de Beer, Paige M. Porrett, Gregory L. Beatty. IL-6/STAT3 activation in hepatocytes drives pro-metastatic niche formation in the liver [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1102.

In vitro evidences of epithelial to mesenchymal transition in low cell-density cultured human fetal hepatocytes

Culturing fetal hepatocytes in high cell-density allowed stabilization of the hepatocyte phenotype up to 8 weeks, including the maintenance of liver-specific functions. On the other hand, when cultured at low cell-density, fetal hepatocytes underwent morphological modifications and acquired fibroblastic morphology. Since a switch from E-cadherin to vimentin expression accompanied these changes, we hypothesized the occurrence of epithelial-to-mesenchymal transition when fetal hepatocytes were cultured at low cell-density. Changes in gene expressionsuch as up-regulation of fibrosis-related geneswere also observed, suggesting that the low cell-density culture system promoted the acquisition of a profibrotic phenotype in cultured hepatocytes. The origin of fibrogenic cells in the liver is not well known, and the role of hepatocytes as a source of fibrogenic cells is controversial. Therefore, we hypothesized that hepatocytes undergoing epithelial-to-mesenchymal transition could have a central role in liver fibrosis as a source of fibrogenic cells.To conclude, the high cell-density culture system could be a useful model for in vitro studies requiring long-term cultures of hepatocytes, such as the development of pharmaceutical drugs and mechanisms of viral infections. The low cell-density culture system may provide additional insights into the origin of fibrogenic cells in the liver, thus contributing to the development of novel therapeutic approaches.

Autologous liver cell transplantation for acute liver failure after massive hepatectomy: role of hepatocytes and liver non parenchymal cells

Autologous liver cell transplantation for acute liver failure after massive hepatectomy: role of hepatocytes and liver non parenchymal cells

Caveolin‐1‐dependent activation of the metalloprotease TACE/ADAM17 by TGF‐β in hepatocytes requires activation of Src and the NADPH oxidase NOX1

Transforming growth factor-β (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, the balance between which decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β. We have previously shown that caveolin-1 (CAV1) is required for activation of the metalloprotease tumour necrosis factor (TNF)-α-converting enzyme/a disintegrin and metalloproteinase 17 (TACE/ADAM17), and hence transactivation of the EGFR pathway. The specific mechanism by which TACE/ADAM17 is activated has not yet been determined. Here we show that TGF-β induces phosphorylation of sarcoma kinase (Src) in hepatocytes, a process that is impaired in Cav1(-/-) hepatocytes, coincident with a decrease in phosphorylated Src in detergent-resistant membrane fractions. TGF-β-induced activation of TACE/ADAM17 and EGFR phosphorylation were blocked using the Src inhibitor PP2. Cav1(+/+) hepatocytes showed early production of reactive oxygen species (ROS) induced by TGF-β, which was not seen in Cav1(-/-) cells. Production of ROS was inhibited by both the NADPH oxidase 1 (NOX1) inhibitor STK301831 and NOX1 knock-down, which also impaired TACE/ADAM17 activation and thus EGFR phosphorylation. Finally, neither STK301831 nor NOX1 silencing impaired Src phosphorylation, but PP2 blocked early ROS production, showing that Src is involved in NOX1 activation. As expected, inhibition of Src or NOX1 increased TGF-β-induced cell death in Cav1(+/+) cells. In conclusion, CAV1 is required for TGF-β-mediated activation of TACE/ADAM17 through a mechanism that involves phosphorylation of Src and NOX1-mediated ROS production.

P0005 : The new strategy of autologous liver cell transplantation for acute liver failure after massive hepatectomy: Role of hepatocytes and liver non parenchymal cells

P0005 : The new strategy of autologous liver cell transplantation for acute liver failure after massive hepatectomy: Role of hepatocytes and liver non parenchymal cells

Caveolin-1 is required for TGF-β-induced transactivation of the EGF receptor pathway in hepatocytes through the activation of the metalloprotease TACE/ADAM17.

Transforming growth factor-beta (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, whose balance decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β in these cells. Caveolin-1 (Cav1) is a structural protein of caveolae linked to TGF-β receptors trafficking and signaling. Previous results have indicated that in hepatocytes, Cav1 is required for TGF-β-induced anti-apoptotic signals, but the molecular mechanism is not fully understood yet. In this work, we show that immortalized Cav1−/− hepatocytes were more sensitive to the pro-apoptotic effects induced by TGF-β, showing a higher activation of caspase-3, higher decrease in cell viability and prolonged increase through time of intracellular reactive oxygen species (ROS). These results were coincident with attenuation of TGF-β-induced survival signals in Cav1−/− hepatocytes, such as AKT and ERK1/2 phosphorylation and NFκ-B activation. Transactivation of the EGFR pathway by TGF-β was impaired in Cav1−/− hepatocytes, which correlated with lack of activation of TACE/ADAM17, the metalloprotease responsible for the shedding of EGFR ligands. Reconstitution of Cav1 in Cav1−/− hepatocytes rescued wild-type phenotype features, both in terms of EGFR transactivation and TACE/ADAM17 activation. TACE/ADAM17 was localized in detergent-resistant membrane (DRM) fractions in Cav1+/+ cells, which was not the case in Cav1−/− cells. Disorganization of lipid rafts after treatment with cholesterol-binding agents caused loss of TACE/ADAM17 activation after TGF-β treatment. In conclusion, in hepatocytes, Cav1 is required for TGF-β-mediated activation of the metalloprotease TACE/ADAM17 that is responsible for shedding of EGFR ligands and activation of the EGFR pathway, which counteracts the TGF-β pro-apoptotic effects. Therefore, Cav1 contributes to the pro-tumorigenic effects of TGF-β in liver cancer cells.

Determination of charactestic blood organic parameters in periparatal and peak lactation dairy cows.

The blood levels of glucose, total proteins, triglycerides, free fatty acids, total cholesterol, total proteins, albumnin, urea, total bilirubin were evaluated in peripartal and peak lactating dairy cows within physiological range.The measure values of the test parameters in puerperal dairy cows were stasticlly different from those in late gestation and peak lacting cows,wehich may be indicative of reduced gluconeogenisis rate in cows. The blood glucose levels in postpartal dairy cows were significantly bellow (p 0.05), respectively, as compared to those in late -gestation and peak lactation cows, clearly indicating their increased accumulation in the liver parenchyma of early lactation cows. The average levels of total proteins, albumin and urea in puerperal cows were stasticlly significantly lower (p<0.05) as compared to those in late gestation and peak lactation cows, clearly suggesting that the fatty infiltration of liver cells induces a reduction in the synthetic capacity of hepatocites in puerpearal cows. The bilirubin levels were statisicaly higher significantly higer in puerperal cows (p<0.01) than in late-gesatation and peak- lactatating cows , being indicative of decreased excretory role of hepatocytes in puerperal cows.

Failure of Fibrotic Liver Regeneration in Mice Is Linked to a Severe Fibrogenic Response Driven by Hepatic Progenitor Cell Activation

Failure of fibrotic liver to regenerate after resection limits therapeutic options and increases demand for liver transplantation, representing a significant clinical problem. The mechanism underlying regenerative failure in fibrosis is poorly understood. Seventy percent partial hepatectomy (PHx) was performed in C57Bl/6 mice with or without carbon tetrachloride (CCl4)-induced liver fibrosis. Liver function and regeneration was monitored at 1 to 14 days thereafter by assessing liver mass, alanine aminotransferase (ALT), mRNA expression, and histology. Progenitor (oval) cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and TWEAK-neutralizing antibody were used to manipulate progenitor cell proliferation in vivo. In fibrotic liver, hepatocytes failed to replicate efficiently after PHx. Fibrotic livers showed late (day 5) peak of serum ALT (3542 ± 355 IU/L compared to 93 ± 65 IU/L in nonfibrotic livers), which coincided with progenitor cell expansion, increase in profibrogenic gene expression and de novo collagen deposition. In fibrotic mice, inhibition of progenitor activation using TWEAK-neutralizing antibody after PHx resulted in strongly down-regulated profibrogenic mRNA, reduced serum ALT levels and improved regeneration. Failure of hepatocyte-mediated regeneration in fibrotic liver triggers activation of the progenitor (oval) cell compartment and a severe fibrogenic response. Inhibition of progenitor cell proliferation using anti-TWEAK antibody prevents fibrogenic response and augments fibrotic liver regeneration. Targeting the fibrogenic progenitor response represents a promising strategy to improve hepatectomy outcomes in patients with liver fibrosis.

MiR-122 regulates hepatic lipid metabolism and tumor suppression

In this issue of JCI, two independent groups describe the effects of germline and liver-specific deletion of Mir122a, the predominant liver miRNA. Their findings reveal a critical role for miR-122 in fat and cholesterol metabolism but suggest that other metabolic actions of the liver are independent of miR-122. Knockout mice also displayed hepatic inflammation, fibrosis, and a high incidence of hepatocellular carcinoma, suggesting that miR-122 has a tumor suppressor role in hepatocytes.

Protein-tyrosine Phosphatase 1B (PTP1B) Deficiency Confers Resistance to Transforming Growth Factor-β (TGF-β)-induced Suppressor Effects in Hepatocytes

Transforming growth factor-β (TGF-β) plays a dual role in hepatocytes, mediating both tumor suppressor and promoter effects. The suppressor effects of the cytokine can be negatively regulated by activation of survival signals, mostly dependent on tyrosine kinase activity. The aim of our work was to study the role of the protein-tyrosine phosphatase 1B (PTP1B) on the cellular responses to TGF-β, using for this purpose immortalized neonatal hepatocytes isolated from both PTP1B(+/+) and PTP1B(-/-) mice. We have found that PTP1B deficiency conferred resistance to TGF-β suppressor effects, such as apoptosis and growth inhibition, correlating with lower Smad2/Smad3 activation. Both responses were recovered in the presence of the general tyrosine kinase inhibitor genistein. PTP1B(-/-) cells showed elevated NF-κB activation in response to TGF-β. Knockdown of the NF-κB p65 subunit increased cell response in terms of Smads phosphorylation and apoptosis. Interestingly, these effects were accompanied by inhibition of Smad7 up-regulation. In addition, lack of PTP1B promoted an altered NADPH oxidase (NOX) expression pattern in response to TGF-β, strongly increasing the NOX1/NOX4 ratio, which was reverted by genistein and p65 knockdown. Importantly, NOX1 knockdown inhibited nuclear translocation of p65, promoted Smad phosphorylation, and decreased Smad7 levels. In summary, our results suggest that PTP1B deficiency confers resistance to TGF-β through Smad inhibition, an effect that is mediated by NOX1-dependent NF-κB activation, which in turn, increases the level of the Smad inhibitor Smad7 and participates in a positive feedback loop on NOX1 up-regulation.