Aconitase Modulates Erythroid Signaling through a Novel MEK-ERK Regulatory Pathway.

Abstract 75

Erythropoiesis is a complex developmental program regulated in part by erythropoietin (EPO) and iron levels. We have previously shown that erythroid iron deprivation causes diminished enzymatic activity of the multifunctional aconitase proteins (Bullock et al., ASH 2007 #707). In addition, pharmacological inhibition of aconitase impairs erythroid growth and maturation in a lineage-selective manner without compromising ATP levels, suggesting a novel, non-metabolic regulatory role for aconitase in erythropoiesis (Talbot et al., ASH 2008 #3865). The nature of this regulatory role was addressed in the present study by examining the connection between aconitase activity and erythroid signal transduction. The impact of aconitase inhibition by fluoroacetate (FA) on several candidate pathways was assessed in primary human erythroid progenitors by standard immunoblotting. This approach consistently implicated the mitogen activated protein kinases (MAPKs) ERK1/2, whose phosphorylation was strongly induced both at early (30 minutes) and late (4 days) time points. In addition, phosphorylation of the other MAPKs p38 and JNK was unchanged, ruling out a non-specific stress response. To determine why aconitase inhibition augmented ERK1/2 phosphorylation, the effects of FA were determined on the upstream kinases MEK1/2. Surprisingly, phosphorylation of MEK1/2 showed no changes at earlier time points (up to 24 hours) and decreases at later time points (2–4 days). Activation of ERK/1/2 in the absence of MEK1/2 activation suggests an unconventional pathway in which aconitase regulates dephosphorylation, scaffolding, or subcellular localization. The inhibition of erythropoiesis by FA could result from increased ERK1/2 activity or diminished MEK1/2 activity. The first mechanism predicts phenotypic rescue by addition of MEK inhibitor (U0126), whereas the second mechanism predicts increased inhibition. In fact, U0126 alone recapitulated the anti-proliferative and anti-differentiative effects of FA on primary erythroid cultures, and the combination of U0126 and FA caused increased inhibition of growth and differentiation, arguing in favor of MEK1/2 repression as a mechanism for FA. Our findings establish that aconitase modulates signaling through the MEK-ERK pathway, perturbation of which may block erythropoiesis. To expand our studies on the in vivo role of aconitase in erythropoiesis, the impact of aconitase blockade was assessed in two additional murine models: phenylhydrazine (PHZ)-induced stress erythropoiesis and Polycythemia vera (PV). In both systems, erythropoiesis was found to be extremely sensitive to aconitase blockade. In the first system, adult C57Bl/6 female mice were treated with continuous FA infusion plus bolus intraperitoneal PHZ. FA-treated mice developed a much more severe anemia in response to PHZ than did non-FA-treated mice despite receiving 50% less PHZ. Their anemia showed a lower nadir (red cell count of 3.56 M/ul vs. 4.75 M/ul, p=0.0013, n=6) and a more prolonged trough. Their reticulocytosis was delayed and diminished. In the second system, PV female mice carrying one copy of the JAK2 V617F allele were treated with continuous FA infusion. Decreased red indices were observed by day 11 of treatment, with a decline in hematocrit from 51.3% to 42.5% in PV mice (p=0.0002, n=4), compared with a decline from 44.6% to 37.9% in wild type mice (p=0.04, n=4). No effects were observed on platelet and leukocyte counts. Interestingly, the rate of decrease of the red cell parameters was greater in the PV mice, suggesting that erythropoiesis driven by JAK2 V617F is highly responsive to changes in aconitase activity levels. We conclude that aconitase functions as a modifier of erythroid signaling to regulate proliferation and differentiation. Indeed, our results indicate that aconitase activity regulates steady state, stress, and JAK2 V617F-driven erythropoiesis, most likely through its novel modulation of MEK-ERK signaling. This novel function may provide an alternate targeting strategy in the treatment of Polycythemia vera.