Mh Schoemaker

Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways.

Ursodeoxycholic acid (UDCA) is used in the treatment of cholestatic liver diseases, but its mechanism of action is not yet well defined. The aim of this study was to explore the protective mechanisms of the taurine‐conjugate of UDCA (tauroursodeoxycholic acid [TUDCA]) against glycochenodeoxycholic acid (GCDCA)‐induced apoptosis in primary cultures of rat hepatocytes. Hepatocytes were exposed to GCDCA, TUDCA, the glyco‐conjugate of UDCA (GUDCA), and TCDCA. The phosphatidylinositol‐3 kinase pathway (PI3K) and nuclear factor‐κB were inhibited using LY 294002 and adenoviral overexpression of dominant‐negative IκB, respectively. The role of p38 and extracellular signal‐regulated protein kinase mitogen‐activated protein kinase (MAPK) pathways were investigated using the inhibitors SB 203580 and U0 126 and Western blot analysis. Transcription was blocked by actinomycin‐D. Apoptosis was determined by measuring caspase‐3, ‐9, and ‐8 activity using fluorimetric enzyme detection, Western blot analysis, immunocytochemistry, and nuclear morphological analysis. Our results demonstrated that uptake of GCDCA is needed for apoptosis induction. TUDCA, but not TCDCA and GUDCA, rapidly inhibited, but did not delay, apoptosis at all time points tested. However, the protective effect of TUDCA was independent of its inhibition of caspase‐8. Up to 6 hours of preincubation with TUDCA before addition of GCDCA clearly decreased GCDCA‐induced apoptosis. At up to 1.5 hours after exposure with GCDCA, the addition of TUDCA was still protective. This protection was dependent on activation of p38, ERK MAPK, and PI3K pathways, but independent of competition on the cell membrane, NF‐κB activation, and transcription. In conclusion, TUDCA contributes to the protection against GCDCA‐induced mitochondria‐controlled apoptosis by activating survival pathways. Supplemental material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/supplmat/index.html). (HEPATOLOGY 2004;39:1563–1573.)

Cytokine regulation of pro- and anti-apoptotic genes in rat hepatocytes: NF-κB-regulated inhibitor of apoptosis protein 2 (cIAP2) prevents apoptosis

BACKGROUND/AIMS In acute liver failure, hepatocytes are exposed to various cytokines that activate both cell survival and apoptotic pathways. NF-kappaB is a central transcription factor in these responses. Recent studies indicate that blocking NF-kappaB causes apoptosis, indicating the existence of NF-kappaB-regulated anti-apoptotic genes. In the present study the relationship between NF-kappaB activation and apoptosis has been investigated in hepatocytes. METHODS Primary rat hepatocytes were exposed to a cytokine mixture of tumor necrosis factor alpha, interleukin-1beta, interferon-gamma and lipopolysaccharide. Modulation of signalling pathways was performed by using dominant negative adenoviral constructs. Apoptosis and NF-kappaB activation were determined by caspase-3 activity, Hoechst staining and electrophoretic mobility shift assay, respectively. Furthermore, expression and regulation of apoptosis-related genes were investigated. RESULTS (1) Inhibition of NF-kappaB activation results in apoptosis. (2) Inhibitor of apoptosis protein (IAP) family members, inhibitor of apoptosis protein1 (cIAP1), and X-chromosome-linked IAP, are expressed in rat hepatocytes. cIAP2 is induced by cytokines in an NF-kappaB-dependent manner and overexpression of cIAP2 inhibits apoptosis. (3) The anti-apoptotic Bcl-2 family member A1/Bfl-1 and the pro-apoptotic members Bak and Bid are induced by cytokines and NF-kappaB-dependent. (4) Nitric oxide inhibits caspase-3 activity in hepatocytes. CONCLUSIONS In inflammatory conditions, hepatocyte survival is dependent on NF-kappaB activation and cIAP2 contributes significantly to this protection.

Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation

BACKGROUND/AIMS To examine the extent and mechanisms of apoptosis in cholestatic liver injury and to explore the role of the transcription factor nuclear factor-kappa B in protection against bile acid-induced apoptosis. METHODS Cholestatic liver injury was induced by bile duct ligation in Wistar rats. Furthermore, primary cultures of rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA), tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA) and to cytokines. Apoptosis was determined by TUNEL-staining, active caspase-3 staining, activation of caspase-8, -9 and -3. RESULTS Limited hepatocyte apoptosis and an increased expression of NF-kappaB-regulated anti-apoptotic genes A1 and cIAP2 were detected in cholestatic rat livers. Bcl-2 expression was restricted to bile duct epithelium. In contrast to TCDCA and TUDCA, GCDCA induced apoptosis in a Fas-associated protein with death domain (FADD)-independent pathway in hepatocytes. Although bile acids do not activate NF-kappaB, NF-kappaB activation by cytokines (induced during cholestasis) protected against GCDCA-induced apoptosis in vitro by upregulating A1 and cIAP2. CONCLUSIONS GCDCA induces apoptosis in a mitochondria-controlled pathway in which caspase-8 is activated in a FADD-independent manner. However, bile acid-induced apoptosis in cholestasis is limited. This could be explained by cytokine-induced activation of NF-kappaB-regulated anti-apoptotic genes like A1 and cIAP2.

Defying death : the hepatocyte's survival kit

Acute liver injury can develop as a consequence of viral hepatitis, drug- or toxin-induced toxicity or rejection after liver transplantation, whereas chronic liver injury can be due to long-term exposure to alcohol, chemicals, chronic viral hepatitis, metabolic or cholestatic disorders. During liver injury, liver cells are exposed to increased levels of cytokines, bile acids and oxidative stress. This results in death of hepatocytes. In contrast, stellate cells become active and are resistant against cell death. Eventually, acute and chronic liver injury is followed by loss of liver function for which no effective therapies are available. Hepatocytes are well equipped with protective mechanisms to prevent cell death. As long as these protective mechanisms can be activated, the balance will be in favour of cell survival. However, the balance between cell survival and cell death is delicate and can be easily tipped towards cell death during liver injury. Therefore understanding the cellular mechanisms controlling death of liver cells is of clinical and scientific importance and can lead to the identification of novel intervention targets. This review describes some of the mechanisms that determine the balance between cell death and cell survival during liver diseases. The strict regulation of apoptotic cell death allows therapeutic intervention strategies. In this light, receptor-mediated apoptosis and mitochondria-mediated cell death are discussed and strategies are provided to selectively interfere with these processes.

PDGF-receptor beta-targeted adenovirus redirects gene transfer from hepatocytes to activated stellate cells

Chronic liver damage may lead to liver fibrosis. In this process, hepatic activated stellate cells are the key players. Thus, activated stellate cells are attractive targets for antifibrotic gene therapy. Recombinant adenovirus is a promising vehicle for delivering therapeutic genes to liver cells. However, this vector has considerable tropism for hepatocytes and Kupffer cells. The aim of this study is therefore to retarget the adenovirus to the activated stellate cells while reducing its affinity for hepatocytes. We constructed a fusion protein with affinity for both the adenovirus and the platelet derived growth factor-receptor beta (PDGF-Rbeta). In contrast to other cells, the PDFG-Rbeta is highly expressed on activated stellate cells. The targeting moiety, the PDGF peptide CSRNLIDC, was cloned in front of the single-chain antibody fragment (S11) directed against the adenoviral knob. This fusion protein enhanced adenoviral gene transfer in both 3T3 fibroblasts and primary isolated activated rat stellate cells by 10-60-fold. A fusion protein with a scrambled PDGF peptide (CIDNLSRC) did not accomplish this effect. Importantly, the PDGF-Rbeta-retargeted adenovirus showed a 25-fold reduced tropism for primary rat hepatocytes. Our novel approach demonstrates that therapeutic genes can be selectively directed to stellate cells. This opens new possibilities for the treatment of liver fibrosis.