Miyuki Ito

Comprehensive analysis of myeloid lineage conversion using mice expressing an inducible form of C/EBPα

CCAAT/enhancer‐binding protein (C/EBP) α is a critical regulator for early myeloid differentiation. Although C/EBPα has been shown to convert B cells into myeloid lineage, precise roles of C/EBPα in various hematopoietic progenitors and stem cells still remain obscure. To examine the consequence of C/EBPα activation in various progenitors and to address the underlying mechanism of lineage conversion in detail, we established transgenic mice expressing a conditional form of C/EBPα. Using these mice, we show that megakaryocyte/erythroid progenitors (MEPs) and common lymphoid progenitors (CLPs) could be redirected to functional macrophages in vitro by a short‐term activation of C/EBPα, and the conversion occurred clonally through biphenotypic intermediate cells. Moreover, in vivo activation of C/EBPα in mice led to the increase of mature granulocytes and myeloid progenitors with a concomitant decrease of hematopoietic stem cells and nonmyeloid progenitors. Our study reveals that C/EBPα can activate the latent myeloid differentiation program of MEP and CLP and shows that its global activation affects multilineage homeostasis in vivo.

TIMP-3 recruits quiescent hematopoietic stem cells into active cell cycle and expands multipotent progenitor pool

Regulating transition of hematopoietic stem cells (HSCs) between quiescent and cycling states is critical for maintaining homeostasis of blood cell production. The cycling states of HSCs are regulated by the extracellular factors such as cytokines and extracellular matrix; however, the molecular circuitry for such regulation remains elusive. Here we show that tissue inhibitor of metalloproteinase-3 (TIMP-3), an endogenous regulator of metalloproteinases, stimulates HSC proliferation by recruiting quiescent HSCs into the cell cycle. Myelosuppression induced TIMP-3 in the bone marrow before hematopoietic recovery. Interestingly, TIMP-3 enhanced proliferation of HSCs and promoted expansion of multipotent progenitors, which was achieved by stimulating cell-cycle entry of quiescent HSCs without compensating their long-term repopulating activity. Surprisingly, this effect did not require metalloproteinase inhibitory activity of TIMP-3 and was possibly mediated through a direct inhibition of angiopoietin-1 signaling, a critical mediator for HSC quiescence. Furthermore, bone marrow recovery from myelosuppression was accelerated by over-expression of TIMP-3, and in turn, impaired in TIMP-3-deficient animals. These results suggest that TIMP-3 may act as a molecular cue in response to myelosuppression for recruiting dormant HSCs into active cell cycle and may be clinically useful for facilitating hematopoietic recovery after chemotherapy or ex vivo expansion of HSCs.

Roundabout 4 is expressed on hematopoietic stem cells and potentially involved in the niche-mediated regulation of the side population phenotype.

Roundabout (Robo) family proteins are immunoglobulin‐type cell surface receptors that are expressed predominantly in the nervous system. The fourth member of this family, Robo4, is distinct from the other family members in that it is expressed specifically in endothelial cells. In this study, we examined the expression of Robo4 in hematopoietic stem cells (HSCs) and its possible role in HSC regulation. Robo4 mRNA was specifically expressed in murine HSCs and the immature progenitor cell fraction but not in lineage‐positive cells or differentiated progenitors. Moreover, flow cytometry showed a correlation between higher expression of Robo4 and immature phenotypes of hematopoietic cells. Robo4high hematopoietic stem/progenitor cells presented higher clonogenic activity or long‐term repopulating activity by colony assays or transplantation assays, respectively. A ligand for Robo4, Slit2, is specifically expressed in bone marrow stromal cells, and its expression was induced in osteoblasts in response to myelosuppressive stress. Interestingly, overexpression of Robo4 or Slit2 in HSCs resulted in their decreased residence in the c‐Kit+Sca‐1+Lineage−‐side population fraction. These results indicate that Robo4 is expressed in HSCs, and Robo4/Slit2 signaling may play a role in HSC homeostasis in the bone marrow niche. STEM CELLS 2009;27:183–190

Wnt modulators, SFRP-1, and SFRP-2 are expressed in osteoblasts and differentially regulate hematopoietic stem cells

Wnt signaling has been implicated in the self-renewal of hematopoietic stem cells (HSCs). Secreted frizzled-related proteins (SFRPs) are a family of soluble proteins containing a region homologous to a receptor for Wnt, Frizzled, and are thought to act as endogenous modulators for Wnt signaling. This study examined the role of SFRPs in HSC regulation. Among the four family members, SFRP-1 and SFRP-2 are specifically induced in the bone marrow in response to myelosuppression, and immunostaining revealed that both proteins were expressed in osteoblasts. Interestingly, SFRP-1 reduced the number of multipotent progenitors in in vitro culture of CD34(-)KSL cells, while SFRP-2 did not. Furthermore, SFRP-1 compromised the long-term repopulating activity of HSCs, whereas SFRP-2 did not affect or even enhanced it in the same setting. These results indicate that although both SFRP-1 and SFRP-2 act as inhibitors for Wnt signaling in vitro, they differentially affect the homeostasis of HSCs.

Activation of CCAAT/Enhancer-Binding Protein α or PU.1 in Hematopoietic Stem Cells Leads to Their Reduced Self-Renewal and Proliferation

Previous studies using loss‐of‐function mutants revealed that CCAAT/enhancer‐binding protein α (C/EBPα) and PU.1 are potential regulators for hematopoietic stem cells (HSCs). To gain further insight into the HSC regulation by C/EBPα or PU.1, we used transgenic mice expressing conditional forms of these transcription factors to examine whether their activation alone is sufficient for modulating HSC functions. The activation of C/EBPα or PU.1 in HSCs in vitro or in vivo led to their suppression of growth, decreased mixed colony formation, and impaired competitive repopulating activities because of their defective self‐renewal. These effects were more prominently observed when C/EBPα was activated, and the differentiation capacity to megakaryocytic lineage was selectively impaired upon C/EBPα activation. Unexpectedly, the expression of Bmi‐1 and HoxB4, well‐known regulators for self‐renewal of HSCs, was not affected by the activation of C/EBPα or PU.1, suggesting that they regulate HSC function through an as yet unknown mechanism. Our data suggest that the activation of C/EBPα or PU.1 is sufficient to repress stem cell capacities in HSCs, and their fine‐tuned regulation is critical for HSC homeostasis.

SHD1 is a novel cytokine-inducible, negative feedback regulator of STAT5-dependent transcription

STAT5 is a critical mediator of a variety of cytokine signaling whose transcriptional activity is regulated by associating with various proteins. During a search for STAT5-interacting proteins, we identified SHD1, a mammalian homologue of yeast gene Sac3, as a potential interacter. SHD1 was localized in the nucleus, and induced by cytokines that activate STAT5, such as erythropoietin, interleukin-2 (IL-2), or IL-3. SHD1 interacted specifically with STAT5A and STAT5B, and interestingly, it specifically repressed STAT5-dependent transcription in vitro without affecting the stability or phosphorylation of STAT5 protein. Gene disruption study revealed that T, B, or bone marrow cells from mice lacking SHD1 were hyperresponsive to T-cell-receptor engagement, or stimulation with various STAT5-activating cytokines. These results suggest that SHD1 is a novel cytokine-inducible negative feedback regulator of STAT5.