技术资料

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs),and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo,but is essential for normal HSC/HPC differentiation. In addition,Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased β1 integrin and Cxcr4 expression. Moreover,deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and,subsequently,improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult. View Publication

Hyperactivation of the transcription factor Stat5 leads to various leukemias. Stat5 activity is regulated by the protein phosphatase SHP-1 in a phospholipase C (PLC)-β3-dependent manner. Thus,PLC-β3-deficient mice develop myeloproliferative neoplasm,like Lyn (Src family kinase)- deficient mice. Here we show that Lyn/PLC-β3 doubly deficient lyn(-/-);PLC-β3(-/-) mice develop a Stat5-dependent,fatal myelodysplastic/myeloproliferative neoplasm,similar to human chronic myelomonocytic leukemia (CMML). In hematopoietic stem cells of lyn(-/-);PLC-β3(-/-) mice that cause the CMML-like disease,phosphorylation of SHP-1 at Tyr(536) and Tyr(564) is abrogated,resulting in reduced phosphatase activity and constitutive activation of Stat5. Furthermore,SHP-1 phosphorylation at Tyr(564) by Lyn is indispensable for maximal phosphatase activity and for suppression of the CMML-like disease in these mice. On the other hand,Tyr(536) in SHP-1 can be phosphorylated by Lyn and another kinase(s) and is necessary for efficient interaction with Stat5. Therefore,we identify a novel Lyn/PLC-β3-mediated regulatory mechanism of SHP-1 and Stat5 activities. View Publication

Ectopic expression of CAAT/enhancer binding protein α (C/EBPα) in p210BCR/ABL-expressing cells induces granulocytic differentiation,inhibits proliferation,and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects,C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding-dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5'-flanking region and enhances the promoter activity of Gfi-1. Moreover,in K562 cells,RNA interference-mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBPα. Ectopic expression of wild-type Gfi-1,but not of a transcriptional repressor mutant (Gfi-1P2A),inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast,Gfi-1 short hairpin RNA-tranduced CD34(+) chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together,these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells. View Publication

Although aldehyde dehydrogenase (ALDH) activity has become a surrogate of hematopoietic stem and progenitor cells (HSPCs),its function during hematopoiesis was unclear. Here,we examined its role in zebrafish hematopoiesis based on pharmacological inhibition and morpholino (MO) knockdown. Zebrafish embryos were treated with diethylaminobenzaldehyde (DEAB,1 μmol/l) between 0- and 48 hour-post-fertilization (hpf). MOs targeting aldhs were injected between 1 and 4-cell stage. The effects on hematopoiesis were evaluated at different stages. DEAB treatment between 0 and 18 hpf increased gene expression associated with HSPC (scl,lmo2),erythropoiesis (gata1,α- and β-eHb) and myelopoiesis (spi1) as well as gfp(+) cells in dissociated Tg(gata1:gfp) embryos. The effects were ameliorated by all-trans retinoic acid (1 nmol/l). Definitive hematopoiesis and the erythromyeloid precursors were unaffected. In all,14 out of 15 zebrafish aldhs were detectable by reverse transcription PCR in 18 hpf embryos,of which only aldh1a2 and aldh16a1 were expressed in sites pertinent to hematopoiesis. Molecular targeting by MOs was demonstrated for 15 aldhs,but none of them,even in combined aldh1a2 and aldh1a3 knockdown,recapitulated the hematopoietic expansion in DEAB-treated embryos. In conclusion,DEAB expands HSPC population during primitive hematopoiesis through inhibition of aldh and retinoic acid synthesis. The specific aldh isoform(s) remains to be determined. View Publication

Injury induces the recruitment of bone marrow-derived cells (BMDCs) that contribute to the repair and regeneration process. The behavior of BMDCs in injured tissue has a profound effect on repair,but the regulation of BMDC behavior is poorly understood. Aberrant recruitment/retention of these cells in wounds of diabetic patients and animal models is associated with chronic inflammation and impaired healing. BMD Gr-1(+)CD11b(+) cells function as immune suppressor cells and contribute significantly to tumor-induced neovascularization. Here we report that Gr-1(+)CD11b(+) cells also contribute to injury-induced neovascularization,but show altered recruitment/retention kinetics in the diabetic environment. Moreover,diabetic-derived Gr-1(+)CD11b(+) cells fail to stimulate neovascularization in vivo and have aberrant proliferative,chemotaxis,adhesion,and differentiation potential. Previously we demonstrated that gene transfer of HOXA3 to wounds of diabetic mice is taken up by and expressed by recruited BMDCs. This is associated with a suppressed inflammatory response,enhanced neovascularization,and accelerated wound healing. Here we show that sustained expression of Hoxa3 in diabetic-derived BMD Gr-1(+)CD11b(+) cells reverses their diabetic phenotype. These findings demonstrate that manipulation of adult stem/progenitor cells ex vivo could be used as a potential therapy in patients with impaired wound healing. View Publication

Endocannabinoids are arachidonic acid derivatives and part of a novel bioactive lipid signaling system,along with their G-coupled cannabinoid receptors (CB�? and CB₂) and the enzymes involved in their biosynthesis and degradation. However,their roles in hematopoiesis and hematopoietic stem and progenitor cell (HSPC) functions are not well characterized. Here,we show that bone marrow stromal cells express endocannabinoids (anandamide and 2-arachidonylglycerol),whereas CB₂ receptors are expressed in human and murine HSPCs. On ligand stimulation with CB₂ agonists,CB₂ receptors induced chemotaxis,migration,and enhanced colony formation of bone marrow cells,which were mediated via ERK,PI3-kinase,and Gαi-Rac1 pathways. In vivo,the CB₂ agonist AM1241 induced mobilization of murine HSPCs with short- and long-term repopulating abilities. In addition,granulocyte colony-stimulating factor -induced mobilization of HSPCs was significantly decreased by specific CB₂ antagonists and was impaired in Cnr2(-/-) cannabinoid type 2 receptor knockout mice. Taken together,these results demonstrate that the endocannabinoid system is involved in hematopoiesis and that CB₂/CB₂ agonist axis mediates repopulation of hematopoiesis and mobilization of HSPCs. Thus,CB₂ agonists may be therapeutically applied in clinical conditions,such as bone marrow transplantation. View Publication

The ability of hematopoietic stem cells (HSCs) to undergo self-renewal is partly regulated by external signals originating from the stem cell niche. Our previous studies with HSCs obtained from fetal liver of mice deficient for the calcium-sensing receptor (CaR) have shown the crucial role of this receptor in HSC lodgment and engraftment in the bone marrow (BM) endosteal niche. Using a CaR agonist,Cinacalcet,we assessed the effects of stimulating the CaR on the function of murine HSCs. Our results show that CaR stimulation increases primitive hematopoietic cell activity in vitro,including growth in stromal cell cocultures,adhesion to extracellular matrix molecules such as collagen I and fibronectin,and migration toward the chemotactic stimulus,stromal cell-derived factor 1α. Receptor stimulation also led to augmented in vivo homing,CXCR4-mediated lodgment at the endosteal niche,and engraftment capabilities. These mechanisms by which stimulating the CaR dictates preferential localization of HSCs in the BM endosteal niche provide additional insights into the fundamental interrelationship between the stem cell and its niche. These studies also have implications in the area of clinical stem cell transplantation,where ex vivo modulation of the CaR may be envisioned as a strategy to enhance HSC engraftment in the BM. View Publication

Normal stem cells and cancer stem cells (CSCs) share similar properties,in that both have the capacity to self-renew and differentiate into multiple cell types. In both the normal stem cell and cancer stem cell fields,there has been a great need for a universal marker that can effectively identify and isolate these rare populations of cells in order to characterize them and use this information for research and therapeutic purposes. Currently,it would appear that certain isoenzymes of the aldehyde dehydrogenase (ALDH) superfamily may be able to fulfill this role as a marker for both normal and cancer stem cells. ALDH has been identified as an important enzyme in the protection of normal hematopoietic stem cells,and is now also widely used as a marker to identify and isolate various types of normal stem cells and CSCs. In addition,emerging evidence suggests that ALDH1 is not only a marker for stem cells,but may also play important functional roles related to self-protection,differentiation,and expansion. This comprehensive review discusses the role that ALDH plays in normal stem cells and CSCs,with focus on ALDH1 and ALDH3A1. Discrepancies in the functional themes between cell types and future perspectives for therapeutic applications will also be discussed. View Publication

OBJECTIVE: To explore the effects of anti-CD44 monoclonal antibody-IM7 on the in vitro adhesion and migration of chronic myeloid leukemia stem cell (CML-LSC) and its mechanism. METHODS: CD34(+)CD38(-)CD123(+) leukemic stem cells (LSC) from 20 newly-diagnosed chronic myeloid leukemia (CML) patients BM cells and CD34(+)CD38(-) hematopoietic stem cells (HSC) from 20 full-term newborn cord blood cells were isolated with EasySep(TM) magnet beads. The CD44 expression of the LSC and HSC was detected by flow cytometry (FCM),and the adhesion and migration ability of the LSC and HSC pre- and post-incubated with IM7 in vitro by MTT assay and transendothelial migration assay,respectively. RESULTS: (1) After incubated with IM7,the LSC and HSC CD44 expression rates were (86.60 ± 2.10)% vs. (25.40 ± 1.70)% (P textless 0.05),respectively. (2) The adhesive ability of the LSC to endothelial cells was decreased markedly after incubated with IM7,the OD value (A(570)) changing from pre-incubation of (0.62 ± 0.11) to post-incubation of (0.34 ± 0.07),while there was little change of A(570) in the HSC group. (3) The migration ability of the LSC group was inhibited evidently after incubated with IM7,the inhibition rate being 46% ∼ 63%,while little change of that in HSC group was detected. (4) The adhesive ability of the LSC group to marrow stromal cells was decreased markedly after incubated with IM7,while little change was found in that of HSC group. CONCLUSION: The anti-CD44 monoclonal antibody-IM7 can effectively inhibit the adhesion and migration abilities of the LSC in vitro,which might provide a theoretical evidence for targeting therapy. View Publication

Polymorphonuclear neutrophils (PMNs) are critical for the formation,maintenance,and resolution of bacterial abscesses. However,the mechanisms that regulate PMN survival and proliferation during the evolution of an abscess are not well defined. Using a mouse model of Staphylococcus aureus abscess formation within a cutaneous wound,combined with real-time imaging of genetically tagged PMNs,we observed that a high bacterial burden elicited a sustained mobilization of PMNs from the bone marrow to the infected wound,where their lifespan was markedly extended. A continuous rise in wound PMN number,which was not accounted for by trafficking from the bone marrow or by prolonged survival,was correlated with the homing of c-kit(+)-progenitor cells from the blood to the wound,where they proliferated and formed mature PMNs. Furthermore,by blocking their recruitment with an antibody to c-kit,which severely limited the proliferation of mature PMNs in the wound and shortened mouse survival,we confirmed that progenitor cells are not only important contributors to PMN expansion in the wound,but are also functionally important for immune protection. We conclude that the abscess environment provides a niche capable of regulating PMN survival and local proliferation of bone marrow-derived c-kit(+)-progenitor cells. View Publication