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.

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.

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. Robo4(high) 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.

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 alpha 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 alpha (C/EBPalpha) and PU.1 are potential regulators for hematopoietic stem cells (HSCs). To gain further insight into the HSC regulation by C/EBPalpha 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/EBPalpha 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/EBPalpha was activated, and the differentiation capacity to megakaryocytic lineage was selectively impaired upon C/EBPalpha 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/EBPalpha or PU.1, suggesting that they regulate HSC function through an as yet unknown mechanism. Our data suggest that the activation of C/EBPalpha or PU.1 is sufficient to repress stem cell capacities in HSCs, and their fine-tuned regulation is critical for HSC homeostasis.

Mammalian twisted gastrulation is essential for skeleto-lymphogenesis.

Dorsoventral patterning depends on the local concentrations of the morphogens. Twisted gastrulation (TSG) regulates the extracellular availability of a mesoderm inducer, bone morphogenetic protein 4 (BMP-4). However, TSG function in vivo is still unclear. We isolated a TSG cDNA as a secreted molecule from the mouse aorta-gonad-mesonephros region. Here we show that TSG-deficient mice were born healthy, but more than half of the neonatal pups showed severe growth retardation shortly after birth and displayed dwarfism with delayed endochondral ossification and lymphopenia, followed by death within a month. TSG-deficient thymus was atrophic, and phosphorylation of SMAD1 was augmented in the thymocytes, suggesting enhanced BMP-4 signaling in the thymus. Since BMP-4 promotes skeletogenesis and inhibits thymus development, our findings suggest that TSG acts as both a BMP-4 agonist in skeletogenesis and a BMP-4 antagonist in T-cell development. Although lymphopenia in TSG-deficient mice would partly be ascribed to systemic effects of runtiness and wasting, our findings may also provide a clue for understanding the pathogenesis of human dwarfism with combined immunodeficiency.

Tyk2 is dispensable for induction of myeloproliferative disease by mutant FLT3.

Internal tandem duplication of FLT3 tyrosine kinase (FLT3-ITD) is the most prevalent mutation found in acute myelogenous leukemia (AML), having been identified in 20% to 30% of all AML patients. We have previously shown that FLT3-ITD signals mainly through the signal transducer and activator of transcription 5 (STAT5) pathway and have suggested the possible involvement of Tyk2 in STAT5 activation by FLT3-ITD. The present study addressed the role of Tyk2 in FLT3-ITD signaling in a murine bone marrow transplantation (BMT) model. Transplantation of wild-type bone marrow cells transduced with the FLT3-ITD gene induced lethal myeloproliferative disease (MPD) in the recipient mice at a median latency of 89 days. Interestingly, some mice presented the proliferation of B- or T-lymphoid blasts in various organs, a presentation that resembled acute lymphoblastic leukemia (ALL). Mice that received Tyk2-deficient bone marrow cells transduced with FLT3-ITD developed lethal MPD with a disease latency (median, 100 days) and pathologic picture similar to those of mice that received wild-type bone marrow cells. These results indicate that (1) Tyk2 is not essential for MPD induction by FLT3-ITD and (2) FLT3-ITD by itself can induce ALL in a murine BMT model.

Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics.

Most of the human genome has now been sequenced and about 30,000 potential open reading frames have been identified, indicating that we use these 30,000 genes to functionally organize our biologic activities. However, functions of many genes are still unknown despite intensive efforts using bioinformatics as well as transgenic and knockout mice. Retrovirus-mediated gene transfer is a powerful tool that can be used to understand gene functions. We have developed a variety of retrovirus vectors and efficient packaging cell lines that have facilitated the development of efficient functional expression cloning methods. In this review, we describe retrovirus-mediated strategies used for investigation of gene functions and function-based screening strategies.

Fusion of MLL and MSF in adult de novo acute myelomonocytic leukemia (M4) with t(11;17)(q23;q25).

The MLL gene at chromosome band 11q23 is frequently rearranged and fused to partner genes in acute leukemias. Previously, the MSF gene, also called AF17q25, has been cloned as a fusion partner of the MLL gene in therapy-related or infant acute myelogenous leukemias with t(11;17)(q23;q25). MSF belongs to the septin family of proteins, which includes other MLL fusion partners, hCDCrel1 and Septin 6, and has also been implicated in the pathogenesis of human ovarian tumor and murine T-cell lymphoma. We describe here a 64-year-old man with de novo acute myelomonocytic leukemia (French-American-British subtype M4) with t(11;17)(q23;q25). His leukemia was successfully induced into a first remission, which, however, lasted only briefly. A second remission was never attained, and the patient died of sepsis 16 months after the diagnosis of leukemia. Examination of his leukemic cells at diagnosis revealed an MLL gene rearrangement, by Southern blotting, and an MLL-MSF fusion transcript, by the reverse transcriptase polymerase chain reaction (RT-PCR) method. Sequence analysis of the RT-PCR product further revealed that MLL exon 5 was fused in-frame to MSF exon 3. Further clinical and molecular analyses of acute leukemias with the MLL-MSF transcript may shed more light on the clinical characteristics and molecular mechanisms of the MLL-septin type leukemias.

Spine formation pattern of adult-born neurons is differentially modulated by the induction timing and location of hippocampal plasticity.

In the adult hippocampus dentate gyrus (DG), newly born neurons are functionally integrated into existing circuits and play important roles in hippocampus-dependent memory. However, it remains unclear how neural plasticity regulates the integration pattern of new neurons into preexisting circuits. Because dendritic spines are major postsynaptic sites for excitatory inputs, spines of new neurons were visualized by retrovirus-mediated labeling to evaluate integration. Long-term potentiation (LTP) was induced at 12, 16, or 21 days postinfection (dpi), at which time new neurons have no, few, or many spines, respectively. The spine expression patterns were investigated at one or two weeks after LTP induction. Induction at 12 dpi increased later spinogenesis, although the new neurons at 12 dpi didn't respond to the stimulus for LTP induction. Induction at 21 dpi transiently mediated spine enlargement. Surprisingly, LTP induction at 16 dpi reduced the spine density of new neurons. All LTP-mediated changes specifically appeared within the LTP-induced layer. Therefore, neural plasticity differentially regulates the integration of new neurons into the activated circuit, dependent on their developmental stage. Consequently, new neurons at different developmental stages may play distinct roles in processing the acquired information by modulating the connectivity of activated circuits via their integration.

Robo4 plays a role in bone marrow homing and mobilization, but is not essential in the long-term repopulating capacity of hematopoietic stem cells.

Roundabout (Robo) family proteins are immunoglobulin-type surface receptors critical for cellular migration and pathway finding of neuronal axons. We have previously shown that Robo4 was specifically expressed in hematopoietic stem and progenitor cells and its high expression correlated with long-term repopulating (LTR) capacity. To reveal the physiological role of Robo4 in hematopoiesis, we examined the effects of Robo4 disruption on the function of hematopoietic stem cells (HSCs) and progenitors. In Robo4-deficient mice, basic hematological parameters including complete blood cell count and differentiation profile were not affected. In contrast to the previous report, HSC/hematopoietic progenitor (HPC) frequencies in the bone marrow (BM) were perfectly normal in Robo4(-/-) mice. Moreover, Robo4(-/-) HSCs were equally competitive as wild-type HSCs in transplantation assays and had normal long-term repopulating (LTR) capacity. Of note, the initial engraftment at 4-weeks after transplantation was slightly impaired by Robo4 ablation, suggesting a marginal defect in BM homing of Robo4(-/-) HSCs. In fact, homing efficiencies of HSCs/HPCs to the BM was significantly impaired in Robo4-deficient mice. On the other hand, granulocyte-colony stimulating factor-induced peripheral mobilization of HSCs was also impaired by Robo4 disruption. Lastly, marrow recovery from myelosuppressive stress was equally efficient in WT- and Robo4-mutant mice. These results clearly indicate that Robo4 plays a role in HSC trafficking such as BM homing and peripheral mobilization, but is not essential in the LTR and self-renewal capacity of HSCs.

Analysis of mouse LMIR5/CLM-7 as an activating receptor: differential regulation of LMIR5/CLM-7 in mouse versus human cells.

We have analyzed leukocyte mono-Ig-like receptor 5 (LMIR5) as an activating receptor among paired LMIRs. Mouse LMIR5 (mLMIR5) is expressed in myeloid cells such as mast cells, granulocytes, macrophages, and dendritic cells. Cross-linking of transduced mLMIR5 in bone marrow-derived mast cells (BMMCs) caused activation events, including cytokine production, cell survival, degranulation, and adhesion to the extracellular matrix. mLMIR5 associated with DAP12 and to a lesser extent with DAP10, and mLMIR5-mediated functions of BMMCs were strongly inhibited by DAP12 deficiency. Importantly, cross-linking of endogenous mLMIR5 induced Syk-dependent activation of fetal liver-derived mast cells. Unlike mLMIR5, cross-linking of human LMIR5 (hLMIR5) induced cytokine production of BMMCs even in the absence of both DAP12 and DAP10, suggesting the existence of unidentified adaptors. Interestingly, hLMIR5 possessed a tyrosine residue (Y188) in the cytoplasmic region. Signaling via Y188 phosphorylation played a predominant role in hLMIR5-mediated cytokine production in DAP12-deficient, but not wild-type BMMCs. In addition, experiments using DAP10/DAP12 double-deficient BMMCs suggested the existence of Y188 phoshorylation-dependent and -independent signals from unidentified adaptors. Collectively, although both mouse and human LMIR5 play activatory roles in innate immunity cells, the functions of LMIR5 were differentially regulated in mouse versus human cells.

Functional analysis of activating receptor LMIR4 as a counterpart of inhibitory receptor LMIR3.

The leukocyte mono-Ig-like receptor (LMIR) belongs to a new family of paired immunoreceptors. In this study, we analyzed activating receptor LMIR4/CLM-5 as a counterpart of inhibitory receptor LMIR3/CLM-1. LMIR4 is expressed in myeloid cells, including granulocytes, macrophages, and mast cells, whereas LMIR3 is more broadly expressed. The association of LMIR4 with Fc receptor-gamma among immunoreceptor tyrosine-based activation motif-bearing molecules was indispensable for LMIR4-mediated functions of bone marrow-derived mast cells, but dispensable for its surface expression. Cross-linking of LMIR4 led to Lyn- and Syk-dependent activation of bone marrow-derived mast cells, resulting in cytokine production and degranulation, whereas that of LMIR3 did not. The triggering of LMIR4 and TLR4 synergistically caused robust cytokine production in accordance with enhanced activation of ERK, whereas the co-ligation of LMIR4 and LMIR3 dramatically abrogated cytokine production. Notably, intraperitoneal administration of lipopolysaccharide strikingly up-regulated LMIR3 and down-regulated LMIR4, whereas that of granulocyte colony-stimulating factor up-regulated both LMIR3 and LMIR4 in granulocytes. Cross-linking of LMIR4 in bone marrow granulocytes also resulted in their activation, which was enhanced by lipopolysaccharide. Collectively, these results suggest that the innate immune system is at least in part regulated by the qualitative and quantitative balance of the paired receptors LMIR3 and LMIR4.

N-terminal region of CCAAT/enhancer-binding protein epsilon is critical for cell cycle arrest, apoptosis, and functional maturation during myeloid differentiation.

CCAAT/enhancer-binding protein epsilon (C/EBPepsilon) plays a critical role in terminal myeloid differentiation. Differentiation is an integrated process of cell cycle arrest, morphological change, functional maturation, and apoptosis. However, the molecular networks underlying these events in C/EBPepsilon-induced differentiation remain poorly understood. To reveal these mechanisms, we performed a detailed molecular analysis of C/EBPepsilon-induced differentiation using an inducible form of C/EBPepsilon. The activation of C/EBPepsilon induced growth arrest, morphological differentiation, the expression of CD11b and secondary granule proteins, and apoptosis in myeloid cell lines. Unlike C/EBPalpha, C/EBPepsilon dramatically up-regulated p27 with a concomitant down-regulation of cdk4/6 and cyclin D2/A/E. Moreover, the anti-apoptotic proteins Bcl-2 and Bcl-x were down-regulated, whereas pro-apoptotic protein Bax remained unchanged. Using a variety of mutants, we revealed that these events were all regulated by the N-terminal activation domain of C/EBPepsilon. Interestingly, some of the differentiation processes such as the induction of secondary granule protein genes were clearly inhibited by c-Myc; however, inhibition of apoptosis by Bcl-x did not affect the entire differentiation processes. These data indicate the N terminus of C/EBPepsilon to be solely responsible for most aspects of myeloid differentiation, and these events were differentially affected by c-Myc.

Comprehensive analysis of myeloid lineage conversion using mice expressing an inducible form of C/EBP alpha.

CCAAT/enhancer-binding protein (C/EBP) alpha is a critical regulator for early myeloid differentiation. Although C/EBPalpha has been shown to convert B cells into myeloid lineage, precise roles of C/EBPalpha in various hematopoietic progenitors and stem cells still remain obscure. To examine the consequence of C/EBPalpha 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/EBPalpha. 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/EBPalpha, and the conversion occurred clonally through biphenotypic intermediate cells. Moreover, in vivo activation of C/EBPalpha 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/EBPalpha can activate the latent myeloid differentiation program of MEP and CLP and shows that its global activation affects multilineage homeostasis in vivo.

Immune suppressor factor confers stromal cell line with enhanced supporting activity for hematopoietic stem cells.

Immune suppressor factor (ISF) is a subunit of the vacuolar ATPase proton pump. We earlier identified a short form of ISF (ShIF) as a stroma-derived factor that supports cytokine-independent growth of mutant Ba/F3 cells. Here, we report that ISF/ShIF supports self-renewal and expansion of primary hematopoietic stem cells (HSCs). Co-culture of murine bone marrow cells with a stromal cell line overexpressing ISF or ShIF (MS10/ISF or MS10/ShIF) not only enhanced their colony-forming activity and the numbers of long-term culture initiating cells, but also maintained the competitive repopulating activity of HSC. This stem cell supporting activity depended on the proton-transfer function of ISF/ShIF. Gene expression analysis of ISF/ShIF-transfected cell lines revealed down-regulation of secreted frizzled-related protein-1 and tissue inhibitor of metalloproteinase-3, and the restoration of their expressions in MS10/ISF cells partially reversed its enhanced LTC-IC supporting activity to a normal level. These results suggest that ISF/ShIF confers stromal cells with enhanced supporting activities for HSCs by modulating Wnt-activity and the extracellular matrix.

The human perforin gene is a direct target of STAT4 activated by IL-12 in NK cells.

IL-12 activates STAT4 by inducing tyrosine phosphorylation, homo-dimerization, and nuclear translocation in NK cells and thereby stimulates proliferation and activation of these cells. The pore-forming protein perforin is a key effector protein for NK cell- and cytotoxic T lymphocyte-mediated cytolysis. Here we demonstrate that IL-12 induces the expression of the perforin gene in human NK cell line, NKL. Electrophoretic mobility shift assays using a probe containing two putative STAT-binding sequences located at -1085 and -1059 in the human perforin gene showed that STAT4 or STAT5 activated by IL-12 or IL-2, respectively, in NKL cells binds this region. Further analyses using various probes with or without mutated STAT-binding sequences showed that, although either of the two tandem STAT-binding sequences binds STAT4 weakly, the presence of both is required for significant binding of activated STAT4 and for formation of the STAT4-DNA-binding complex with lower electrophoretic mobility. Furthermore, mutation of either of the tandem STAT-binding sequences abolished the IL-12-induced activation of the perforin gene promoter in reporter gene assays. These results indicate that the IL-12-induced expression of the perforin gene in NK cells is directly regulated by STAT4, which binds, most likely as a homo-tetramer, to the tandem STAT-binding sequences in the perforin gene promoter.