Iron-sulfur (Fe-S) clusters are cofactors essential for the activity of numerous enzymes including DNA polymerases, helicases, and glycosylases. They are synthesized in the mitochondria as Fe-S intermediates and are exported to the cytoplasm for maturation by the mitochondrial transporter ABCB7. Here, we demonstrate that ABCB7 is required for bone marrow B cell development, proliferation, and class switch recombination, but is dispensable for peripheral B cell homeostasis in mice. Conditional deletion of ABCB7 using Mb1-cre resulted in a severe block in bone marrow B cell development at the pro-B cell stage. The loss of ABCB7 did not alter expression of transcription factors required for B cell specification or commitment. While increased intracellular iron was observed in ABCB7-deficient pro-B cells, this did not lead to increased cellular or mitochondrial reactive oxygen species, ferroptosis, or apoptosis. Interestingly, loss of ABCB7 led to replication-induced DNA damage in pro-B cells, independent of VDJ recombination, and these cells had evidence of slowed DNA replication. Stimulated ABCB7-deficient splenic B cells from CD23-cre mice also had a striking loss of proliferation and a defect in class switching. Thus, ABCB7 is essential for early B cell development, proliferation, and class switch recombination.
NKAP is a multi-functional nuclear protein that has been shown to be essential for hematopoiesis. Deletion of NKAP in hematopoietic stem cells was previously found to result in rapid lethality and hematopoietic failure. NKAP deficient cells also exhibited diminished proliferation and increased expression of the cyclin dependent kinase inhibitors (CDKIs) p19 Ink4d and p21 Cip1. To determine how dysregulation of CDKII expression contributes to the effects of NKAP deficiency, NKAP was deleted in mice also deficient in p19 Ink4d or p21 Cip1 using poly-IC treatment to induce Mx1-cre. Hematopoietic failure and lethality were not prevented by deficiency in either CDKI when NKAP was deleted. Inducible deletion of NKAP in cultured hematopoietic progenitors ex vivo resulted in a senescent phenotype and altered expression of numerous cell cycle regulators including the CDKI p16 INK4a. Interestingly, while combined deficiency in p16 INK4a and p21 Cip1 did not reverse the effect of NKAP deficiency on hematopoiesis in vivo, it did shift the consequence of NKAP deficiency from senescence to apoptosis in ex vivo cultures. These results suggest that NKAP may limit cellular stress that can trigger cell cycle withdrawal or cell death, a role critical for the maintenance of a viable pool of hematopoietic progenitors.
The hematopoietic marrow microenvironment is composed of multiple cell types embedded in an extracellular matrix (ECM). We have explored marrow ECM using mass spectrometry and found dermatopontin (DPT), a small non-collagenous ECM protein, to be present. We found that DPT cooperates with other ECM proteins to promote hematopoietic cell adherence in vitro on plastic as well as OP9 stromal cells. We generated constitutional DPT-/- mice that were viable and had no peripheral lympho-hematopoietic abnormalities. The composition of the marrow of wild-type and DPT-/- mice was equivalent in terms of cellularity, CFU-C, LSK (Lineage-, SCA-1+, KIT+), and LSK-SLAM (LSK, CD48-, CD150+) frequencies. These data suggest that DPT fosters adherence but is not required for steady-state hematopoiesis in vivo. There are likely overlapping cellular adhesion mechanisms that can compensate to maintain the hematopoietic niche in the absence of DPT.
Abstract Iron-sulfur (Fe-S) clusters are cofactors essential for the activity of numerous enzymes including DNA polymerases, helicases, and glycosylases. They are synthesized in the mitochondria as Fe-S intermediates and are exported to the cytoplasm for maturation by the mitochondrial transporter ABCB7. Here, we demonstrate that ABCB7 is required for bone marrow B cell development, proliferation, and class switch recombination, but is dispensable for peripheral B cell homeostasis in mice. Conditional deletion of ABCB7 using Mb1-cre resulted in a severe block in bone marrow B cell development at the pro-B cell stage. The loss of ABCB7 did not alter expression of transcription factors required for B cell specification or commitment. While increased intracellular iron was observed in ABCB7-deficient pro-B cells, this did not lead to increased cellular or mitochondrial reactive oxygen species, ferroptosis, or apoptosis. Interestingly, loss of ABCB7 led to replication-induced DNA damage in pro-B cells, independent of VDJ recombination, and these cells had evidence of slowed DNA replication. Interestingly, ABCB7 appeared to be dispensable for splenic B cell homeostasis, as CD23-cre ABCB7 cKO mice had normal splenic B cell populations. However, stimulated ABCB7-deficient splenic B cells from CD23-cre ABCB7 cKO mice had a striking loss of proliferation and a defect in class switching. Thus, ABCB7 is essential for early B cell development, proliferation, and class switch recombination. Supported by 1R21 AI157328-01 to V.S.S., T32AI007425 to M.J.L.
Abstract ABCB7 is a mitochondrial transporter that exports iron-sulfur (Fe-S) cluster intermediates from the mitochondria to the cytoplasm. Using CD2-iCre ABCB7 cKO mice, we found ABCB7 is required for early B cell development. These mice have a severe block in pro-B cell development at the Fr. C to Fr. C′ transition, with reduced expression of intracellular μ and pre-BCR. CD19-cre and CD23-cre ABCB7 cKO mice have normal B cell development and peripheral B cell populations, indicating ABCB7 is required only during early B cell development. We observed reduced expression of Runx1, Ikaros, Foxo1, E47 (E2A), and Pax5 in CD2-iCre ABCB7 cKO pro-B cells. Interestingly, we also observed decreased BACH2 expression, which indicates elevated heme synthesis is occurring in the absence of ABCB7. We hypothesize that in the absence of ABCB7, developing B cells enhance heme synthesis to dispose of accumulating mitochondrial iron. Using a hen egg lysozyme BCR transgene, we were able to rescue B cell development in CD2-iCre ABCB7 cKO mice. Intriguingly, at the age of 5–8 weeks old, CD2-iCre ABCB7 cKO mice become lethargic and hunched. CBCs reveal an increase in peripheral blood neutrophils. Both the spleen and bone marrow become highly infiltrated by CD11b+Ly6Glow/+Ly6C− cells that are immature and mature neutrophils. As other knockout mice with pro-B cell blocks do not have neutrophilia, we considered if increased iron or heme is responsible for our observation. As heme can stimulate TLR4, we propose that excess heme from developing ABCB7-deficent B cells activates macrophages. In support of this, hemin induced GM-CSF production in BMDM cultures. We are currently exploring the mechanism by which excess heme production can stimulate the production of neutrophils.
Abstract NKAP is a multifunctional nuclear protein that associates with the histone deacetylase HDAC3. Although both NKAP and HDAC3 are critical for hematopoietic stem cell (HSC) maintenance and survival, it was not known whether these two proteins work together. To assess the importance of their association in vivo, serial truncation and alanine scanning was performed on NKAP to identify the minimal binding site for HDAC3. Mutation of either Y352 or F347 to alanine abrogated the association of NKAP with HDAC3, but did not alter NKAP localization or expression. Using a linked conditional deletion/re-expression system in vivo, we demonstrated that re-expression of the Y352A NKAP mutant failed to restore HSC maintenance and survival in mice when endogenous NKAP expression was eliminated using Mx1-cre and poly-IC, whereas re-expression of wild type NKAP maintained the HSC pool. However, Y352A NKAP did restore proliferation in murine embryonic fibroblasts when endogenous NKAP expression was eliminated using ER-cre and tamoxifen. Therefore, Y352 in NKAP is critical for association with HDAC3 and for HSC maintenance and survival but is not important for proliferation of murine embryonic fibroblasts, demonstrating that NKAP functions in different complexes in different cell types.
Abstract Patients with iron overload disorders often develop lymphocyte deficiencies, indicating iron homeostasis is critical for lymphopoiesis. Loss of ABCB7, a mitochondrial iron transporter, leads to mitochondrial iron accumulation and rapid hematopoietic failure. We found that ABCB7 is required for lymphopoiesis, as CD2-icre ABCB7 cKO mice have a severe block in both T and B cell development. The block in T cell development occurs at the DN3 stage. This DN3 block is not due to aberrant β-selection, proliferation, or viability. The block in B cell development occurs at the Fr. C to Fr. C′ transition. Pre-BCR expression was reduced in developing B cells and they exhibited reduced proliferation compared to WT B cells. Because loss of ABCB7 causes mitochondrial iron accumulation, which is toxic to cells, we hypothesized that lymphoid cells increase heme synthesis in order to detoxify the accumulating iron. We found that HO-1, a surrogate marker for intracellular heme levels, was elevated in these lymphocytes. Currently, we are examining the mechanism responsible for the block in lymphocyte development upon loss of ABCB7.
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