TNF-α Hypersensitivity of Fanconi Anemia Type C Deficient Cells Is Dependent on the Apoptosis Signal-Regulating Kinase 1 Signaling Pathway.

Fanconi anemia (FA) is a chromosomal instability disorder characterized by a progressive bone marrow (BM) aplasia. Previous studies from our lab and others suggest that enhanced oxidant- and inhibitory cytokine-mediated apoptosis of hematopoietic stem/progenitor cells are important mechanisms in the pathogenesis of BM failure in FA. Recently, we showed that oxidant-induced apoptosis in Fancc −/− murine embryonic fibroblasts (MEFs) was mediated through hyperactivation of the redox-dependent protein, apoptosis signal-regulating kinase 1 (ASK1, Saadatzadeh et al, JBC 2004). Here, we tested whether alterations in ASK1 signaling also participate in the pro-apoptotic phenotype of primary Fancc −/− MEFs and progenitors in response to the inhibitory cytokine, TNF-α. To determine whether Fancc −/− MEFs exhibit altered ASK1 signaling after TNF-α treatment, we examined ASK1 activity using in vitro kinase assays. These studies demonstrated a 2–3 fold increase in TNF-α-induced ASK1 activity in Fancc −/− MEFs compared to WT (n=5). Consistent with ASK1 hyperactivation, we observed an increase in activation of the downstream stress kinases, c-jun N-terminal kinase and p38, in Fancc −/− MEFs compared to WT (n=5). Utilizing a dominant negative ASK1 cDNA to decrease ASK1 activity, we determined that TNF-α-induced apoptosis in Fancc −/− MEFs was ASK1 dependent (n=3, p<0.05). In addition, TNF-α-induced apoptosis in Fancc −/− MEFs was completely blocked by both antioxidants (selenomethionine and N-acetylcysteine, n=3, p<0.001) and the p38 inhibitor SB 203580 (n=6, p<0.001). To examine whether ASK1 has a critical role in the hypersensitivity of Fancc −/− progenitors to TNF-α-induced apoptosis, we crossed ASK1 knockout mice with Fancc mice to generate the following four experimental genotypes; WT, Fancc +/+;ASK1 −/−, Fancc −/−;ASK1 +/+, and Fancc −/−;ASK1 −/−. BM cellularity and progenitors/femur were similar between all genotypes (n=3). Interestingly, TNF-α hypersensitivity of Fancc −/− progenitors was restored to WT levels when ASK1 was deleted (n=3, p<0.05), suggesting that TNF-α-induced hypersensitivity of Fancc −/− progenitors is mediated through ASK1, similar to Fancc −/− MEFs. In addition, TNF-α-induced apoptosis in Fancc −/− progenitors was restored to WT levels after culturing with the p38 inhibitor SB 203580 (n=6, p<0.005). Since ASK1 activity is directly regulated by the cellular redox state, these data support the idea that aberrant redox regulation may be involved in the observed pro-apoptotic phenotype of Fancc −/− cells after exposure to inhibitory cytokines such as TNF-α. Collectively, these data suggest that inhibiting the ASK1 apoptotic pathway in Fancc −/− cells via antioxidants or small molecule inhibitors may protect Fancc −/− hematopoeitic stem/progenitors from an apoptotic fate and delay BM failure.