M Homan

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.

Opposite effect of oxidative stress on inducible nitric oxide synthase and haem oxygenase-1 expression in intestinal inflammation: anti-inflammatory effect of carbon monoxide.

Inducible nitric oxide synthase (iNOS) is expressed in intestinal epithelial cells (IEC) of patients with active inflammatory bowel disease (IBD) and in IEC of endotoxaemic rats. The induction of iNOS in IEC is an element of the NF‐κB‐mediated survival pathway. Haem oxygenase‐1 (HO‐1) is an AP‐1‐regulated gene that is induced by oxidative stress. The enzyme produces carbon monoxide (CO), which may attenuate the inflammatory response. The aim of this study was to investigate the regulation and interaction of iNOS and HO‐1 in response to inflammation and oxidative stress. Male Wistar rats were treated with the thiol‐modifying agent diethylmaleate (DEM) to induce oxidative stress and rendered endotoxaemic by LPS injection. Human colonic biopsies and the human colon carcinoma cell line DLD‐1 were treated with DEM and the lipid peroxidation end‐product 4‐hydroxynonenal to induce oxidative stress and exposed to cytokine mix (CM) to mimic inflammation. In some experiments, cells were incubated with 250–400 ppm CO prior to and during stimulation with CM. HO‐1 and iNOS expression was evaluated by RT‐PCR, western blotting, and immunohistology. NF‐κB activation was evaluated by EMSA. LPS induced iNOS but not HO‐1 in epithelial cells of the ileum and colon. Oxidative stress strongly induced HO‐1 in epithelial and inflammatory cells. Combined oxidative stress and endotoxaemia decreased iNOS expression but strongly induced HO‐1 expression. Similarly, CM induced iNOS but not HO‐1 in colonic biopsies and DLD‐1 cells. Oxidative stress prevented iNOS induction in an NF‐κB‐dependent manner but increased HO‐1 expression in CM‐exposed DLD‐1 cells. CO inhibited iNOS mRNA induction in CM‐stimulated DLD‐1 cells. These data demonstrate opposite regulation of iNOS and HO‐1 in intestinal epithelial cells in response to cytokine exposure and oxidative stress. These findings suggest that iNOS (NF‐κB driven) and HO‐1 (AP‐1 driven) represent mutually exclusive survival mechanisms in intestinal epithelial cells. Copyright

Diffuse and intestinal type gastric carcinomas differ in their expression of apoptosis related proteins

Background: Gastric carcinomas can be divided into intestinal and diffuse types, with the last type having a worse prognosis. Aims: To investigate whether specific patterns in the expression of apoptosis related proteins correlate with carcinoma type and/or prognosis Methods: The expression of Fas, Bcl-2, Bax, Bcl-xl, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) was studied immunohistochemically and the extent of apoptosis and proliferation was investigated in 11 cases of intestinal type and in eight cases of diffuse type carcinoma. Results: Fas was expressed in all intestinal type and in one diffuse type carcinoma. Bcl-xl was expressed in 10 of 11 intestinal type and in one of eight diffuse type carcinomas. Bcl-2 was expressed in lamina propria immune cells. iNOS was expressed in six of 11 intestinal type and in four of eight diffuse type carcinomas, and COX-2 was expressed in eight of 11 intestinal type and in six of eight diffuse type carcinomas. Conclusion: Fas and Bcl-xl expression can differentiate between intestinal type and diffuse type gastric carcinomas. No differences in apoptosis and proliferation between intestinal type and diffuse type gastric carcinomas were observed.