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

Sabina C. Cosulich2, Neil H. James1, Maurice R.C. Needham, Peter P. Newham, Ken R. Bundell and Ruth A. Roberts1

AstraZeneca Pharmaceuticals and
1 Zeneca Central Toxicology Laboratory, 3G8 Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK


    Abstract
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 Abstract
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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-ß1. Tumour necrosis factor {alpha} (TNF-{alpha}) and the transcription factor NF{kappa}B have been implicated in the mode of action of PPs. TNF-{alpha} signalling to NF{kappa}B is thought to be responsible for many of the effects elicited by this cytokine. NF{kappa}B regulates gene expression in immunity, stress responses and the inhibition of apoptosis. Activation of NF{kappa}B requires the successive action of NF{kappa}B-inducing kinase and the phosphorylation of NF{kappa}B inhibitory proteins (I{kappa}B) by an I{kappa}B kinase (IKK) complex. The IKK2 subunit of I{kappa}B kinase is thought to be essential for NF{kappa}B 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.

Abbreviations: GFP, green fluorescent protein; PPs, peroxisome proliferators; rIAP-1, rat inhibitor of apoptosis-1; TGF-ß1, transforming growth factor-ß1; TNF-{alpha}, tumour necrosis factor {alpha}; TNFR1, tumor necrosis factor {alpha} receptor 1.


    Introduction
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 Abstract
 Introduction
 References
 
Peroxisome proliferators (PPs) are a group of structurally diverse compounds that cause proliferation of hepatic peroxisomes, liver enlargement, hepatocyte hyperplasia and hepatic tumours (1,2). Stimulation of cell proliferation caused by PPs is accompanied by suppression of spontaneous apoptosis and that induced by transforming growth factor-ß1 (TGF-ß1) (3,4). The mechanisms by which PPs perturb liver growth may involve the pro-inflammatory cytokine tumour necrosis factor {alpha} (TNF-{alpha}) and activation of the transcription factor NF{kappa}B (57). Kupffer cells, the resident hepatic macrophages, are a rich source of cytokines such as TNF-{alpha} (8) and have been suggested to play a key role in the induction of hepatocyte proliferation caused by PPs (9,10). Moreover, the PP Wy-14,643 activates NF{kappa}B in Kupffer cells (7). NF{kappa}B consists of homo- and heterodimeric complexes of related proteins that control the expression of genes involved in mediating immune and inflammatory responses, growth, differentiation and development (reviewed in refs 11,12). Activation and nuclear localization of NF{kappa}B in response to diverse stimuli, such as inflammatory cytokines, mitogens and bacterial lipopolysaccharide, occurs as a result of the sequential phosphorylation, ubiquitination and degradation of I{kappa}B proteins (13,14). The I{kappa}B kinase complex is composed of three subunits, IKK{alpha} (IKK1), IKKß (IKK2) and IKK{gamma} (15,16). Biochemical and genetic evidence has indicated that despite their similar structures and in vitro kinase activities, IKKs have different physiological functions. Disruption of the IKK1 locus does not affect activation of IKK by pro-inflammatory stimuli, but interferes with multiple morphogenic events, including limb and skeletal patterning as well as proliferation and differentiation of epidermal keratinocytes (17). In contrast, mice lacking the IKK2 gene die at mid-gestation from liver apoptosis (18), a phenotype which resembles that of mice deficient of the p65 subunit of NF{kappa}B (19). These findings are consistent with a role for IKK2 and NF{kappa}B in hepatocyte survival (2022). Thus, NF{kappa}B activates a number of genes that function to suppress TNF-{alpha}-mediated apoptosis (2325).

To address the mechanism of suppression of apoptosis by PPs and the involvement of TNF-{alpha}-mediated signalling pathways, we infected primary rat hepatocyte cultures with adenoviral constructs containing a wild-type or dominant negative form of IKK2 (K44A). Conditions required to infect primary hepatocytes with adenovirus constructs were determined by monitoring expression of green fluorescent protein (GFP) as a marker for virus infection. Approximately 60–80% of hepatocytes were found to express GFP when infected with virus at a m.o.i. of 10 p.f.u./cell (Figure 1A and BGo). Under these conditions, cells transduced with the IKK2wt and IKK2dn adenoviruses expressed similar levels of IKK2 protein (Figure 1CGo) and FLAG sequence (Figure 1DGo) for up to 72 h after infection. We therefore examined whether IKK2 expression affected downstream events, such as NF{kappa}B-mediated gene transcription. Upon treatment of primary rat hepatocytes with TNF-{alpha}, we observed an increase in inhibitor of apoptosis-1 (rIAP-1), an NF{kappa}B-inducible gene (25,26; Figure 2Go). To further examine whether expression of downstream genes would be affected in hepatocytes transduced with IKK2wt or IKK2dn adenoviruses alone, we analysed rIAP-1 expression by RT–PCR. Transduction with IKK2wt alone caused an increase in rIAP-1 expression compared to LacZ- or IKK2dn-transduced hepatocytes (Figure 2CGo). We next examined whether expression of IKK2wt or IKK2dn affected hepatocyte apoptosis. Upon infection with IKK2wt, no increase in spontaneous apoptosis was observed in the presence or absence of exogenous TNF-{alpha} (Figure 3Go). In contrast, infection with adenovirus encoding IKK2dn caused apoptosis in primary rat hepatocyte cultures (Figure 3Go). Furthermore, addition of TNF-{alpha} did not lead to a further increase in the level of apoptosis in IKK2dn-transduced hepatocytes (Figure 3Go).



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Fig. 1. Adenovirus infection of primary rat hepatocytes. IKK2wt, IKK2dn and AdLacZ were a kind gift from AstraZeneca Pharmaceuticals. Briefly, IKK2 constructs were cloned into the NotI and XbaI sites of pAdTrack-CMV downstream of the cytomegalovirus (CMV) promoter. Recombinant adenoviruses were generated as described in He et al. (30). Primary rat hepatocytes were prepared as described previously (31) and cultured in Williams E medium supplemented with 10% foetal calf serum, insulin (10 µg/ml), hydrocortisone (0.1 mM), L-glutamine (2 mM), penicillin (100 U/ml), BSA (0.01%) and streptomycin (100 µg/ml). (A and B) Twenty-four hours after plating, hepatocytes were infected with the IKK2wt construct at a m.o.i. of 10. Viral infection was detected using GFP, encoded by a gene incorporated into the viral backbone to allow direct observation of the efficiency of transfection and infection (30). Expression of GFP was analysed 24 h after infection using an Olympus inverted microscope. (A) Light microscopy; (B) fluorescence microscopy. (C and D) Primary rat hepatocyte cultures were infected with IKK2wt and IKK2dn. Twenty-four hours later, cells were lysed and subjected to SDS–PAGE (Novex) followed by western blotting using (C) anti-IKK2 antibodies (Santa Cruz) or (D) anti-FLAG antibodies (AstraZeneca Pharmaceuticals).

 


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Fig. 2. Rat hepatocytes were cultured in Williams E medium as described for Figure 1Go, before RNA extraction using Total RNA Isolation Reagent (Advanced Biotechnologies). RNA was then subject to RT–PCR. Primers used for rIAP-1 were 5'-ACAGTCCCACTGAGAAGC-3' and 5'-TCCAGCACTTGCAAGCTG-3'. All RT–PCR reactions were subject to amplification with primers for acidic ribophosphoprotein (ARPP) (5'-GATGTGCAGCTGATCAAGACTGGAG-3' and 5'-ATTCCTCCGACTCTTCCTTTGCTTC-3'). C, untreated control. (A and B) Primary rat hepatocyte cultures were treated with 25 ng/ml recombinant TNF-{alpha} (Insight Biotechnologies) for the times indicated before RNA extraction and RT–PCR to detect rIAP-1 or ARPP. (C and D) Primary rat hepatocyte cultures were infected with recombinant adenoviruses as indicated for 24 h prior to RNA extraction and RT–PCR to detect rIAP-1 or ARPP. C, untreated control.

 


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Fig. 3. IKK2dn causes apoptosis in primary hepatocytes. Primary rat hepatocyte cultures were infected with IKK2wt or IKK2dn in the presence or absence of 25 ng/ml TNF-{alpha}. Twenty-four hours later, cells were treated with (5 ng/ml) TGF-ß1 for a further 24 h prior to fixing and staining with Hoechst 33258 (5 ng/ml). A minimum of 1000 cells were counted for each flask and percentage apoptosis scored by morphology. Data represent means of three independent experiments and are expressed as fold induction ± SD with respect to the value obtained for AdLacZ-transduced cultures (typical values 0.6–3%) treated with solvent (dimethylformamide).

 
We have previously demonstrated that the PP nafenopin suppresses spontaneous and TGF-ß1-induced apoptosis in primary rat hepatocyte cultures (3,4). To establish whether IKK2 expression affected PP-induced suppression of apoptosis, we treated adenovirus-infected primary hepatocyte cultures with 50 µM nafenopin. Expression of IKK2wt protected hepatocytes from TGF-ß1-induced apoptosis (Figure 4Go). This suppression was not affected by addition of nafenopin (Figure 4Go). IKK2dn-induced apoptosis, however, was unaffected by addition of nafenopin (Figure 4Go). Moreover, in cells expressing IKK2dn, nafenopin no longer suppressed TGF-ß1-induced apoptosis (Figure 4Go), demonstrating a requirement for IKK2 activity in the suppression of apoptosis by this PP.



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Fig. 4. Nafenopin requires IKK2 activity to suppression apoptosis in primary hepatocytes. Primary rat hepatocyte cultures were infected with IKK2wt or IKK2dn in the presence or absence of 50 µM nafenopin and/or 5 ng/ml TGF-ß1 prior to fixing and staining with Hoechst 33258 (5 ng/ml). A minimum of 1000 cells were counted for each flask. Data are expressed as fold induction ± SD with respect to the value obtained for AdLacZ-transduced cultures (typical values 0.6–3%) treated with solvent (dimethylformamide).

 
The IKK2 subunit of I{kappa}B kinase has been shown to be essential for NF{kappa}B activation and prevention of apoptosis (2225). The results shown herein support a role for IKK2 in hepatocyte survival. As low levels of TNF-{alpha} may be present in primary hepatocyte cultures (27), inhibition of IKK2 (and presumably NF{kappa}B) activity using IKK2dn viruses would lead to activation of caspases upon ligation of tumor necrosis factor {alpha} receptor 1 (TNFR1) by TNF-{alpha} (28). These effects would not have been observed in studies which used neutralization of TNF-{alpha} or TNFR1 (5,6), as recruitment of the signalling complex downstream of TNFR1 which leads to activation of caspases would have been prevented. In the liver, NF{kappa}B may also function to protect from apoptosis induced by other factors, such as TGF-ß1. In murine hepatocyte cell lines, TGF-ß1-mediated induction of apoptosis involves inactivation of NF{kappa}B through I{kappa}B hypophosphorylation (29). In this system, ectopic expression of a subunit of NF{kappa}B reduced the extent of cell death induced by TGF-ß1. In agreement with these findings, we show that overexpression of IKK2wt causes suppression of both spontaneous and TGF-ß1-induced apoptosis in primary rat hepatocytes.

The ability of the PP class of rodent hepatocarcinogens to cause hepatic cell proliferation and survival has been extensively described. However, the molecular mechanisms involved in this growth perturbation are still unclear. This study provides evidence that the mechanism by which PPs suppress hepatocyte apoptosis involves signalling downstream of TNF-{alpha} and requires IKK2 activity. Furthermore, it provides evidence that overexpression of IKK2 suppresses apoptosis and that IKK2 directly promotes hepatocyte survival.


    Notes
 
2 To whom correspondence should be addressed Email: sabina.cosulich{at}astrazeneca.com Back


    Acknowledgments
 
We would like to thank Hazel Weir, Michelle Mobbs, Christine Harding and Georgia Cerillo for providing the IKK2 constructs and adenoviruses.


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Received December 23, 1999; revised May 15, 2000; accepted May 25, 2000.