Introduction Progression of simple steatosis to NASH is attributed to inflammation and oxyradical overload (“oxidative stress”). Expression of the zinc finger transcription factor, KLF6, which up-regulates p21 and glucokinase, is increased in whole liver as disease progresses. KLF6 is regulated by alternative splicing and we propose that the balance of KLF6 alternative splice forms in different cellular compartments within the liver may influence the rate of NAFLD progression. We have therefore studied the impact of oxidative stress on KLF6 splice isoform expression.
Methods HepG2 cells were treated with either tertiary butyl hydrogen peroxide or angiotensin II, in the presence/absence of the anti-oxidant N-acteyl-L-cysteine (NAC). Reactive oxygen species were quantified by DCFDA and lucigenin chemiluminescence. KLF6 isoform expression and subcellular localisation was determined using variant specific real-time PCR/western blotting and immunofluorescence (IF).
Results While mRNA expression of both KLF6 full length (KLF6-FL) and its SV1/SV2 variants was markedly increased (10–50-fold) after exposure to oxidative stress, this occurred at 8 h and was unaffected by NAC. Expression of the little characterised KLF6-SV3 variant, however, was dramatically increased by over 30-fold at just 15 min. Furthermore, this increase was significantly attenuated by 50% in the presence of NAC. Western blotting confirmed protein accumulation of both KLF6-FL and KLF6-SV3 at 30 min, falling after 4 h, with a sixfold increase in the KLF6 target gene, p21, at 4–8 h. KLF6 IF studies confirmed that both KLF6-FL and KLF6-SV3 in HepG2 cells translocate to the nucleus after exposure to oxidative stress. In human NAFLD, a modest 1.5–2-fold increase in KLF6-FL mRNA contrasted sharply with a dramatic ninefold increase in KLF6-SV3 mRNA (p<0.01) in tissues with inflammation (n=16) compared to those without (n=7).
Conclusion While KLF6-FL clearly accumulates in the nucleus of HepG2 cells in response to oxidative stress, it is the little characterised KLF6-SV3 isoform which is redox sensitive at the level of transcription and which is dramatically increased in association with inflammation in NAFLD. This variant retains the NLS and the first zinc finger of the DNA binding domain. Further characterisation of its functions and targets will help us to understand its role in NAFLD progression.
Competing interests None declared.
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