Abstract
The underlying molecular mechanisms that promote bone marrow failure in Fanconi anemia (FA) are incompletely understood. Evidence from our lab and others suggest that enhanced oxidant and TNF-α mediated apoptosis of hematopoietic stem and progenitor cells is a major contributing factor. Previously, we showed that enhanced apoptosis of FA type C deficient (Fancc −/−) progenitors involves aberrant activation of the stress kinase, p38MAPK. Given the importance of c-Jun N-terminal kinase (JNK) in the stress response, we hypothesized that enhanced TNF-α-induced apoptosis in Fancc −/− cells would also involve altered JNK activation. The aims of the current study were to determine whether Fancc −/− cells exhibit altered JNK activation and to examine whether inhibition of JNK and/or p38MAPK kinase would enhance Fancc −/− hematopoietic stem cell (HSC) repopulating ability. In Fancc −/− murine embryonic fibroblasts (MEFs), TNF-α induced a 2-fold increase in JNK in vitro kinase activity compared to WT (n=4, p<0.05). Use of either a JNK inhibitor (n=3, p<0.003) or knockdown of JNK1/JNK2 expression using siRNAs (n=3, p<0.001) protected Fancc −/− MEFs from TNF-α induced apoptosis. Importantly, TNF-α induced apoptosis of Fancc −/− ckit+ low-density bone marrow cells was also restored to WT levels by culturing with a JNK inhibitor (n=3, p<0.01), and clonogenic hematopoietic progenitor assays demonstrated that JNK inhibition improved Fancc −/− colony formation in the presence of TNF-α (n=3, p<0.002). Taken together these data suggest that the predisposition of Fancc −/− MEFs and hematopoietic progenitors to TNF-α induced apoptosis involves JNK activation. Based on these data and previous studies demonstrating a role for p38MAPK in enhanced Fancc −/− progenitor apoptosis, competitive repopulation assays were conducted to examine whether culturing Fancc −/− cells with JNK and/or p38MAPK inhibitors would enhance HSC reconstituting function. Surprisingly, in two separate experiments Fancc −/− donor cells cultured with the JNK inhibitor had levels of donor chimerism that were equivalent to Fancc −/− donor cells cultured with the vehicle control. In contrast, culturing Fancc −/− cells with a p38MAPK inhibitor (SB203580) significantly increased repopulating ability compared to Fancc −/− cells cultured with vehicle control in two separate transplants (total n=12–14 recipients/transplant group, p<0.01). Interestingly, a similar increase in donor chimerism was observed in WT donor cells cultured with the p38MAPK inhibitor (total n=12–14 recipients/transplant group, p<0.002), supporting an integral role of p38MAPK in maintaining HSC function. The improvement in reconstitution was sustained over 12 months, and multilineage analysis revealed enhanced lymphoid and myeloid reconstitution, supporting an increase in HSC function with inhibition of p38MAPK. Twelve months post-transplantation all mice exhibited normal peripheral blood counts, BM and spleen histology. Taken together these data suggest that p38MAPK, but not JNK has a critical role in maintaining survival of WT and Fancc −/− reconstituting cells under conditions of stress.
Disclosures: No relevant conflicts of interest to declare.
Author notes
Corresponding author