Nuclear factor κB in proliferation, activation, and apoptosis in rat hepatic stellate cells (original) (raw)
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Journal of Hepatology, 1997
Background/Aims: Hepatic stellate cells represent the principal matrix-synthesising cells of damaged liver and are targets of a number of cytokines currently under investigation. The study analyses the effects of tumour necrosis factor-a and interferon-y on proliferation, "activation" and protein synthesis of hepatic stellate cells. M&z&: Primary cultures of hepatic stellate cells were exposed to tmnour necrosis factor-a and interferon-y. Cell proliferation was studied by 3H-thymidine and bromo-deoxy-uridine incorporation. Protein synthesis was analysed using immunoprecipitation, Western-and Northern blotting techniques. Results: Proliferation of hepatic stellate cells was reduced by tumor necrosis factor-a and interferon-y, while "activation" of hepatic stellate cells as assessed by expression of smooth muscle a-actin and of TGF-/?/activin type I receptor was induced by tmnour necrosis factor-a but downregulated by interferon-y. Tumour necrosis factor-a increased the synthesis of dis-IVER FIBROSIS is characterised by increased and alter-L ed deposition of newly formed extracellular matrix components (ECM), resulting from a shifted balance between connective tissue production and degradation (for reviews see (l-3). Fibrogenesis is initiated by hepatocyte damage, leading to a recruitment of inflammatory blood cells and platelets as well as activation of Kupffer cells with subsequent release of cytokines and growth factors. Hepatic stellate cells (HSC), also known as Ito cells, fat-storing cells, or lipocytes, seem to be the primary target cells for mitogenic and
Laboratory …, 2010
Hepatocellular apoptosis, hepatic inflammation, and fibrosis are prominent features in chronic liver diseases. However, the linkage among these processes remains mechanistically unclear. In this study, we examined the apoptosis and activation of Kupffer cells (KCs) as well as their pathophysiological involvement in liver fibrosis process. Hepatic fibrosis was induced in rats by dimethylnitrosamine (DMN) or carbon tetrachloride (CCl4) treatment. KCs were isolated from normal rats and incubated with lipopolysaccharide (LPS) or from fibrotic rats. The KCs were stained immunohistochemically with anti-CD68 antibody, a biomarker for KC. The level of expression of CD68 was analyzed by western blot and real-time PCR methods. The apoptosis and pathophysiological involvement of KCs in the formation of liver fibrosis were studied using confocal microscopy. The mRNA and protein expression of CD68 were significantly increased in DMNand CCL4-treated rats. Confocal microscopy analysis showed that CD68-positive KCs, but not a-smooth muscle actin (SMA)-positive cells, underwent apoptosis in the liver of DMN-and CCL4-treated rats. It was also revealed that the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and CD68-double-positive apoptotic KCs located in the portal or fibrotic septa area were situated next to hepatic stellate cells (HSCs). Tumor necrosis factor-a (TNF-a) and KC co-localized in the liver in the neighbor of HSCs. The double a-SMAand collagen type I-positive cells predominantly existed in fibrotic septa, and those cells were co-localized clearly with CD68-positive cells. Interestingly, some CD68 and Col (1) double positive, but completely negative for a-SMA, were found in the portal areas and hepatic sinusoids; this phenomenon was also validated in primary isolated KCs after 6 h LPS exposure or fibrotic rats in vitro. These results show that KCs are associated with hepatocellular apoptosis, inflammation, and fibrosis process in a liver fibrosis models.
Hepatology, 1999
containing medium underwent a rapid (3-hour) classical induction of p50:p65 and p65:p65 nuclear factor-B (NF-B) dimers. Subsequent culturing was associated with prolonged expression of active p50:p65 and persistent induction of a high-mobility NF-B DNA binding complex consisting of potentially novel Rel-like protein(s). Formation of the latter complex was competed for by specific double-stranded oligonucleotides, was up-regulated by treatment of HSCs with tumor necrosis factor ␣ (TNF-␣), and was maintained at basal levels of expression by a soluble HSC-derived factor. An NF-B-responsive CAT reporter gene was highly active in early cultured HSCs but was also trans-activated at a lower but significant level in longerterm cultured cells and could be completely suppressed by expression of dominant negative IB-␣. Physiological significance of the lower persistent NF-B activities was also demonstrated by the ability of long-term cultured HSCs to support the activity of the NF-B-dependent human intercellular adhesion molecule-1 (ICAM-1) promoter. Freshly isolated HSCs expressed high levels of IB-␣ and IB-. Culture activation was accompanied by a long-term reduction in levels of IB-␣ with no detectable expression in the nuclear fraction of cells, under these conditions p50:p65 was detected in the nucleus. IB- expression was transiently reduced and, upon replenishment, was associated Abbreviations: HSC, hepatic stellate cell; ␣-SMA, ␣-smooth muscle actin; NF-B, nuclear factor-B; TNF-␣, tumor necrosis factor ␣; ICAM-1, intercellular adhesion molecule-1; PBS, phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; EMSA, electrophoretic mobility shift assay; SDS, sodium dodecyl sulfate; CAT, chloramphenicol acetyltransferase.
2021
Background: Tumor necrosis factor-α (TNFα) is a pleiotropic cytokine involved in nuclear factor kappa B (NF-κB) mediated cell survival as well as cell death. High serum TNFα levels correlate with liver fibrosis and participate in enhancing hepatic stellate cell (HSC) viability. However, the regulatory role of cellular inhibitor of apoptosis-1/2 (cIAP1/2) in TNFα induced NF-κB signaling in activated HSCs is largely unknown. Methods and Results: Activated HSCs treated with cIAP1/2 inhbitior SMAC mimetic BV6 and Birinapant in presence of TNFα and macrophage secretome. TNFα cytokine increased cIAP2 expression in activated HSCs and enhanced cell viability through canonical NF-κB signaling in activated HSCs. cIAP2 inhibition via BV6 decreased the TNFα induced canonical NF-κB signaling and reduced cell viability in activated HSCs. SMAC mimetic, Birinapant alone did not affect the cell viability and was incompetent in inducing cell death. However, pre-treatment of TNFα sensitized HSCs and B...
American Journal of Physiology-gastrointestinal and Liver Physiology, 2002
Hepatic stellate cells (HSC), particularly activated HSC, are thought to be the principle matrix-producing cell of the diseased liver. However, other cell types of the fibroblast lineage, especially the rat liver myofibroblasts (rMF), also have fibrogenic potential. A major difference between the two cell types is the different life span under culture conditions. Although nearly no spontaneous apoptosis could be shown in rMF cultures, 18 Ϯ 2% of the activated HSC (day 7) were apoptotic. Compared with activated HSC, CD95R was expressed in 70% higher amounts in rMF. CD95L could only be detected in activated HSC. Stimulation of the CD95 system by agonistic antibodies (1 ng/ml) led to apoptosis of all rMF within 2 h, whereas activated HSC were more resistant (5.3 h/ 40% of total cells). Although transforming growth factor- downregulated apoptosis in both activated HSC and rMF, tumor necrosis factor-␣ (TNF-␣) upregulated apoptosis in rMF. Lack of spontaneous apoptosis and CD95L expression in rMF and the different reaction on TNF-␣ stimulation reveal that activated HSC and rMF belong to different cell populations.
Expression and Regulation of Cell Adhesion Molecules by Hepatic Stellate Cells (HSC) of Rat Liver
The American Journal of Pathology, 1999
Hepatic stellate cells (HSC) , a pericyte-like nonparenchymal liver cell population , are regarded as the principal matrix-synthesizing cells of fibrotic liver. They might also play a role during liver inflammation. The present study analyzed (i) expression of cell adhesion molecules (CAMs) mediating cell infiltration , like intercellular adhesion molecule-1 (I-CAM-1) and vascular cell adhesion molecule-1 (V-CAM-1) , by HSC, (ii) CAM regulation in HSC by growth factors and inflammatory cytokines , and (iii) CAM expression in situ during liver inflammation , using immunochemistry and Northern blot analysis. I-CAM-1 and V-CAM-1 expression was present in HSC in vitro and in cells located in the sinusoidal/perisinusoidal area of normal liver. Growth factors , eg , transforming growth factor-1 , down-regulated I-CAM-1-and V-CAM-1-coding mRNAs and stimulated N-CAM expression of HSC. In contrast , inflammatory cytokines like tumor necrosis factor-␣ reduced N-CAM-coding mRNAs, whereas induction of I-CAM-1-and V-CAM-1-specific transcripts increased severalfold. In situ, messengers specific for I-CAM-1 and V-CAM-1 were induced 3 hours after CCl 4 treatment (thereby preceding mononuclear cell infiltration starting at 12 hours) , were expressed at maximal levels 9 -12 hours after CCl 4 application , and decreased afterwards. I-CAM-1 and V-CAM-1 immunoreactivity increased in a linear fashion starting 3 hours after CCl 4 -induced liver injury, was detected in highest amounts at 24 -48 hours characterized by maximal cell infiltration , and returned to baseline values at 96 hours. Interestingly , the induction/repression of CAM-specific messengers paralleled the time kinetics of tumor necrosis factor-␣/ transforming growth factor-1 expression in injured liver. HSC might be important during the onset of hepatic tissue injury as proinflammatory elements and might interact with I-CAM-1 and V-CAM-1 ligandbearing cells, namely lymphocyte function-associated antigen-1-or Mac-1/very late activation antigen-4-positive inflammatory cells, thereby modulating the recruitment and migration of mononuclear cells within the perisinusoidal space of diseased livers.
Hepatology, 1998
in tissue injury and inflammation is a key pathogenetic event in liver fibrogenesis leading to an expanded pool of matrix producing myofibroblasts (MFB) which represent the transformed counterpart of HSC. We hypothesize that expansion of the pool of MFB might also be accomplished by modulation of apoptosis, which plays an opposite and complementary role to mitosis in the cellular homeostasis. We characterized the susceptibility of HSC in primary culture and of MFB in secondary culture to apoptosis induced by the soluble Fas ligand (sFasL) and related the effects to the expression levels of Fas (APO-1/CD95) and some major proapoptotic and contra-apoptotic protooncogenes. MFB showed a dose-dependent apoptotic reaction upon exposure to sFasL as evidenced by a strong increase of nucleosomal DNA fragments, loss of cellular DNA, positive TUNEL reaction, and annexin staining. The effect was found only if protein synthesis (cycloheximide) or RNA synthesis (actinomycin D) were arrested. HSC maintained for various times in primary culture were completely resistant to sFasL in combination with cycloheximide, but in late primary cultures (day 7 onward) an increasing susceptibility to sFasL-mediated apoptosis was developed. By semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and alkaline phosphatase-antialkaline phosphatase staining Fas receptor was identified both in HSC and MFB at comparable expression levels. The expression of the contra-apoptotic protooncogenes bcl-2 and bcl-xl was found to be much stronger in early HSC than in late HSC and MFB as shown by ribonuclease protection assay. The expression of bcl-2 was additionally confirmed by semiquantitative RT-PCR and immunoblotting. Proapoptotic bax was found in comparable quantities at the RNA level in HSC and MFB but at the protein level MFB showed increased bax expression. It is concluded that transformation of HSC to MFB is paralleled by an increasing sensitivity to sFasL-mediated apoptosis, which might be related to a strong decrease of bcl-2 and bcl-xl expression, leading to a preponderance of proapoptotic gene expression in MFB. Modulation of apoptotic susceptibility of transforming HSC could be an important complementary pathway in the pathogenesis of fibrosis. (HEPATOLOGY 1998;28:492-502.)
Hepatology, 2013
We have developed a novel model for depleting mouse hepatic stellate cells (HSCs) that has allowed us to clarify their contributions to hepatic injury and fibrosis. Transgenic (Tg) mice expressing the herpes simplex virus thymidine kinase gene (HSV-Tk) driven by the mouse GFAP promoter were used to render proliferating HSCs susceptible to killing in response to ganciclovir (GCV). Effects of GCV were explored in primary HSCs and in vivo. Panlobular damage was provoked to maximize HSC depletion by combining CCl 4 (centrilobular injury) with allyl alcohol (AA) (periportal injury), as well as in a bile duct ligation (BDL) model. Cell depletion in situ was quantified using dual immunofluorescence (IF) for desmin and GFAP. In primary HSCs isolated from both untreated wild-type (WT) and Tg mice, GCV induced cell death in 5050% of HSCs from Tg, but not WT, mice. In TG mice treated with CCl 4 1AA1GCV, there was a significant decrease in GFAP and desmin-positive cells, compared to WT mice (5065% reduction; P < 0.01), which was accompanied by a decrease in the expression of HSC-activation markers (alpha smooth muscle actin, beta platelet-derived growth factor receptor, and collagen I). Similar results were observed after BDL. Associated with HSC depletion in both fibrosis models, there was marked attenuation of fibrosis and liver injury, as indicated by Sirius Red/Fast Green, hematoxylin and eosin quantification, and serum alanine/aspartate aminotransferase. Hepatic expression of interleukin-10 and interferon-gamma was increased after HSC depletion. No toxicity of GCV in either WT or Tg mice accounted for the differences in injury. Conclusion: Activated HSCs significantly amplify the response to liver injury, further expanding this cell type's repertoire in orchestrating hepatic injury and repair. (HEPATOLOGY 2013;57:340-350)