Oxidative stress modulates heme synthesis and induces peroxiredoxin-2 as a novel cytoprotective response in  -thalassemic erythropoiesis

Abstract

The online version of this article has a Supplementary Appendix. Backgroundb-thalassemic syndromes are inherited red cell disorders characterized by severe ineffective erythropoiesis and increased levels of reactive oxygen species whose contribution to b-thalassemic anemia is only partially understood. Design and MethodsWe studied erythroid precursors from normal and b-thalassemic peripheral CD34+ cells in twophase liquid culture by proteomic, reverse transcriptase polymerase chain reaction and immunoblot analyses. We measured intracellular reactive oxygen species, heme levels and the activity of d-aminolevulinate-synthase-2. We exposed normal cells and K562 cells with silenced peroxiredoxin-2 to H2O2 and generated a recombinant peroxiredoxin-2 for kinetic measurements in the presence of H2O2 or hemin. ResultsIn b-thalassemia the increased production of reactive oxygen species was associated with down-regulation of heme oxygenase-1 and biliverdin reductase and up-regulation of peroxiredoxin-2. In agreement with these observations in b-thalassemic cells we found decreased heme levels related to significantly reduced activity of the first enzyme of the heme pathway, d-aminolevulinate synthase-2 without differences in its expression. We demonstrated that the activity of recombinant d-aminolevulinate synthase-2 is inhibited by both reactive oxygen species and hemin as a protective mechanism in b-thalassemic cells. We then addressed the question of the protective role of peroxiredoxin-2 in erythropoiesis by exposing normal cells to oxidative stress and silencing peroxiredoxin-2 in human erythroleukemia K562 cells. We found that peroxiredoxin-2 expression is up-regulated in response to oxidative stress and required for K562 cells to survive oxidative stress. We then showed that peroxiredoxin-2 binds heme in erythroid precursors with high affinity, suggesting a possible multifunctional cytoprotective role of peroxiredoxin-2 in b-thalassemia. ConclusionsIn b-thalassemic erythroid cells the reduction of d-aminolevulinate synthase-2 activity and the increased expression of peroxiredoxin-2 might represent two novel stress-response protective systems.Key words: b-thalassemia, heme biosynthesis, oxidative stress, ROS, peroxiredoxin-2.Citation: De Franceschi L, Bertoldi M, De Falco L, Santos Franco S, Ronzoni L, Turrini F, Colancecco A, Camaschella C, Cappellini MD, and Iolascon A. Oxidative stress modulates heme synthesis and induces peroxiredoxin-2 as a novel cytoprotective response in b-thalassemic erythropoiesis. Haematologica 2011;96(11):1595-1604. doi:10.3324/haematol.2011 This is an open-access paper.Oxidative stress modulates heme synthesis and induces peroxiredoxin-2 as a novel cytoprotective response in b-thalassemic erythropoiesis

Erythroid cells generated in the absence of specific 1-integrin heterodimers accumulate reactive oxygen species at homeostasis and are unable to mount effective antioxidant defenses

“…49 Since in our mice proliferative responses in the spleen appear to be dependent on α5β1 and not α4β1, it follows that after engagement of α5β1 integrins, the FAKrecruited complexes and their downstream initiated cascades are likely involved (Online Supplementary Figure S3B). Erythropoiesis in mice with specific integrin deficiencies haematologica | 2013; 98 (11)The fact that treatment of normal mice with an anti-FAK inhibitor partially phenocopies the response of β1 D/D mice post stress supports this view (Online Supplementary Figure S3A). It is also intriguing that mice with deletion of Src kinase Lyn, SHP1 and SHIP-1 phosphatases 50 have a similar phenotype to that of α4 −/− mice, suggesting that progenitor expansion is not dependent on Src signaling.…”

“…to combat increased ROS formation which, if left unbalanced, can affect plasma membrane proteins, lipid peroxidation and compromise RBC viability resulting in intra-and extra-vascular lysis. 3,4 Therefore, it is not surprising that mice with reduced cellular anti-oxidant responses [5][6][7][8][9][10][11][12][13][14] have a poorly compensated anemia and decreased erythroid responses to stress. In addition to genetic impairments in direct anti-oxidant responses (i.e.…”

Membrane association of peroxiredoxin-2 in red cells is mediated by the N-terminal cytoplasmic domain of band 3

“…PRDX2 (4 ȝM, 0.09 mg/ml)) intrinsic fluorescence spectrum was measured by exciting the enzyme at 295 nm, at which tryptophan residues have their absorbance maximum, and recording spectra in the interval from 300 to 500 nm with a Jasco FP750 spectrofluorimeter in 50 mM sodium phosphate buffer, 0.1 M NaCl, pH 7.4, or in 50 mM Peroxidase activity. PRDX2 activity was measured as previously reported [25].…”

“…To further validate the binding of PRDX2 to band 3 on the red cell membrane, we performed cross-linking studies, using recombinant PRDX2 [25] labeled with a photoactivatable crosslinking probe (sulfo-SBED). The labeled probe was incubated with KI-IOVs as previously described by Anong et al [23].…”

Hemopexin Therapy Improves Cardiovascular Function by Preventing Heme-Induced Endothelial Toxicity in Mouse Models of Hemolytic Diseases

“…3,23,24 Moreover, severe forms of SCD and β-thalassemia require a blood transfusion regimen that further increases the amount of circulating Hb/heme, thus exacerbating oxidative stress. 25,26 Although an iron chelation therapy is routinely associated with a transfusion regimen, 27,28 no heme chelation therapy has been developed to date that specifically prevents heme-induced endothelial damage and oxidative stress.…”