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Ecotropic viral integration site 1 (Evi1) is one of the master regulators in the development of acute myeloid leukemia (AML) and myelodysplastic syndrome. High expression of Evi1 is found in 10% of patients with AML and indicates a poor outcome. Several recent studies have indicated that Evi1 requires collaborative factors to induce AML. Therefore, the search for candidate factors that collaborate with Evi1 in leukemogenesis is one of the key issues in uncovering the mechanism of Evi1-related leukemia. Previously, we succeeded in making a mouse model of Evi1-related leukemia using a bone marrow transplantation (BMT) system. In the Evi1-induced leukemic cells, we identified frequent retroviral integrations near the CCAAT/enhancer-binding protein β (C/EBPβ) gene and overexpression of its protein. These findings imply that C/EBPβ is a candidate gene that collaborates with Evi1 in leukemogenesis. Cotransduction of Evi1 and the shortest isoform of C/EBPβ, liver inhibitory protein (LIP), induced AML with short latencies in a mouse BMT model. Overexpression of LIP alone also induced AML with longer latencies. However, excision of all 3 isoforms of C/EBPβ (LAP*/LAP/LIP) did not inhibit the development of Evi1-induced leukemia. Therefore, isoform-specific intervention that targets LIP is required when we consider C/EBPβ as a therapeutic target.

Ecotropic viral integration site 1 (Evi1), a transcription factor of the SET/PR domain protein family, is essential for the maintenance of hematopoietic stem cells (HSCs) in mice and is overexpressed in several myeloid malignancies. Here, we generate reporter mice in which an internal ribosome entry site (IRES)-GFP cassette is knocked-in to the Evi1 locus. Using these mice, we find that Evi1 is predominantly expressed in long-term HSCs (LT-HSCs) in adult bone marrow, and in the hematopoietic stem/progenitor fraction in the aorta-gonad-mesonephros, placenta, and fetal liver of embryos. In both fetal and adult hematopoietic systems, Evi1 expression marks cells with long-term multilineage repopulating activity. When combined with conventional HSC surface markers, sorting according to Evi1 expression markedly enhances purification of cells with HSC activity. Evi1 heterozygosity leads to marked impairment of the self-renewal capacity of LT-HSCs, whereas overexpression of Evi1 suppresses differentiation and boosts self-renewal activity. Reintroduction of Evi1, but not Mds1-Evi1, rescues the HSC defects caused by Evi1 heterozygosity. Thus, in addition to documenting a specific relationship between Evi1 expression and HSC self-renewal activity, these findings highlight the utility of Evi1-IRES-GFP reporter mice for the identification and sorting of functional HSCs.

Lineage-specific transcription factors must be precisely regulated during stem cell self-renewal and lineage commitment decisions. The role of specific transcription factors in hematopoietic stem cell (HSC) fate decisions has derived largely from genetic strategies, primarily gene-targeting and transgenic or retroviral overexpression experiments. From the previous experimental results, several transcription factors have been found to play critical roles in HSC physiology. Among them, we focus two transcription factors, Runx1/AML1 and Evi-1, in this review. During embryogenesis, both Runx1 and Evi-1 are essential for HSCs whereas in the adult, Runx1 and Evi-1 regulate HSCs negatively and positively, respectively., (2009 Wiley-Liss, Inc.)

The Notch proteins constitute a family of transmembrane receptors that play a pivotal role in cellular differentiation, proliferation and apoptosis. Although it has been recognized that excess Notch signaling is potentially tumorigenic, little is known about precise mechanisms through which dysregulated Notch signaling induces neoplastic transformation. Here we demonstrate that Notch signaling has a transcriptional cross-talk with transforming growth factor-beta (TGF-beta) signaling, which is well characterized by its antiproliferative effects. TGF-beta-mediated transcriptional responses are suppressed by constitutively active Notch1, and this inhibitory effect is canceled by introduction of transcriptional coactivator p300. We further show that this blockade of TGF-beta signaling is executed by the sequestration of p300 from Smad3. Moreover, in a human cervical carcinoma cell line, CaSki, in which Notch1 is spontaneously activated, suppression of Notch1 expression with small interfering RNA significantly restores the responsiveness to TGF-beta. Taken together, we propose that Notch oncoproteins promote cell growth and cancer development partly by suppressing the growth inhibitory effects of TGF-beta through sequestrating p300 from Smad3.

Acute myelogenous leukemia 1 (AML1; runt-related transcription factor 1 [Runx1]) is a member of Runx transcription factors and is essential for definitive hematopoiesis. Although AML1 possesses several subdomains of defined biochemical functions, the physiologic relevance of each subdomain to hematopoietic development has been poorly understood. Recently, the consequence of carboxy-terminal truncation in AML1 was analyzed by the hematopoietic rescue assay of AML1-deficient mouse embryonic stem cells using the gene knock-in approach. Nonetheless, a role for specific internal domains, as well as for mutations found in a human disease, of AML1 remains to be elucidated. In this study, we established an experimental system to efficiently evaluate the hematopoietic potential of AML1 using a coculture system of the murine embryonic para-aortic splanchnopleural (P-Sp) region with a stromal cell line, OP9. In this system, the hematopoietic defect of AML1-deficient P-Sp can be rescued by expressing AML1 with retroviral infection. By analysis of AML1 mutants, we demonstrated that the hematopoietic potential of AML1 was closely related to its transcriptional activity. Furthermore, we showed that other Runx transcription factors, Runx2/AML3 or Runx3/AML2, could rescue the hematopoietic defect of AML1-deficient P-Sp. Thus, this experimental system will become a valuable tool to analyze the physiologic function and domain contribution of Runx proteins in hematopoiesis.

Stem cell leukemia (SCL) protein has been shown to be an essential transcription factor during hematopoietic development in the embryo. In adult hematopoiesis, however, the role for SCL has remained largely unknown, whereas it is expressed in bone marrow hematopoietic stem cells (HSCs). In this study, we performed HSC transplantation and an in vitro HSC differentiation assay using retrovirally transduced HSCs with wild-type (WT) and dominant-negative (DN) SCL. The transplantation experiments showed that SCL does not affect the long-term repopulating capacity of HSCs but that WT SCL and DN SCL increase the short-term contribution of the transduced HSCs in myeloid and lymphoid lineages, respectively. An in vitro single-cell assay using a fetal thymus organ culture system further demonstrated that WT SCL facilitates HSCs to differentiate into the myeloid lineage but that DN SCL facilitates HSCs to differentiate into the lymphoid lineage. We conclude that the up-regulation or down-regulation of SCL directs HSCs toward myeloid or lymphoid lineage, respectively, although SCL does not affect their long-term repopulating capacity.

In the transforming growth factor beta (TGF-beta) superfamily, activin A, TGF-beta1, and bone morphogenic protein 4 (BMP-4) have various effects on hematopoiesis, including early mesodermo-hematogenesis. After these cytokines bind to their respective receptor, a regulatory Smad is phosphorylated and becomes associated with Smad4, the common Smad, and the resulting complex translocates to the nucleus to regulate transcription. DLX1 is the product of a member of the distal-less homeobox gene family, which is known to have important roles in embryogenesis, particularly in craniofacial development, and in GABAergic neurogenesis. DLX1 has been reported to be temporally and spatially coexpressed with BMP-4 during embryogenesis in selected contexts. We report here that, in addition to the previously reported regions/cells, DLX1 is expressed in hematopoietic cells in a lineage-dependent manner and that DLX1 interacts with Smad4 through its homeodomain. We show that it blocks multiple signals from TGF-beta superfamily cytokines such as activin A, TGF-beta1, and BMP-4, including differentiation of a hematopoietic cell line by activin A. Taken together, these data suggest that DLX1 may function as a regulator of multiple signals from TGF-beta superfamily members in broad biological contexts during blood production.

Notch signaling is involved in cell fate decisions in many systems including hematopoiesis. It has been shown that expression of an activated form of Notch1 (aNotch1) in 32D mouse myeloid progenitor cells inhibits the granulocytic differentiation induced by granulocyte colony-stimulating factor (G-CSF). Results of the current study show that aNotch1, when expressed in F5-5 mouse erythroleukemia cells, also inhibits erythroid differentiation. Comparison of the expression levels of several transcription factors after stimulation for myeloid and erythroid differentiation, in the presence or absence of aNotch1, revealed that aNotch1 did not change its regulation pattern with any of the transcription factors examined, except for GATA-2, despite its inhibitory effect on differentiation. GATA-2 was down-regulated when the parental 32D and F5-5 were induced to differentiate into granulocytic and erythroid lineages, respectively. In these induction procedures, however, the level of GATA-2 expression was sustained when aNotch1 was expressed. To ascertain whether maintenance of GATA-2 is required for the Notch-induced inhibition of differentiation, the dominant-negative form of GATA-3 (DN-GATA), which acted also against GATA-2, or transcription factor PU.1, which was recently shown to be the repressor of GATA-2, was introduced into aNotch1-expressing 32D (32D/aNotch1) cells that do not express GATA family proteins other than GATA2. Both DN-GATA and PU.1 reversed the phenotype of 32D/aNotch1 inducing its differentiation when G-CSF was added. Furthermore, enforced expression of HES-1, which is involved in Notch signaling, delayed differentiation of 32D, and again this phenotype was neutralized by DN-GATA. These results indicate that GATA-2 activity is necessary for the Notch signaling in hematopoietic cells.

Antigen-induced eosinophil recruitment into the airways of sensitized mice is mediated by CD4(+) T cells and their cytokines, especially IL-5. In this study, we found that the antigen-induced airway eosinophilia was diminished in Stat5a-deficient (Stat5a(-/-)) mice and Stat5b-deficient (Stat5b(-/-)) mice. We also found that antigen-induced CD4(+) T-cell infiltration and IL-5 production in the airways were diminished in Stat5a(-/- )mice and Stat5b(-/-) mice. Moreover, antigen-induced proliferation of splenocytes was diminished in Stat5a(-/- )mice and Stat5b(-/-) mice, suggesting that the generation of antigen-primed T cells may be compromised in Stat5a(-/-) mice and Stat5b(-/-) mice and this defect may account for the diminished antigen-induced T-cell infiltration into the airways. Interestingly, IL-4 and IL-5 production from anti-CD3-stimulated splenocytes was diminished in Stat5a(-/-) mice and Stat5b(-/-) mice. However, antigen-specific IgE and IgG1 production was diminished in Stat5a(-/-) mice but not in Stat5b(-/-) mice, whereas antigen-specific IgG2a production was increased in Stat5a(-/-) mice, suggesting the enhanced Th1 responses in Stat5a(-/-) mice. Finally, we found that eosinophilopoiesis induced by the administration of recombinant IL-5 was also diminished in Stat5a(-/-) mice and Stat5b(-/-) mice. Together, these results indicate that both Stat5a and Stat5b are essential for induction of antigen-induced eosinophil recruitment into the airways and that the defects in antigen-induced eosinophil recruitment in Stat5a(-/-) mice and Stat5b(-/-) mice result from both impaired IL-5 production in the airways and diminished IL-5 responsiveness of eosinophils. (Blood. 2000;95:1370-1377)

TGF-beta plays an important role in lung fibrosis, which is a major cause of suffering and death seen in pulmonary disease. Smad7 has been recently identified as an antagonist of TGF-beta signaling. To investigate whether this novel molecule can be exploited for therapy of lung fibrosis, we determined the effect of exogenous Smad7, introduced by a recombinant human type 5 adenovirus vector, on bleomycin-induced lung fibrosis in mice. C57BL/6 mice with bleomycin-induced lungs received an intratracheal injection of a recombinant adenovirus carrying mice Smad7 cDNA. These mice demonstrated suppression of type I precollagen mRNA, reduced hydroxyproline content, and no morphological fibrotic responses in the lungs when compared with mice administered adenovirus carrying Smad6 cDNA. In addition, we found that expression of Smad7 transgene blocked Smad2 phosphorylation induced by bleomycin in mouse lungs. These data indicated that gene transfer of Smad7 (but not Smad6) prevented bleomycin-induced lung fibrosis, suggesting that Smad7 may have applicability in the treatment of pulmonary fibrosis.