Microphthalmia transcription factor expression contributes to bone marrow failure in Fanconi anemia
Alessia Oppezzo, Julie Bourseguin, Emilie Renaud, Patrycja Pawlikowska, Filippo Rosselli
Alessia Oppezzo, Julie Bourseguin, Emilie Renaud, Patrycja Pawlikowska, Filippo Rosselli
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Research Article Cell biology Hematology

Microphthalmia transcription factor expression contributes to bone marrow failure in Fanconi anemia

Abstract

Hematopoietic stem cell (HSC) attrition is considered the key event underlying progressive BM failure (BMF) in Fanconi anemia (FA), the most frequent inherited BMF disorder in humans. However, despite major advances, how the cellular, biochemical, and molecular alterations reported in FA lead to HSC exhaustion remains poorly understood. Here, we demonstrated in human and mouse cells that loss-of-function of FANCA or FANCC, products of 2 genes affecting more than 80% of FA patients worldwide, is associated with constitutive expression of the transcription factor microphthalmia (MiTF) through the cooperative, unscheduled activation of several stress-signaling pathways, including the SMAD2/3, p38 MAPK, NF-κB, and AKT cascades. We validated the unrestrained Mitf expression downstream of p38 in Fanca–/– mice, which display hallmarks of hematopoietic stress, including loss of HSC quiescence, DNA damage accumulation in HSCs, and reduced HSC repopulation capacity. Importantly, we demonstrated that shRNA-mediated downregulation of Mitf expression or inhibition of p38 signaling rescued HSC quiescence and prevented DNA damage accumulation. Our data support the hypothesis that HSC attrition in FA is the consequence of defects in the DNA-damage response combined with chronic activation of otherwise transiently activated signaling pathways, which jointly prevent the recovery of HSC quiescence.

Authors

Alessia Oppezzo, Julie Bourseguin, Emilie Renaud, Patrycja Pawlikowska, Filippo Rosselli

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Figure 2

Mitf expression in FA is dependent on cell-secreted factor(s).

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Mitf expression in FA is dependent on cell-secreted factor(s). (A) Repr...
(A) Representative Western blots illustrating Mitf expression in extracts from WT, Fanca–/–, or Fancc–/– MEFs collected 24 hours after seeding in fresh or 72-hour conditioned (old) medium from WT or Fanca–/– MEF cultures. β-Actin was used as a loading control (n = 3). (B) qRT-PCR of Mitf expression in WT or Fanca–/– MEFs. MiTF expression in each cell line was first normalized to that of Oaz1 (internal control) and then to the Mitf/Oaz1 ratio in WT MEFs, which was set as 1 in each experiment. Data are shown as mean ± SEM of n experiments (for WT cells, n = 10 [fresh] and n = 3 [old WT and old Fanca–/–]; for Fanca–/– cells, n = 10 [Fresh], n = 2 [Old WT] and n = 7 [Old Fanca–/–]). (C and D) TGF-β (C) or activin A (D) concentrations (pg/mL) measured by ELISA in the supernatants of WT, Fanca–/–, or Fancc–/– MEFs maintained for 48 hours in medium without serum. Data are shown as mean ± SEM (n = 5). (E) Representative Western blots illustrating pSmad2/3 or p-p38 expression in the same extracts and conditions described in A. Since these are the same cell extracts analyzed in A, the β-actin blot is the same. (F) Representative Western blots of WT and Fanca–/– MEFs treated with the indicated concentrations of TGF-β or activin A and collected 24 hours later. β-Actin was used as a loading control (n = 3). (G) Mitf expression evaluated by qRT-PCR in Fanca–/– MEFs 2 hours following treatment with solvent (DMSO), p38 inhibitors (SB203580 [10 μM] or BIRB-796 [10 μM]), or pSmad2/3 inhibitors (SIS3 [2.5 μM] or SD208 [10 μM]). Data are shown as mean ± SEM of n = 5 (NT, DMSO, SB203580) or n = 3 (BIRB-796, SIS3, and SD208) experiments. Statistical significance was assessed using an unpaired 2-tailed t test with Welch’s correction (B) or 1-way ANOVA with Dunnett’s correction (C, D, and G). *P < 0.05; **P < 0.01; ***P < 0.001.