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Abstract
NF-κB is an important regulator of both differentiation and function of lineage-committed hematopoietic cells. Targeted deletion of IκB kinase (IKK) β results in altered cytokine signaling and marked neutrophilia. To investigate the role of IKKβ in regulation of hematopoiesis, we employed Mx1-Cre mediated IKKβ conditional knockout mice. As previously reported, deletion of IKKβ in hematopoietic cells results in neutrophilia, and we now also noted decreased monocytes and modest anemia. Granulocyte-macrophage progenitors (GMPs) accumulated markedly in bone marrow of IKKβ deleted mice whereas the proportion and number of megakaryocyte-erythrocyte progenitors (MEP) decreased. Accordingly, we found a significantly reduced frequency of proerythroblasts and basophilic and polychromatic erythroblasts, and IKKβ-deficient bone marrow cells yielded a significantly decreased number of BFU-E compared to wild type. These changes are associated with elevated expression of C/EBPα, Gfi1, and PU.1 and diminished Gata1, Klf1, and SCL/Tal1 in IKKβ deficient Lineage-Sca1+c-Kit+ (LSK) cells. In contrast, no effect on erythropoiesis or expression of lineage-related transcription factors was found in marrow lacking NF-κB p65. Bone marrow from IKKβ knockout mice has elevated numbers of phenotypic long and short term hematopoietic stem cells (HSC). A similar increase was observed when IKKβ was deleted after marrow transplantation into a wild type host, indicating cell autonomous expansion. Myeloid progenitors from IKKβ- but not p65-deleted mice demonstrate increased serial replating in colony-forming assays, indicating increased cell autonomous self-renewal capacity. In addition, in a competitive repopulation assay deletion of IKKβ resulted in a stable advantage of bone marrow derived from IKKβ knockout mice. In summary, loss of IKKβ resulted in significant effects on hematopoiesis not seen upon NF-κB p65 deletion. These include increased myeloid and reduced erythroid transcription factors, skewing differentiation towards myeloid over erythroid differentiation, increased progenitor self-renewal, and increased number of functional long term HSCs. These data inform ongoing efforts to develop IKK inhibitors for clinical use.
Highlights
Hematopoiesis is a tightly regulated process in which pluripotent hematopoietic stem cells (HSC) differentiate into lineage specific progenitors and mature subsequently into specialized hematopoietic cells [1]
Using primers designed to anneal at the excised region (Table 1) efficient deletion of the Ikbkb or RelA genes could be demonstrated at the genomic DNA level (Fig 1B)
Neutrophilia was observed in the marrow of IKKβΔ/Δ and p65Δ/Δ mice and these mice each had decreased marrow monocytes (Fig 1C)
Summary
Hematopoiesis is a tightly regulated process in which pluripotent hematopoietic stem cells (HSC) differentiate into lineage specific progenitors and mature subsequently into specialized hematopoietic cells [1]. HSCs maintain a balance between self-renewal and differentiation to support hematopoiesis throughout the lifetime of the organism. Five members are recognized in mammalian cells: RelA/p65, Rel B, c-Rel, NF-κB1/p50 and NF-κB2/p52; all sharing a Rel homology domain that mediates dimerization and DNA binding. IB is phosphorylated by IB kinase (IKK), ubiquitinated, and undergoes proteasomal degradation to allow active dimers to translocate into the nucleus and bind target DNA κB sites. While IKKα regulates non-canonical NF-κB signaling, IKKβ participates in the regulation of the canonical pathway, which typically culminates in translocation of p50:p65 dimers to the nucleus. In addition to phosphorylating IB proteins, other IKK substrates modulate immune response, chromatin remodeling, and autophagy [3,4,5,6,7,8]
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