Abstract

Fanconi anemia (FA) is characterized by chromosomal instability and bone marrow hypoplasia. To explore the mechanism of FA-related bone marrow failure, we studied erythropoietin (Epo)-dependent proliferation and differentiation of erythroid progenitors expanded from mice lacking the Fanca or Fancg gene. Erythroid progenitors can be expanded in serum-free medium supplemented with Epo, Stem Cell Factor (SCF) and glucocorticoids. The expansion capacity of Fanca−/− and Fancg−/− erythroid progenitors was reduced and the progenitors differentiated to mature erythrocytes instead after 10 days of culture. When Epo-induced signal transduction was compared between progenitors expanded for 6 days from fetal livers of Fanca−/−, Fancg−/− and wt littermates, Epo-induced phosphorylation of STAT5 and ERK1/2 appeared to be severely reduced and delayed in progenitors lacking Fanca or Fancg. Both in wt, Fanca−/− and Fancg−/− progenitors the phosphorylation of STAT5 and ERK1/2 increased with rising Epo-concentrations, but at low concentrations 4-fold more Epo was required to obtained comparable Stat5 phosphorylation in FancA−/− or FancG−/− cells, while efficient phosphorylation at high concentrations was never achieved in Fanca or Fancg-deficient cells. As a consequence, the regulation of Epo-target genes like p21WAF, Gilz, SOCS3, Spi2.1 and BclXL was impaired. In contrast to downstream signaling intermediates, Epo-induced phosphorylation of the Epo-receptor and its associated kinase JAK2 was not affected. Stem Cell Factor-induced c-Kit activation and downstream Erk1/2 phosphorylation was also equally efficient in wt, Fanca−/− and Fancg−/− progenitors. Thus, the signaling defect specifically seems to affect targets downstream of Jak2. Surprisingly, Epo-induced activation of Stat5 was unaffected in freshly isolated bone marrow cells. Comparison of Epo-induced Stat5 phosphorylation in erythroid progenitors cultured at atmospheric (20%) and physiologic (3%) oxygen revealed that Stat5 phosphorylation was only lost upon expansion of the progenitors at atmospheric (hyperoxic) conditions. Hyperoxia can damage many biomolecules among which DNA. In contrast to wt cells, Fanca−/− and Fancg−/− progenitors may not be able to repair DNA-damage induced by atmospheric oxygen. To examine whether increased DNA damage could impair Epo-induced signal transduction, we expanded erythroid progenitors from fetal livers of mice lacking the DNA-repair enzyme Ercc1. Ercc1 is directly involved in repair of DNA crosslinks, while a defective FA pathway renders cells hypersensitive to DNA-crosslinks. Epo-induced phosphorylation of Stat5 was impaired in Ercc1−/− progenitors, indicating that deficient repair of DNA damage, rather than FA-pathway specific mechanisms may cause the observed signaling defect. To substantiate this finding we treated wt and p53−/− progenitors with increasing concentrations of mitomycin C (MMC). Sublethal concentrations of MMC abrogated the renewal capacity and impaired Epo-induced phosporylation of Stat5 and Erk1/2 in wt but not in p53−/− erythroid progenitors. Together the data suggest that impaired progenitor expansion and Stat5 phosphorylation is not a specific aspect of cells lacking the fanconi pathway, but a general aspect of a p53-dependent response to genomic stress.

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