Fibrocytes are not an essential source of type I collagen during lung fibrosis.

Progressive fibrosis involves accumulation of activated collagen-producing mesenchymal cells. Fibrocytes are hematopoietic-derived cells with mesenchymal features that potentially have a unique and critical function during fibrosis. Fibrocytes have been proposed as an important direct contributor of type I collagen deposition during fibrosis based largely on fate-mapping studies. To determine the functional contribution of hematopoietic cell-derived type I collagen to fibrogenesis, we use a double-transgenic system to specifically delete the type I collagen gene across a broad population of hematopoietic cells. These mice develop a robust fibrotic response similar to littermate genotype control mice injured with bleomycin indicating that fibrocytes are not a necessary source of type I collagen. Using collagen-promoter GFP mice, we find that fibrocytes express type I collagen. However, fibrocytes with confirmed deletion of the type I collagen gene have readily detectable intracellular type I collagen indicating that uptake of collagen from neighboring cells account for much of the fibrocyte collagen. Collectively, these results clarify several seemingly conflicting reports regarding the direct contribution of fibrocytes to collagen deposition.

Mutation of the 5'-untranslated region stem-loop structure inhibits α1(I) collagen expression in vivo.

Type I collagen is a heterotrimeric extracellular matrix protein consisting of two α1(I) chains and one α2(I) chain. During liver fibrosis, activated hepatic stellate cells (HSCs) are the major source of the type I collagen that accumulates in the damaged tissue. Expression of α1(I) and α2(I) collagen mRNA is increased 60-fold compared with quiescent stellate cells and is due predominantly to post-transcriptional message regulation. Specifically, a stem-loop structure in the 5'-untranslated region of α1(I) collagen mRNA may regulate mRNA expression in activated HSCs through its interaction with stem-loop binding proteins. The stem-loop may also be necessary for efficient production and folding of the type I collagen heterotrimer. To assess the role of the stem-loop in type I collagen expression in vivo, we generated a knock-in mouse harboring a mutation that abolished the stem-loop structure. Heterozygous and homozygous knock-in mice exhibited a normal phenotype. However, steady-state levels of α1(I) collagen mRNA decreased significantly in homozygous mutant MEFs as well as HSCs; intracellular and secreted type I collagen protein levels also decreased. Homozygous mutant mice developed less liver fibrosis. These results confirm an important role of the 5' stem-loop in regulating type I collagen mRNA and protein expression and provide a mouse model for further study of collagen-associated diseases.

Kupffer cell and interleukin-12-dependent loss of natural killer T cells in hepatosteatosis.

UNLABELLED: Hepatosteatosis is associated with increased expression of tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-12, major T helper (Th) 1 cytokines, and reduced hepatic natural killer T (NKT) cell numbers. The relationship between lipid accumulation, cytokine expression, and hepatic NKT cells is not known. This study was conducted to assess the role of IL-12 in the development of hepatic steatosis and its potential impact on liver NKT cells. Male C57Bl/6 wildtype (WT) and IL-12-deficient (IL-12(-/-)) mice were fed a choline-deficient diet (CDD) for 0, 10, or 20 weeks. CDD led to marked hepatosteatosis, reduced hepatic but not splenic NKT cell numbers and function, and increased hepatic expression of the T(h)1-type cytokines IL-12, interferon gamma (IFN-gamma), and TNF-alpha in WT mice. The absence of IL-12 resulted in similar CDD-induced hepatosteatosis, but preserved hepatic NKT cells and significantly reduced hepatic IFN-gamma and TNF-alpha expression. Treatment of CDD-fed mice with lipopolysaccharide led to a significant increase in hepatic IL-12 expression, and Kupffer cell (KC) depletion reduced liver IL-12 expression and restored NKT cells in CDD-induced fatty liver. Interestingly, KCs from CDD-fed mice failed to produce increased quantities of IL-12 upon activation in vitro when compared to similarly treated KCs from control fed mice, suggesting that secondary factors in vivo promote heightened IL-12 production. Finally, human livers with severe steatosis showed a substantial decrease in NKT cells. CONCLUSION: Hepatosteatosis reduces the numbers of hepatic NKT cells in a KC-and IL-12-dependent manner. Our results suggest a pivotal and multifunctional role of KC-derived IL-12 in the altered immune response in steatotic liver, a process that is likely active within human nonalcoholic fatty liver disease.

S-adenosyl-L-methionine inhibits collagen secretion in hepatic stellate cells via increased ubiquitination.

BACKGROUND: Liver fibrosis is the excessive accumulation of extracellular matrix (ECM) components that disrupt normal liver microcirculation and lead to organ injury. Hepatic stellate cells (HSCs), following transdifferentiation, are the central mediators of hepatic fibrosis through increased secretion of ECM components, including type I collagen. AIMS: The mechanism(s) by which the antioxidant S-adenosyl-L-methionine (SAMe) acts to modulate type I collagen secretion in activated HSCs was examined. METHODS: Hepatic stellate cells were culture-activated for 13-15 days and treated with SAMe. Type I collagen, proteasomal activity and resident endoplasmic reticulum (ER) protein [78-kDa glucose-regulated protein (Grp78) and protein disulphide isomerase (PDI)] expression were measured. Nuclear factor-κB (NF-κB) activity, and its role in SAMe-mediated collagen inhibition, was determined. Type I collagen polyubiquitination was examined. RESULTS: S-adenosyl-L-methionine significantly inhibited type I collagen secretion without significant changes in type I collagen mRNA expression. SAMe also increased NF-κB activity, and blocking NF-κB activity using a dominant-negative IκBα abolished the SAMe-mediated type I collagen secretion. Examination of the post-transcriptional fate of procollagen demonstrated that SAMe treatment led to intracellular type I collagen polyubiquitination accompanied by diminution of proteasomal activity. Expression of Grp78 and PDI (resident ER proteins) were significantly decreased by SAMe treatment. CONCLUSIONS: S-adenosyl-L-methionine inhibits collagen processing leading to increased ubiquitination and decreased secretion. These findings represent a novel mechanism for modulating type I collagen expression in activated HSCs.

Inhibition of transforming growth factor-beta/Smad signaling improves regeneration of small-for-size rat liver grafts.

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of cell proliferation. This study investigated whether overexpression of Smad7, which blocks TGF-beta-induced activation of Smad2/3, could prevent the suppression of regeneration of small-for-size liver grafts. Rats were intravenously given adenoviruses (2 x 10(10) pfu/rat) carrying the LacZ gene or the Smad7 gene (Ad-Smad7) 3 days prior to liver harvesting. Half-size livers were implanted into recipients of the same weight or twice the donor weight, and this resulted in half-size or quarter-size liver grafts. Cell proliferation, detected by 5-bromo-2'-deoxyuridine (BrdU) incorporation, increased to 23% in half-size grafts at 38 hours after implantation but was only 4% in quarter-size grafts. Graft weight did not increase after 38 hours in full-size and quarter-size grafts but increased 28% in half-size grafts. Ad-Smad7 restored BrdU labeling to 32%, and the graft weight increased to 43% in quarter-size grafts. Serum total bilirubin increased approximately 30-fold after the implantation of quarter-size grafts. Ad-Smad7 blunted hyperbilirubinemia by 80%. The basal hepatic TGF-beta(1) level was 7 ng/g of liver wet weight, and this increased to 30 ng/g at 1.5 hours after the transplantation of full-size grafts but decreased rapidly afterwards. After the transplantation of quarter-size grafts, however, TGF-beta(1) progressively increased to 159 ng/g in 38 hours. Nuclear phosphorylated Smad2/3 was barely detectable, and p21Cip1 expression was negligible in full-size grafts but increased markedly in quarter-size grafts. Ad-Smad7 blocked Smad2/3 activation and expression of p21Cip1. Together, these data show that TGF-beta is responsible, at least in part, for the defective liver regeneration in small-for-size grafts by activating the Smad signaling pathway.

Inhibition of phosphatidylinositol 3-kinase signaling in hepatic stellate cells blocks the progression of hepatic fibrosis.

UNLABELLED: The hepatic stellate cell (HSC) is the primary cell type in the liver responsible for excess collagen deposition during fibrosis. Following a fibrogenic stimulus the cell changes from a quiescent vitamin A-storing cell to an activated cell type associated with increased extracellular matrix synthesis and increased cell proliferation. The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been shown to regulate several aspects of HSC activation in vitro, including collagen synthesis and cell proliferation. Using a targeted approach to inhibit PI3K signaling specifically in HSCs, we investigated the role of PI3K in HSCs using a rodent model of hepatic fibrosis. An adenovirus expressing a dominant negative form of PI3K under control of the smooth muscle alpha-actin (alphaSMA) promoter was generated (Ad-SMAdnPI3K). Transducing HSCs with Ad-SMAdnPI3K resulted in decreased proliferation, migration, collagen expression, and several additional profibrogenic genes, while also promoting cell death. Inhibition of PI3K signaling was also associated with reduced activation of Akt, p70 S6 kinase, and extracellular regulated kinase signaling as well as reduced cyclin D1 expression. Administering Ad-SMAdnPI3K to mice following bile duct ligation resulted in reduced HSC activation and decreased extracellular matrix deposition, including collagen expression. A reduction in profibrogenic mediators, including transforming growth factor beta, tissue inhibitor of metalloproteinase 1, and connective tissue growth factor was also noted. However, liver damage, assessed by alanine aminotransferase levels, was not reduced. CONCLUSION: Inhibition of PI3K signaling in HSCs during active fibrogenesis inhibits extracellular matrix deposition, including synthesis of type I collagen, and reduces expression of profibrogenic factors. These data suggest that targeting PI3K signaling in HSCs may represent an effective therapeutic target for hepatic fibrosis.

Accumulation of hedgehog-responsive progenitors parallels alcoholic liver disease severity in mice and humans.

BACKGROUND & AIMS: Improving outcomes in alcoholic liver disease (ALD) necessitates better understanding of how habitual ethanol (EtOH) consumption alters normal regenerative mechanisms within the liver. Hedgehog (Hh) pathway activation promotes expansion of progenitor populations in other tissues. We evaluated the hypothesis that chronic EtOH exposure activates Hh signaling in liver. METHODS: Hh signaling, liver progenitors, transforming growth factor (TGF)-beta induction, and liver damage were compared in mice fed chow, high-fat diets (HF), or HF + EtOH for 4 weeks. Susceptibility to TGF-beta-mediated apoptosis was compared in Hh-responsive liver cells (eg, immature cholangiocytes and oval cells) and mature hepatocytes (which are unresponsive to Hh). Hepatic accumulation of Hh-responsive cells were compared in controls and ALD patients and correlated with a discriminant function (DF) that predicts subacute mortality. RESULTS: Hh signaling and numbers of Hh-responsive cells were increased in HF mice and greatest in HF+EtOH mice. In both, progenitor and stromal cell populations harbored Hh-responsive cells. More ductular-type progenitors and fibrosis markers were noted in HF+EtOH mice than in HF mice. The former also expressed more TGF-beta-1. TGF-beta-1 treatment selectively promoted the viability of Hh-responsive immature liver cells and caused mature hepatocytes that survived to produce Hh ligands. Hh-responsive cells were increased in ALD patients. Lobular accumulation of Hh-responsive immature ductular cells was greater in those with a DF >32 than those with a DF <32. CONCLUSIONS: Hh signaling is increased in ALD and may influence ALD outcomes by promoting hepatic accumulation of immature ductular cells.

TNFalpha is required for cholestasis-induced liver fibrosis in the mouse.

TNFalpha, a mediator of hepatotoxicity in several animal models, is elevated in acute and chronic liver diseases. Therefore, we investigated whether hepatic injury and fibrosis due to bile duct ligation (BDL) would be reduced in TNFalpha knockout mice (TNFalpha-/-). Survival after BDL was 60% in wild-type mice (TNFalpha+/+) and 90% in TNFalpha-/- mice. Body weight loss and liver to body weight ratios were reduced in TNFalpha-/- mice compared to TNFalpha+/+ mice. Following BDL, serum alanine transaminases (ALT) levels were elevated in TNFalpha+/+ mice (268.6+/-28.2U/L) compared to TNFalpha-/- mice (105.9U/L+/-24.4). TNFalpha-/- mice revealed lower hepatic collagen expression and less liver fibrosis in the histology. Further, alpha-smooth muscle actin, an indicator for activated myofibroblasts, and TGF-beta mRNA, a profibrogenic cytokine, were markedly reduced in TNFalpha-/- mice compared to TNFalpha+/+ mice. Thus, our data indicate that TNFalpha induces hepatotoxicity and promotes fibrogenesis in the BDL model.

Pivotal role of Smad3 in a mouse model of T cell-mediated hepatitis.

UNLABELLED: Transforming growth factor beta (TGFbeta) promotes hepatocellular apoptosis and suppresses hepatic lymphocyte responses in part through activation of Smad3. The purpose of the current study was to determine the importance of Smad3 signaling in an experimental model of autoimmune hepatitis induced by concanavalin A (ConA), a process involving T cell activation and hepatocellular apoptosis. C57Bl/6 wild-type (Wt) or Smad3-deficient (Smad3(-/-)) mice were injected intravenously with 15 mg/kg ConA or vehicle. Nine hours post ConA injection, Wt mice presented with severe hepatitis as assessed by increased liver transferases. This injury was associated with eosinophil accumulation and preceded at 3 hours post-injection by significant increases in hepatic T helper 1 (interferon gamma) and T helper 2 (interleukin-4) cytokine production. Absence of Smad3 significantly blunted hepatocellular injury 9 hours post ConA injection, which was associated with reduced early T helper 1 and T helper 2 cytokine production and eosinophil accumulation. Smad3(-/-) livers also showed significant reductions in hepatocellular apoptosis as assessed by terminal UTP nick-end labeling when compared to ConA-treated Wt mice in conjunction with reduced caspase 3 cleavage, which was likely mediated by a Smad3-dependent inhibition of the survival factor extracellular signal-regulated kinase 1/2. In vitro, Smad3(-/-) hepatocytes were resistant to TGFbeta-induced apoptosis, and this protection was dependent on extracellular signal-regulated kinase activation. CONCLUSION: Together, these results show, for the first time, the significance of Smad3 signaling in autoimmune hepatitis, underlining the control of Smad3-dependent TGFbeta signaling on proinflammatory cytokine production, eosinophil recruitment, and hepatocellular apoptosis. Interruption of this pathway could be beneficial clinically to limit acute fulminant liver pathologies.

The tumor suppressor protein PTEN inhibits rat hepatic stellate cell activation.

BACKGROUND: Following a fibrogenic stimulus, the hepatic stellate cell (HSC) transforms from a quiescent to an activated cell type associated with increased proliferation, collagen and smooth muscle alpha-actin (alphaSMA) expression. Phosphatase and Tensin Homolog Deleted on Chromosome Ten (PTEN), a tumor suppressor phosphatase, has been shown to play a role in several nonmalignant diseases. Here, we investigated the role of PTEN during HSC activation. METHODS: Rat HSCs 2 days after isolation were transduced with adenoviruses expressing either the wild-type (WT) or a dominant negative form of PTEN, and culture-associated activation of HSCs, including morphological changes, expression of alphaSMA and alpha1(I) collagen, and cell proliferation, were evaluated. Apoptosis of HSCs was detected by measuring activity of caspase 3/7. Phosphorylation status of Akt, p70(S6K), and Erk was detected by Western blotting. RESULTS: Overexpression of WT-PTEN inhibited phenotypic changes were associated with HSC activation, including morphological changes, expression of alphaSMA and alpha1(I) collagen, and HSC proliferation, including cyclin D1 expression. WT-PTEN expression also induced apoptosis in HSCs with increased caspase 3/7 activity. Expression of WT-PTEN also caused decreased activation of Akt, p70(S6K), and Erk signaling pathways. CONCLUSIONS: Taken together, these findings show that PTEN represents an important negative regulator for transactivation of HSCs. This may have important implications for the design of therapeutic strategies to prevent the progression of liver fibrosis.

Transforming growth factor beta mediates hepatocyte apoptosis through Smad3 generation of reactive oxygen species.

TGFbeta induces hepatocyte apoptosis via reactive oxygen species (ROS) generation, the mitochondrial permeability transition (MPT), and caspase activation. The role of the Smad pathway in these events is unknown. In this study primary hepatocytes were isolated from Smad3 wild-type (+/+) and knockout (-/-) mice, and were treated with TGFbeta (5ng/ml) and/or trolox (2mM). ROS generation, MPT, TGFbeta-dependent transcription, and apoptosis were assessed in the presence or absence of Smad3 wild-type (WT) and dominant-negative (DN) plasmids. With TGFbeta treatment, Smad3 (-/-) hepatocytes did not generate ROS activity, exhibit MPT, activate caspases, or undergo apoptosis when compared to Smad 3 (+/+) hepatocytes. Similarly, transfection of Smad3 (+/+) hepatocytes with DN-Smad3 inhibited TGFbeta-mediated transcription, ROS generation, MPT, and apoptosis. However, Smad3 (-/-) cells transfected with WT-Smad3 and treated with TGFbeta demonstrated increased transcriptional activity, the MPT, and TGFbeta-induced apoptosis. TGFbeta-mediated ROS generation occurred through an NADPH-like oxidase pathway since diphenyleneiodonium chloride inhibited ROS induction. In conclusion, TGFbeta-induced hepatocyte apoptosis occurs through Smad3 dependent activation of ROS with subsequent activation of the MPT and caspases.

Mechanisms of liver fibrosis.

Liver fibrosis represents a significant health problem worldwide of which no acceptable therapy exists. The most characteristic feature of liver fibrosis is excess deposition of type I collagen. A great deal of research has been performed to understand the molecular mechanisms responsible for the development of liver fibrosis. The activated hepatic stellate cell (HSC) is the primary cell type responsible for the excess production of collagen. Following a fibrogenic stimulus, HSCs change from a quiescent to an activated, collagen-producing cell. Numerous changes in gene expression are associated with HSC activation including the induction of several intracellular signaling cascades, which help maintain the activated phenotype and control the fibrogenic and proliferative state of the cell. Detailed analyses in understanding the molecular basis of collagen gene regulation have revealed a complex process offering the opportunity for multiple potential therapeutic strategies. However, further research is still needed to gain a better understanding of HSC activation and how this cell maintains its fibrogenic nature. In this review we describe many of the molecular events that occur following HSC activation and collagen gene regulation that contribute to the fibrogenic nature of these cells and provide a review of therapeutic strategies to treat this disease.

Methods for assessing the molecular mechanisms controlling gene regulation.

Regulation of gene expression is a complex process that can be controlled at several steps,including transcription, pre-mRNA splicing and export, mRNA stability, translation, protein modification, and protein half-life. Because transcriptional regulation often involves DNA-protein interactions, several techniques are used, including nuclear run-off assays, DNase I footprinting analysis, and mobility shift assays. Together these assays can determine transcriptional rates, as well as locate, identify, and characterize DNA-protein interactions. Functional analyses to assess the role of specific regulatory regions in gene regulation often requires the introduction of reporter genes under the control of regulatory elements being investigated into mammalian cells. This is often accomplished using transient transfections or, more recently, adenovirally mediated gene delivery. Adenovirus-mediated gene delivery is useful for cells that are difficult to transfect with conventional methods, such as hepatic stellate cells, and when close to 100% of transfection efficiency is needed. Posttranscriptional regulation is often involved in regulating gene expression and may involve mRNA stabilization or translational regulation. Together, these techniques can provide information about which step a particular gene is predominantly regulated. This chapter will detail common methodology used to assess molecular mechanisms involved in controlling gene regulation.

Liver fibrosis: signals leading to the amplification of the fibrogenic hepatic stellate cell.

Liver fibrosis represents a major medical problem with significant morbidity and mortality. Worldwide hepatitis viral infections represent the major cause liver fibrosis; however, within the United States chronic ethanol consumption is the leading cause of hepatic fibrosis. Other known stimuli for liver fibrosis include helminthic infection, iron or copper overload and biliary obstruction. Fibrosis can be classified as a wound healing response to a variety of chronic stimuli that is characterized by an excessive deposition of extracellular matrix proteins of which type I collagen predominates. This excess deposition of extracellular matrix proteins disrupts the normal architecture of the liver resulting in pathophysiological damage to the organ. If left untreated fibrosis can progress to liver cirrhosis ultimately leading to organ failure and death if left untreated. This review will discuss the molecular events leading to liver fibrosis. The discussion will include collagen gene regulation and proliferative signals that contribute to the amplification of the hepatic stellate cell, the primary fibrogenic cell type that resides in the liver.

PPAR Gamma and Hepatic Stellate Cells.

Activation of Hepatic stellate cells (HSC) in fibrogenesis involves distinct morphological and biochemical changes. This activation requires the coordinated changes in activity of several transcription factors. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is one such factor whose activity is decreased in activated HSC. PPAR gamma ligands suppress several markers of HSC activation such as expression of collagen and alpha smooth muscle actin (alpha-SMA), cell proliferation and migration. Expression of PPAR gamma, per se, also inhibits HSC activation. These findings support the role of PPAR gamma in reversion of activated HSC toward their quiescent state.

TNF alpha-induced hepatocyte apoptosis is associated with alterations of the cell cycle and decreased stem loop binding protein.

BACKGROUND: Inhibition of nuclear factor kappa B (NF kappa B) during liver regeneration induces hepatocyte apoptosis associated with normal DNA synthesis but decreased mitosis, suggesting that inhibition of NF kappa B impairs progression from S-phase through the G(2)/M phase of the cell cycle. Our aim was to determine if inhibition of NF kappa B alters cell cycle characteristics in hepatocytes treated with tumor necrosis factor alpha (TNF alpha). METHODS: Primary hepatocytes from BALB/c mice were infected with adenoviruses expressing luciferase (control; AdLuc) or the I kappa B super-repressor (AdI kappa B) and treated with or without TNF alpha (30 ng/ml). Flow cytometry was performed (0 to 40 hours) to determine apoptosis and cell cycle progression. Reverse transcriptase-polymerase chain reaction and immunoblots assessed changes in cell cycle mediators and antiapoptotic factors. RESULTS: Primary hepatocytes treated with AdI kappa B and TNF alpha demonstrated significantly more S-phase cells (14% +/- 3% vs 6% +/- 2%, P<.05) at 14 hours compared with controls. Inhibition of NF kappa B with or without TNFalpha was associated with decreased expression of stem loop bind protein, a marker of cell cycle progression through S-phase. The NF kappa B-induced antiapoptotic proteins, iNOS and TRAF2, had decreased message at 9 and 12 hours, respectively, in TNF alpha- and AdI kappa B-treated cells. CONCLUSION: Inhibition of NF kappa B in TNF alpha-treated primary mouse hepatocytes is associated with increased S-phase cell cycle retention and decreased stem loop bind protein.

Transforming growth factor-beta1 induces hepatocyte apoptosis by a c-Jun independent mechanism.

BACKGROUND: During hepatic regeneration, transforming growth factor (TGF)-beta1 messenger RNA increases after the initial cycle of DNA synthesis, and it may control hepatocyte growth by inducing apoptosis. TGF-beta1 also induces c-Jun, a potential proapoptotic transcription factor. We hypothesized that autocrine expression of activated TGF-beta1 (Ad5aTGF-beta1) would increase c-jun expression in rat liver and limit hepatic regeneration by inducing apoptosis. METHODS: Male rats (175 to 200 g) received portal venous injections with adenoviruses expressing either luciferase (Ad5Luc), as a control, or Ad5aTGF-beta1 at a dose of 6 x 10(9) plaque-forming units. Livers were harvested 24 or 48 hours after injection and nuclear extracts and total RNA isolated. TGF-beta1 expression was confirmed by Northern blot analysis in all TGF-beta1-injected rats. RESULTS: A 2.5-fold increase in c-jun mRNA expression was detected in Ad5aTGF-beta1-infected rats compared with control rats. Transcriptional activity was assessed with an AP-1-responsive-reporter gene that increased 3-fold in rat primary hepatocytes infected with Ad5aTGF-beta1. C-Jun N-terminal kinase activity also increased 6- to 7-fold in Ad5aTGF-beta1-treated rats 24 and 48 hours after injection. Ad5aTGF-beta1-injected rats demonstrated increased AP-1 binding activity compared with Ad5Luc rats. Hepatocytes infected in vitro with Ad5aTGF-beta1 demonstrated increased apoptosis compared with Ad5Luc-infected hepatocytes (47% vs 27%) 36 hours after infection. Dual adenoviral infection with Ad5aTGF-beta1 and a dominant-negative c-Jun (Ad5TAM67) decreased AP-1-induced Ad5Luc activity but not hepatocyte apoptosis (46% with dominant-negative c-Jun and 47% without). CONCLUSIONS: These data demonstrate that TGF-beta1 induces c-Jun, but c-Jun is not proapoptotic in hepatocytes.

Cholestasis induces murine hepatocyte apoptosis and DNA synthesis with preservation of the immediate-early gene response.

BACKGROUND: Major hepatic resection in patients with unrelieved obstructive jaundice carries an increased risk of postoperative liver failure. We hypothesized that cholestasis induces hepatocyte apoptosis and impairs hepatic regeneration by inhibiting up-regulation of the known immediate-early response genes, nuclear factor kappa B (NF-kappaB) and activating protein-1 (AP-1). The aim of this study was to determine whether the immediate-early gene response in hepatic regeneration remains intact in extrahepatic cholestasis. METHODS: Eight-week-old BALB/c mice underwent either sham operation (SO) or common bile duct ligation (BDL). Two-thirds partial hepatectomy (PH) was performed at 4 and 7 days, with remnant liver harvested 0, 15, 30, or 60 minutes after PH. Serum analysis for markers of cholestasis and histopathology was obtained. Proliferating cell nuclear antigen and terminal deoxyuridine triphosphate nick end labeling (TUNEL) immunohistochemistry for detection of DNA synthesis and apoptosis, respectively, was performed 4, 7, or 10 days after SO or BDL. Liver samples from 0, 15, 30, or 60 minutes after PH were analyzed for NF-kappaB and AP-1 DNA binding activity by using electrophoretic mobility shift assays. RESULTS: Increased serum bilirubin level and hematoxylin-eosin-stained liver sections confirmed cholestasis in BDL mice. BDL induced marked DNA synthesis and hepatocyte apoptosis in prehepatectomy liver at both 4 and 7 days. Substantially higher basal levels of both NF-kappaB and AP-1 binding activity were present in BDL compared with SO mice. Fold induction of NF-kappaB and AP-1, however, was similar between BDL and SO mice. Cholestasis induced hepatocyte DNA synthesis and apoptosis. Basal NF-kappaB and AP-1 DNA binding activity was increased in BDL mice, but fold induction of these immediate-early genes did not differ from controls. CONCLUSIONS: Although basal NF-kappaB and AP-1 DNA binding is increased in cholestasis, the immediate-early gene response to PH remains intact in BDL mice.

Distinct populations of hepatic stellate cells in the mouse liver have different capacities for retinoid and lipid storage.

UNLABELLED: Hepatic stellate cell (HSC) lipid droplets are specialized organelles for the storage of retinoid, accounting for 50-60% of all retinoid present in the body. When HSCs activate, retinyl ester levels progressively decrease and the lipid droplets are lost. The objective of this study was to determine if the HSC population in a healthy, uninjured liver demonstrates heterogeneity in its capacity for retinoid and lipid storage in lipid droplets. To this end, we utilized two methods of HSC isolation, which leverage distinct properties of these cells, including their vitamin A content and collagen expression. HSCs were isolated either from wild type (WT) mice in the C57BL/6 genetic background by flotation in a Nycodenz density gradient, followed by fluorescence activated cell sorting (FACS) based on vitamin A autofluorescence, or from collagen-green fluorescent protein (GFP) mice by FACS based on GFP expression from a GFP transgene driven by the collagen I promoter. We show that GFP-HSCs have: (i) increased expression of typical markers of HSC activation; (ii) decreased retinyl ester levels, accompanied by reduced expression of the enzyme needed for hepatic retinyl ester synthesis (LRAT); (iii) decreased triglyceride levels; (iv) increased expression of genes associated with lipid catabolism; and (v) an increase in expression of the retinoid-catabolizing cytochrome, CYP2S1. CONCLUSION: Our observations suggest that the HSC population in a healthy, uninjured liver is heterogeneous. One subset of the total HSC population, which expresses early markers of HSC activation, may be "primed" and ready for rapid response to acute liver injury.