A dominant negative form of IKK2 prevents suppression of apoptosis by the peroxisome proliferator nafenopin.

Peroxisome proliferators (PPs) are a class of non-genotoxic chemicals that cause rodent liver enlargement and hepatocarcinogenesis. In primary rat hepatocyte cultures, PPs suppress spontaneous apoptosis and that induced by a number of pro-apoptotic stimuli such as transforming growth factor-beta(1). Tumour necrosis factor alpha (TNF-alpha) and the transcription factor NFkappaB have been implicated in the mode of action of PPs. TNF-alpha signalling to NFkappaB is thought to be responsible for many of the effects elicited by this cytokine. NFkappaB regulates gene expression in immunity, stress responses and the inhibition of apoptosis. Activation of NFkappaB requires the successive action of NFkappaB-inducing kinase and the phosphorylation of NFkappaB inhibitory proteins (IkappaB) by an IkappaB kinase (IKK) complex. The IKK2 subunit of IkappaB kinase is thought to be essential for NFkappaB activation and prevention of apoptosis. To determine whether IKK2 plays a role in the suppression of apoptosis by PPs, we expressed a dominant negative form of IKK2 (IKK2dn) in primary rat hepatocyte cultures. Infection with an adenovirus construct expressing IKK2dn caused apoptosis in control primary rat hepatocytes in the absence of exogenous TNF-alpha. Moreover, IKK2dn-induced apoptosis could not be rescued by addition of TNF-alpha or the peroxisome proliferator nafenopin. These results demonstrate a requirement for intracellular signalling pathways mediated by IKK2 in the suppression of apoptosis by the PP class of hepatocarcinogens.

Role for tumor necrosis factor alpha receptor 1 and interleukin-1 receptor in the suppression of mouse hepatocyte apoptosis by the peroxisome proliferator nafenopin.

Peroxisome proliferators (PPs) cause rodent liver enlargement and tumors. In vitro, PPs induce rat and mouse hepatocyte DNA synthesis and suppress apoptosis, a response mimicked by exogenous tumor necrosis factor alpha (TNFalpha). Here, we determine the role of TNF receptor 1 (TNFR1), TNF receptor 2 (TNFR2), and nuclear factor kappa beta (NFkappaB) in the response of mouse hepatocytes to the PP, nafenopin. Nafenopin (50 micromol/L) induced DNA synthesis as measured by bromodeoxyuridine (BrdU) incorporation, suppressed cell death as measured by Hoechst 33258 staining, induced peroxisomal beta-oxidation as measured by cyanide insensitive palmitoyl CoA oxidation (PCO) and caused activation of nuclear factor kappa beta (NFkappaB) as determined by electrophoretic mobility gel shift assay (EMSA). The induction of DNA synthesis and the suppression of apoptosis in response to nafenopin was abrogated completely by blocking antibodies to TNFR1 but not to TNFR2. In contrast, the induction of peroxisomal beta-oxidation by nafenopin was not blocked by the anti-TNFR1 antibody. Next, we evaluated the response of hepatocytes to interleukin-1 (IL-1), another proinflammatory cytokine. IL-1alpha (2.5 ng/mL) and, to a lesser extent, IL-1beta (5 ng/mL), shared the ability of TNFalpha to induce DNA synthesis and suppress apoptosis. In addition, anti-IL-1 receptor, type 1/p80 (IL-1R) antibodies were able to abrogate the response to nafenopin. IL-1alpha was still able to perturb hepatocyte growth in the presence of the anti-TNFR1 antibody suggesting that IL-1alpha acts independently rather than by elaborating TNFalpha. In summary, these data provide additional evidence for a role for hepatic cytokines in the perturbation of hepatocyte growth by PPs such as nafenopin.