HOXB4's road map to stem cell expansion

Abstract

Homeodomain-containing transcription factors are important regulators of stem cell behavior. HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis with enriched adult HSCs and embryonic derivatives expressing inducible HOXB4. Thereby, we identified a set of overlapping genes that likely represent "universal" targets of HOXB4. A substantial number of loci are involved in signaling pathways important for controlling self-renewal, maintenance, and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-alpha. This protection likely contributes to the competitive repopulation advantage of HOXB4-expressing HSCs observed in vivo. The concept of TNF-alpha inhibition may also prove beneficial for patients undergoing bone marrow transplantation. Furthermore, we demonstrate that HOXB4 activity and FGF signaling are intertwined. HOXB4-mediated expansion of adult and ES cell-derived HSCs was enhanced by specific and complete inhibition of FGF receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day 4-6 embryoid bodies was blunted in the presence of basic FGF (FGF2), indicating a dominant negative effect of FGF signaling on the earliest hematopoietic cells. In summary, our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance toward stem cell self-renewal.

Fig. 1.

qRT-PCR confirms changes in gene expression observed in the microarray study. Gene expression changes were measured in triplicate analysis of RNA samples from purified HOXB4ER⁺LSK cells that had been incubated with and without TMX for 4 h to induce HOXB4 in the presence or absence of CHX. Relative differences in gene expression between TMX-induced and noninduced HOXB4ER samples were calculated by using the 2^−ΔΔCT method (13) by normalizing the CT values for each gene to the CT values of the housekeeping gene, actin. Values are shown as log2-fold induction (mean ± SD) in the absence (green bars) and presence of CHX (red bars). ** indicates direct gene targets of HOXB4, displaying differential gene expression in the presence of CHX.

Fig. 2.

Selected regulatory pathways affected by HOXB4 in adult HSCs/HPCs and EBs. HOXB4 influences the expression of genes involved in pivotal cell-intrinsic pathways such as regulation of cell cycle, differentiation, and apoptosis. It also modulates the response to multiple conserved extrinsic signals provided by the microenvironment. Affiliations of the genes with the indicated pathways are based on published reports in various mammalian systems. Arrows indicate up-/down-regulation of gene expression as a consequence of HOXB4 induction.

Fig. 3.

Induction of HOXB4 activity protects cultivated HSCs from the negative effects TNF-α on stem cell self-renewal. (A) Experimental design to test the effect of TNF-α on the multilineage hematopoietic reconstitution capacity of HOXB4ER-expressing LSK cells in a competitive transplant setting. The progeny (expansion equivalent) of 2,000 HOXB4ER-transduced and 2,000 control vector (tCD34)-transduced LSK cells were mixed and cocultured, as indicated, in serum-free cytokine-supplemented medium in the presence or absence of TNF-α or FCS ± HOXB4 induction with TMX. After 7 days, the frequency of HOXB4ER-expressing and tCD34-expressing LSK cells in the resulting cell populations was determined by flow cytometry, and the cell samples were transplanted into cohorts of lethally irradiated recipient mice. (B) Reconstituting activity of HOXB4ER-expressing (■) and tCD34-expressing (▵) cells cultivated for 7 days, as indicated. Small horizontal lines indicate the median percentage of HOXB4ER⁺ or tCD34⁺ cells in the peripheral blood of the mice 11 weeks after transplantation.

Fig. 4.

HOXB4 and FGF signaling are intertwined. Inducible HOXB4 ES cells were treated from days 4 to 6 of EB formation with and without Dox in the absence or presence of either the FGF receptor inhibitor, SU5402, or bFGF (FGF2) as indicated. EBs were collected on day 6 and processed for methylcellulose-CFU assays or for RNA extraction and subsequent semiquantitative RT-PCR analysis. (A) Inhibition of FGF signaling enhances HOXB4-mediated expansion early HPCs. (B) HOXB4 down-regulates the expression of Dusp6 and Etv5, a key player in the activation of FGF target genes. (C) bFGF inhibits HOXB4-mediated expansion of ES cell-derived early HPCs. Ery, definitive erythroid; GM, granulocyte macrophage. Results are presented as mean ± SD (n = 3). P values were determined by Student's t test.