Abstract
Gene targeting via homologous recombination can occasionally result in incomplete disruption of the targeted gene. Here, we show that a widely used Nur77-deficient transgenic mouse model expresses a truncated protein encoding for part of the N-terminal domain of nuclear receptor Nur77. This truncated Nur77 protein is absent in a newly developed Nur77-deficient mouse strain generated using Cre-Lox recombination. Comparison of these two mouse strains using immunohistochemistry, flow cytometry, and colony-forming assays shows that homologous recombination-derived Nur77-deficient mice, but not WT or Cre-Lox-derived Nur77-deficient mice, suffer from liver immune cell infiltrates, loss of splenic architecture, and increased numbers of bone marrow hematopoietic stem cells and splenic colony-forming cells with age. Mechanistically, we demonstrate that the truncated Nur77 N-terminal domain protein maintains the stability and activity of hypoxia-inducible factor (HIF)-1, a transcription factor known to regulate bone marrow homeostasis. Additionally, a previously discovered, but uncharacterized, human Nur77 transcript variant that encodes solely for its N-terminal domain, designated TR3β, can also stabilize and activate HIF-1α. Meta-analysis of publicly available microarray data sets shows that TR3β is highly expressed in human bone marrow cells and acute myeloid leukemia samples. In conclusion, our study provides evidence that a transgenic mouse model commonly used to study the biological function of Nur77 has several major drawbacks, while simultaneously identifying the importance of nongenomic Nur77 activity in the regulation of bone marrow homeostasis.
Highlights
In response to inflammatory stimuli and other danger signals, the bone marrow can give rise to a wide range of special
hematopoietic stem and progenitor cell (HSPC) reside in specific areas of the bone marrow, termed the HSPC niche, where their survival is supported by low oxygen tension and concomitant induction of the transcription factor hypoxia-inducible factor (HIF)-1 (2)
A dual deficiency of Nur[77] and its family member NOR-1 leads to rapid development of acute myeloid leukemia (AML), with bone marrow differentiation being heavily skewed toward myeloid progeny (7)
Summary
Classical Nur77-KO mice, but not WT or complete Nur77-KO mice, exhibit liver immune cell infiltrates and loss of splenic architecture at old age. We considered the possibility that the N-terminal domain– encoding Nur77(1–117) protein expressed in classical Nur77-KO mice regulates HIF-1␣ stability and thereby causes the bone marrow, spleen, and liver phenotypes observed in these mice Consistent with this hypothesis, lentiviral overexpression of Nur77(1–117) protein in BMDMs from complete Nur77-KO mice resulted in increased HIF-1␣ protein expression in response to the hypoxia mimetic cobalt chloride (Fig. 3A). Gene expression of Egfr and Vcam[1], two factors that promote HSPC retention in the bone marrow (23, 24), was significantly lower in both classical Nur77-KO and complete Nur77-KO mice compared with WT mice (Fig. 3F) These results show that Nur77(1–117) can stabilize and activate HIF-1␣ both in vitro and in vivo. These results indicate that the human NUR77 transcript variant TR3 is strongly associated with high bone marrow expression and AML
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