Association of filamin A and vimentin with hepatitis C virus proteins in infected human hepatocytes.

Chronic hepatitis C (CHC) infection caused by hepatitis C virus (HCV) is a major cause of liver disease and remains a major therapeutic challenge. A variety of host proteins interact with HCV proteins. The definitive role of cytoskeletal (CS) proteins in HCV infection remains to be determined. In this study, our aim was to determine the expression profile of differentially regulated and expressed selected CS proteins and their association with HCV proteins in infected hepatocytes as possible therapeutic targets. Using proteomics, qRT-PCR, Western blot and immunofluorescence techniques, we revealed that filamin A (fila) and vimentin (vim) were prominently increased proteins in HCV-expressing human hepatoma cells compared with parental cells and in liver biopsies from patients with CHC vs controls. HCV nonstructural (NS) 3 and NS5A proteins were associated with fila, while core protein partially with fila and vim. Immunoprecipitation showed interactions among fila and NS3 and NS5A proteins. Cells treated with interferon-α showed a dose- and time-dependent decrease in CS and HCV proteins. NS proteins clustered at the perinuclear region following cytochalasin b treatment, whereas disperse cytoplasmic and perinuclear distribution was observed in the no-treatment group. This study demonstrates and signifies that changes occur in the expression of CS proteins in HCV-infected hepatocytes and, for the first time, shows the up-regulation and interaction of fila with HCV proteins. Association between CS and HCV proteins may have implications in future design of CS protein-targeted therapy for the treatment for HCV infection.

Anti-Fas induces hepatic chemokines and promotes inflammation by an NF-kappa B-independent, caspase-3-dependent pathway.

Agonistic antibodies against the Fas receptor, when administered to mice in vivo, cause significant apoptosis in the liver. In this study we show that anti-Fas antibody not only causes apoptosis of liver cells but also provokes hepatic inflammation. Two hours after injection of anti-Fas, when mice displayed evidence of caspase-3 activation and apoptosis, we found significant hepatic induction of the CXC chemokines macrophage inflammatory protein-2 and KC. Coincident with the chemokine induction was infiltration of the hepatic parenchyma by neutrophils. Neutralization experiments identified that chemokines were the cause of Fas-induced hepatic inflammation, with KC having the predominant effect. Chemokine induction in the livers of anti-Fas-treated mice was not associated with activation of NF-kappa B. Instead, it coincided with nuclear translocation of activator protein-1 (AP-1). AP-1 activation in liver was detected 1-2 h after anti-Fas treatment, suggesting a connection to the onset of apoptosis. When apoptosis was prevented by pretreating mice with a caspase-3 inhibitor, AP-1 activation and hepatic chemokine production were both significantly reduced. Hepatic inflammation was also reduced by 70%. Taken together, these findings indicate that Fas ligation can induce inflammation in the liver in vivo. Inflammation does not arise from Fas-mediated signaling through NF-kappa B; rather, it represents an indirect effect, requiring activation of caspase-3 and nuclear translocation of AP-1.

Monocyte chemoattractant protein-1 expression by osteoblasts following infection with Staphylococcus aureus or Salmonella.

Two common pathogens of bone, Staphylococcus aureus and Salmonella, were investigated for their ability to induce chemokine expression in bone-forming osteoblasts. Cultured mouse or human osteoblasts could rapidly respond to bacterial infection by upregulating the mRNA encoding the chemokine, monocyte chemoattractant protein-1 (MCP-1). This rapid induction occurred on infection with either the gram-positive pathogen, S. aureus, or the gram-negative pathogen, Salmonella. Increased mRNA expression translated into MCP-1 secretion by cultured mouse or human osteoblasts in response to viable bacteria, whereas UV-killed bacteria were less effective in stimulating chemokine secretion. There was a dose-response relationship observed between the amount of input bacteria and increases in MCP-1 secretion. Immunohistochemical staining of infected osteoblasts indicated that the majority of cells could express MCP-1, with some osteoblasts having a higher intensity of staining than others. Organ cultures of mouse calvaria (skullcap) bone showed increases in MCP-1 immunostaining following bacterial infection. The immunoreactive MCP-1 in infected calvaria localized to areas containing active osteoblasts. Taken together, these studies demonstrate a conserved osteoblast-derived MCP-1 response to two very different pathogens of bone.

Attenuation of CCl(4)-induced hepatic fibrosis by GdCl(3) treatment or dietary glycine.

The role of Kupffer cells in CCl(4)-induced fibrosis was investigated in vivo. Male Wistar rats were treated with phenobarbital and CCl(4) for 9 wk, and a group of rats were injected with the Kupffer cell toxicant gadolinium chloride (GdCl(3)) or were fed glycine, which inactivates Kupffer cells. After CCl(4) alone, the fibrosis score was 3.0 +/- 0.1 and collagen protein and mRNA expression were elevated, but GdCl(3) or glycine blunted these parameters. Glycine did not alter cytochrome P-450 2E1, making it unlikely that glycine affects CCl(4) metabolism. Treatment with GdCl(3) or glycine prevented CCl(4)-induced increases in transforming growth factor (TGF)-beta 1 protein levels and expression. CCl(4) treatment increased alpha-smooth muscle actin staining (score 3.0 +/- 0.2), whereas treatment with GdCl(3) and glycine during CCl(4) exposure blocked this effect (1.2 +/- 0.5); there was no staining with glycine treatment. These results support previous in vitro data and demonstrate that treatment of rats with the selective Kupffer cell toxicant GdCl(3) prevents stellate cell activation and the development of fibrosis.

Autocrine expression of activated transforming growth factor-beta(1) induces apoptosis in normal rat liver.

The aim of this study was to determine the differential effects of latent and activated transforming growth factor (TGF)-beta(1) in growth control of normal and proliferating hepatocytes in vivo. Rats were injected with adenoviruses expressing control transgenes (Ctrl), latent TGF-beta(1) [TGF-beta(L)], or activated TGF-beta(1) [TGF-beta(A)]. Additional animals underwent two-thirds partial hepatectomy (PH) 24 h after injection. Increased hepatocyte apoptosis was observed in TGF-beta(A)-injected but not TGF-beta(L)-injected animals 24 h postinjection (10.5%) compared with Ctrl animals (0.37%). The percent of apoptotic cells increased to 32.1% in TGF-beta(A)-injected animals 48 h after injection. Furthermore, TGF-beta(A)-injected rats did not survive 24 h after PH. Four hours after PH, 0.25 and 14.1% apoptotic hepatocytes were seen in Ctrl- and TGF-beta(A)-injected rats, respectively. TGF-beta(A)-induced apoptosis in primary rat hepatocytes was blocked with a pancaspase inhibitor. Thus autocrine expression of TGF-beta(A) but not TGF-beta(L) induces hepatocyte apoptosis in the normal rat liver. Rats overexpressing TGF-beta(A) do not survive two-thirds PH due to hepatic apoptosis. Thus activation of TGF-beta(1) may be a critical step in the growth control of normal and proliferating rat hepatocytes.

Expression of small heat shock protein alphaB-crystallin is induced after hepatic stellate cell activation.

Using the differential PCR display method to select cDNA fragments that are differentially expressed after hepatic stellate cell (HSC) activation, we have isolated from activated HSCs a cDNA that corresponds to rat alphaB-crystallin. Northern blots confirmed expression of alphaB-crystallin in culture-activated HSCs but not in quiescent HSCs. Western blot analysis and immunocytochemical staining confirmed expression of alphaB-crystallin protein in activated but not quiescent HSCs. alphaB-crystallin is induced as early as 6 h after plating HSCs on plastic and continues to be expressed for 14 days in culture. Expression of alphaB-crystallin was also induced in vivo in activated HSCs from experimental cholestatic liver fibrosis. Confocal microscopy demonstrated a cytoplasmic distribution of alphaB-crystallin in a cytoskeletal pattern. Heat shock treatment resulted in an immediate perinuclear redistribution that in time returned to a normal cytoskeletal distribution. The expression pattern of alphaB-crystallin was similar to that of HSP25, another small heat shock protein, but differed from the classic heat shock protein HSP70. Therefore, alphaB-crystallin represents an early marker for HSC activation.

Peroxisome proliferator-activated receptors and hepatic stellate cell activation.

The present study examined the roles of peroxisome proliferator-activated receptors (PPAR) in activation of hepatic stellate cells (HSC), a pivotal event in liver fibrogenesis. RNase protection assay detected mRNA for PPARgamma1 but not that for the adipocyte-specific gamma2 isoform in HSC isolated from sham-operated rats, whereas the transcripts for neither isoforms were detectable in HSC from cholestatic liver fibrosis induced by bile duct ligation (BDL). Semi-quantitative reverse transcriptase-polymerase chain reaction confirmed a 70% reduction in PPARgamma mRNA level in HSC from BDL. Nuclear extracts from BDL cells showed an expected diminution of binding to PPAR-responsive element, whereas NF-kappaB and AP-1 binding were increased. Treatment of cultured-activated HSC with ligands for PPARgamma (10 microm 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)); 0.1 approximately 10 microm BRL49653) inhibited DNA and collagen synthesis without affecting the cell viability. Suppression of HSC collagen by 15dPGJ(2) was abrogated 70% by the concomitant treatment with a PPARgamma antagonist (GW9662). HSC DNA and collagen synthesis were inhibited by WY14643 at the concentrations known to activate both PPARalpha and gamma (>100 microm) but not at those that only activate PPARalpha (<10 microm) or by a synthetic PPARalpha-selective agonist (GW9578). 15dPGJ(2) reduced alpha1(I) procollagen, smooth muscle alpha-actin, and monocyte chemotactic protein-1 mRNA levels while inducing matrix metalloproteinase-3 and CD36. 15dPGJ(2) and BRL49653 inhibited alpha1(I) procollagen promoter activity. Tumor necrosis factor alpha (10 ng/ml) reduced PPARgamma mRNA, and this effect was prevented by the treatment with 15dPGJ(2). These results demonstrate that HSC activation is associated with the reductions in PPARgamma expression and PPAR-responsive element binding in vivo and is reversed by the treatment with PPARgamma ligands in vitro. These findings implicate diminished PPARgamma signaling in molecular mechanisms underlying activation of HSC in liver fibrogenesis and the potential therapeutic value of PPARgamma ligands for liver fibrosis.

c-Jun does not mediate hepatocyte apoptosis following NFkappaB inhibition and partial hepatectomy.

Inhibition of the transcription factor nuclear factor kappa B (NFkappaB) induces marked hepatocyte apoptosis and liver dysfunction after partial hepatectomy (PH) in rats. Hepatocyte apoptosis may be due to direct inhibition of NFkappaB-induced hepatocyte survival genes or due to indirect increased signaling through the stress-activated protein kinase pathway (SAPK), resulting in increased c-Jun. c-Jun, an AP-1 transcription factor, induces apoptosis in fibroblasts. Our aim was to determine if hepatocyte apoptosis following inhibition of NFkappaB and partial hepatectomy in rats is due to increased c-Jun. Adult male Sprague-Dawley rats (200 g) were injected intraportally with 6 x 10(9) PFU adenoviral vector containing luciferase (Ad5Luc) or superrepressor IkappaB (Ad5IkappaB) transgene that inhibits NFkappaB translocation into the nucleus. Two-thirds PH was performed 24 h after vector administration, and the remnant liver was harvested 30 min or 24 h after PH. Northern and Western blots were performed to examine the presence of IkappaB and c-Jun. A GST c-Jun kinase assay was used to examine Jun-N-terminal kinase (JNK) activity. AP-1 DNA binding activity was assessed by electrophoretic mobility shift assay. TUNEL assay was performed to assess apoptosis. All rats receiving adenoviral vectors expressed the luciferase or superrepressor IkappaB transgenes. c-Jun mRNA, protein levels, and DNA binding activity were not increased in rats treated with Ad5IkappaB at 30 min after PH compared to rats injected with Ad5Luc. Jun kinase activity increased following partial hepatectomy, but activity was similar in Ad5Luc- and Ad5IkappaB-treated animals. AP-1 DNA binding activity was not altered substantially in rats treated with Ad5IkappaB. The percentage of apoptotic hepatocytes was similar between Ad5Luc- and Ad5IkappaB-injected animals at 0 h, but livers from Ad5IkappaB-treated rats had increased apoptosis at 24 h compared to Ad5Luc rats (24% vs. 4%) after PH. Hepatocyte apoptosis after NFkappaB inhibition and PH is not mediated by increased JNK activity or c-Jun.

Interleukin-6 increases rat metalloproteinase-13 gene expression through stimulation of activator protein 1 transcription factor in cultured fibroblasts.

The role of IL-6 in collagen production and tissue remodeling is controversial. In Rat-1 fibroblasts, we measured the effect of IL-6 on matrix metalloproteinase-13 (MMP-13), c-jun, junB, and c-fos gene expression, binding of activator protein 1 (AP1) to DNA, amount of AP1 proteins, immunoreactive MMP-13 and TIMP-1 proteins, and Jun N-terminal kinase activity. We show that IL-6 increased MMP-13-mRNA and MMP-13 protein. These effects were exerted by acting on the AP1-binding site of the MMP-13 promoter, as shown by transfecting cells with reporter plasmids containing mutations in this element. Mobility shift assays demonstrated that IL-6 induced the DNA binding activity of AP1. This effect was accompanied by a marked increase in c-Jun, JunB, and c-Fos mRNA, as well as in c-Jun protein and its phosphorylated form. The latter is not due to increased Jun N-terminal kinase activity but to a decreased serine/threonine phosphatase activity. We conclude that IL-6 increases interstitial MMP-13 gene expression at the promoter level. This effect seems to be mediated by the induction of c-jun, junB, and c-fos gene expression, by the binding of AP1 to DNA, by increasing phosphorylated c-Jun, and by the inhibition of serine/threonine phosphatase activity. These effects of IL-6 might contribute to remodeling connective tissue.

NF-kappaB inhibits expression of the alpha1(I) collagen gene.

Fibrosis results from an increase in the synthesis and deposition of type I collagen. Fibrosis is frequently associated with inflammation, which is accompanied by increased levels of tumor necrosis factor-alpha (TNFalpha) and activation of the transcription factor NF-kappaB. However, several agents known to activate NF-kappaB, such as phorbol 12-myristate 13-acetate (PMA) and TNFalpha, result in decreased expression of type I collagen. Therefore, we directly examined the effects of NF-kappaB on alpha1(I) collagen gene expression in two collagen-producing cells, NIH 3T3 fibroblasts and hepatic stellate cells (HSCs). Transient transfections of NIH 3T3 cells or HSCs using NF-kappaB p50, p65, and c-Rel expression plasmids with collagen reporter gene plasmids demonstrated a strong inhibitory effect on transcription of the collagen gene promoter. Dose-response curves showed that p65 was a stronger inhibitor of collagen gene expression than was NF-kappaB p50 or c-Rel (maximum inhibition 90%). Transient transfections with reporter gene plasmids containing one or two Spl binding sites demonstrated similar inhibitory effects of NF-kappaB p65 on the activity of these reporter genes, suggesting that the inhibitory effects of NF-kappaB p65 are mediated through the critical Spl binding sites in the alpha1(I) collagen gene promoter. Cotransfection experiments using either a super-repressor I[ke]B or Spl partially blocked the inhibitory effects of p65 on collagen reporter gene activity. Coimmunoprecipitation experiments demonstrated that NF-kappaB and Spl do interact in vivo. Nuclear run-on assays showed that NF-kappaB p65 inhibited transcription of the endogenous alpha1(I) collagen gene. Together, these results demonstrate that NF-kappaB decreases transcription of the alpha1(I) collagen gene.

Expression of interleukin-10 by in vitro and in vivo activated hepatic stellate cells.

Activated hepatic stellate cells (HSC) participate in matrix remodeling and deposition in liver fibrosis. The present study demonstrates that interleukin (IL)-10 is expressed by HSC upon activation in vitro or in vivo and that autocrine effects of this cytokine include inhibition of collagen production. Culture activation of HSC caused a distinct increase in IL-10 mRNA level compared with freshly isolated quiescent HSC. Treatment of cultured HSC with tumor necrosis factor-alpha, transforming growth factor-beta, or lipopolysaccharide further increased IL-10 mRNA by 2-fold and resulted in the release of IL-10 protein into the medium. HSC isolated from rats after bile duct ligation (BDL) showed prominent increases in IL-10 mRNA (x 100) and protein (x 30) levels at 7 days after BDL, but such induction disappeared in advanced liver fibrosis (19 days after BDL). IL-10 expression correlated positively with mRNA expression of interstitial collagenase and inversely with that of alpha1(I) collagen. Addition of anti-IL-10 IgG to cultured HSC caused enhanced collagen production under a basal or stimulated condition with TGF-beta, tumor necrosis factor-alpha, or lipopolysaccharide. These effects were associated with increased alpha1(I) collagen mRNA and reciprocally reduced collagenase mRNA levels. Co-transfection of HSC with an IL-10 expression vector and collagen reporter genes showed a 40% inhibition of alpha1(I) collagen promoter activity. These results demonstrate that activation of HSC causes enhanced autocrine expression of IL-10 which possesses a negative autoregulatory effect on HSC collagen production mediated at least in part by alpha1(I) collagen transcriptional inhibition and stimulation of collagenase expression. These findings, along with the demonstrated early induction of HSC IL-10 expression and its late disappearance during biliary liver fibrosis, suggest its in vivo role in matrix remodeling and a possibility that failure for HSC to sustain IL-10 expression underlies pathologic progression to liver cirrhosis.

Stability of Escherichia coli sodA mRNA and identification of the transcriptional start site(s) under different environmental and oxidative stresses.

Manganese-containing superoxide dismutase (MnSOD-sodA) in Escherichia coli (E. coli) is regulated at the transcriptional level as observed in studies using both operon and gene fusions. In this paper we examine the regulation of sodA gene at the level of mRNA. We examine the effects of several aerobic inducing conditions (i.e., nalidixic acid, paraquat, or 2,2'-dipyridyl) on mRNA stability, transcription initiation, and translation. The half-life of sodA mRNA was found to be approximately 3-4 min, showing no differences in mRNA stability between induced and uninduced cells. We also found, by reverse transcriptase, that the second putative promoter is not functional under normal or stress conditions, and the amount of mRNA was found to be proportional to active MnSOD. Thus, these results indicate that under oxidative stress/inducing conditions, the increase in aerobic transcription of sodA occurs from only one transcription start site without affecting the stability of sodA mRNA. In addition, the 1:1 ratio found between increases in sodA mRNA and active MnSOD suggests that no translational regulation occurs aerobically.

Roles of manganese and iron in the regulation of the biosynthesis of manganese-superoxide dismutase in Escherichia coli.

Aerobic life-style offers both benefits and risks to living cells. The major risk comes from the formation of reactive oxygen intermediates (i.e. superoxide radical, O2-; hydrogen peroxide, H2O2; and hydroxyl radical, OH.) during normal oxygen metabolism. However, living cells are able to cope with oxygen toxicity by virtue of a unique set of antioxidant enzymes that scavenge O2- and H2O2, and prevent the formation OH.. Superoxide dismutases (SODs; EC 1.15.1.1) are metalloenzymes essential for aerobic survival. Escherichia coli contains two forms of this enzyme: an iron-containing enzyme (FeSOD) and a manganese-containing enzyme (MnSOD). In E. coli, MnSOD biosynthesis is under rigorous control. The enzyme is induced in response to a variety of environmental stress conditions including exposure to oxygen, redox cycling compounds such as paraquat which exacerbate the level of intracellular superoxide radicals, iron chelation (i.e. iron deprivation), and oxidants. A model for the regulation of the MnSOD has been proposed in which the MnSOD gene (sodA) is negatively regulated at the level of transcription by an iron-containing redox-sensitive repressor protein. The effect of iron-chelation most probably results in removal of the iron necessary for repressor activity. Recent studies have shown that sodA expression is regulated by three iron-dependent regulatory proteins, Fur (ferric uptake regulation), Fnr (fumarate nitrate regulation) and SoxR (superoxide regulon), and by the ArcA/ArcB (aerobic respiration control) system. The potential Fur-, Fnr- and ArcA-binding sites in the sodA promoter region have been identified by using different cis-acting regulatory mutations that caused anaerobic derepression of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of fur on the transcriptional and post-transcriptional regulation of MnSOD gene (sodA) in Escherichia coli.

Earlier studies have shown that the Fur (ferric uptake regulation) protein acts as an anaerobic/aerobic repressor of MnSOD (sodA) expression. We found that the aerobic expression of sodA::lacZ fusion in a Fur- background to be threefold higher than in a Fur+ background. This effect of fur mutation was not seen in a strain harboring the sodA+ gene instead of the sodA::lacZ fusion. However, we observed a proportionate increase in the concentrations of sodA::lacZ and sodA+ mRNAs in response to a mutation in the fur gene. These data suggest that the formation of active MnSOD is dependent on a functional fur gene. Indeed, we found that in a fur mutant iron was incorporated into SodA in place of manganese, thus creating inactive and/or partially active forms of the enzyme (i.e., Fe2SodA and/or Mn,FeSodA, respectively), resulting in little or no increase in total MnSOD activity. Thus, Fur plays the role of a repressor at the transcriptional level, but it also plays an indirect role at the post-transcriptional level where it affects the maturation of SodA into a fully active enzyme, Mn2SodA (MnSOD).

Transcriptional activation of Mn-superoxide dismutase gene (sodA) of Escherichia coli by MnCl2.

Transcription of the manganese-superoxide dismutase gene (sodA) in Escherichia coli was shown to be activated by manganese. Addition of MnCl2 increased the expression of beta-galactosidase from a sodA::lacZ protein fusion and increased the concentration of mRNA transcribed from sodA+ and sodA::lacZ constructs. The stimulatory affect of manganese on the expression of sodA::lacZ was greatly reduced (i.e., > 90%) in a strain harboring a fur mutation. We also found that manganese was capable of altering DNA topology. These results show that Mn2+ causes activation of sodA transcription.

Transcriptional regulation of Mn-superoxide dismutase gene (sodA) of Escherichia coli is stimulated by DNA gyrase inhibitors.

The expression of manganese-containing superoxide dismutase (sodA) in Escherichia coli using sodA::lacZ gene fusion was found to be stimulated by DNA gyrase inhibitors, nalidixic acid, or coumermycin A1. Aerobically, the gyrase inhibitors increased the expression of sodA::lacZ in the presence or absence of either paraquat or the iron chelator 2,2'-dipyridyl. The concentrations of the inhibitors used were found to reduce DNA supercoiling. Treatment of wild-type cells (sodA+) with nalidixic acid increased the transcription of MnSOD mRNA. Anaerobically, the expression of sodA::lacZ in wild-type cells was not affected by nalidixic acid. However, nalidixic acid had a stimulatory effect on the anaerobic expression of sodA::lacZ in cells preinduced by the iron chelator as well as in mutants derepressed in sodA expression by virtue of their lacking the trans-acting repressor proteins or the cis-acting regulatory elements needed for sodA regulation. The results indicate that the effect of DNA gyrase inhibitors is secondary to the cis- and trans-regulatory elements of sodA and suggest that changes in DNA topology may affect transcriptional regulation of sodA.