技术资料

In our investigations of the bone marrow (BM) of PECAM-1 null (knockout,KO) mice,we observed that the trabecular bone volume and number of trabeculae were significantly reduced in femoral and tibial long bones. Further studies in vitro revealed increased numbers and size of osteoclasts,enhanced bone resorption on dentin substrates,and hypersensitivity to macrophage CSF and receptor activator of NF-kappaB ligand in BM-derived osteoclast precursor cultures from KO mice. Associations among PECAM-1,Syk,and SHP-1 were found in wild-type BM monocyte derived osteoclast-like cells. The absence of PECAM-1 and SHP-1 interactions in the KO cells leads to the dysregulation of Syk kinases and/or phosphatases,possibly SHP-1. Indeed,KO derived osteoclast-like cells exhibited increased Syk tyrosine phosphorylation levels compared with WT cells. Lastly,WT mice engrafted with marrow from KO kindred showed loss of trabecular bone analogous to KO mice,consistent with increased osteoclastogenesis. View Publication

Poor recovery of cryopreserved human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells is a significant impediment to progress with pluripotent stem cells. In this study,we demonstrate that Y-27632,a specific inhibitor of Rho kinase (ROCK) activity,significantly enhances recovery of hES cells from cryopreserved stocks when cultured with or without a growth inactivated feeder layer. Furthermore,treatment with the ROCK inhibitor for several days increased the number of colonies and colony size of hES cells compared to shorter exposures. Remarkably,hES cells that had formed relatively few colonies 5 days after thawing exhibited rapid growth upon addition of Y-27632. Additionally,we determined that Y-27632 significantly improves the recovery of cryopreserved human iPS cells and their growth upon subculture. Thus,Y-27632 provides a means to kick-start" slow-growing human pluripotent stem cells� View Publication

Protein modification by small ubiquitin-related modifier proteins (SUMOs) controls diverse cellular functions. Dysregulation of SUMOylation or deSUMOylation processes has been implicated in the development of cancer and neurodegenerative diseases. However,no small-molecule inhibiting protein SUMOylation has been reported so far. Here,we report inhibition of SUMOylation by ginkgolic acid and its analog,anacardic acid. Ginkgolic acid and anacardic acid inhibit protein SUMOylation both in vitro and in vivo without affecting in vivo ubiquitination. Binding assays with a fluorescently labeled probe showed that ginkgolic acid directly binds E1 and inhibits the formation of the E1-SUMO intermediate. These studies will provide not only a useful tool for investigating the roles of SUMO conjugations in a variety of pathways in cells,but also a basis for the development of drugs targeted against diseases involving aberrant SUMOylation. View Publication

Reprogramming of somatic cells to pluripotency,thereby creating induced pluripotent stem (iPS) cells,promises to transform regenerative medicine. Most instances of direct reprogramming have been achieved by forced expression of defined factors using multiple viral vectors. However,such iPS cells contain a large number of viral vector integrations,any one of which could cause unpredictable genetic dysfunction. Whereas c-Myc is dispensable for reprogramming,complete elimination of the other exogenous factors is also desired because ectopic expression of either Oct4 (also known as Pou5f1) or Klf4 can induce dysplasia. Two transient transfection-reprogramming methods have been published to address this issue. However,the efficiency of both approaches is extremely low,and neither has been applied successfully to human cells so far. Here we show that non-viral transfection of a single multiprotein expression vector,which comprises the coding sequences of c-Myc,Klf4,Oct4 and Sox2 linked with 2A peptides,can reprogram both mouse and human fibroblasts. Moreover,the transgene can be removed once reprogramming has been achieved. iPS cells produced with this non-viral vector show robust expression of pluripotency markers,indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimaeric mice. When the single-vector reprogramming system was combined with a piggyBac transposon,we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This system minimizes genome modification in iPS cells and enables complete elimination of exogenous reprogramming factors,efficiently providing iPS cells that are applicable to regenerative medicine,drug screening and the establishment of disease models. View Publication

Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro,as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In this study,we employed an OP9 differentiation system to characterize the hematopoietic and endothelial differentiation potential of seven human iPSC lines obtained from human fetal,neonatal,and adult fibroblasts through reprogramming with POU5F1,SOX2,NANOG,and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC,H1,H7,H9,H13,and H14). Similar to hESCs,all iPSCs generated CD34(+)CD43(+) hematopoietic progenitors and CD31(+)CD43(-) endothelial cells in coculture with OP9. When cultured in semisolid media in the presence of hematopoietic growth factors,iPSC-derived primitive blood cells formed all types of hematopoietic colonies,including GEMM colony-forming cells. Human induced pluripotent cells (hiPSCs)-derived CD43(+) cells could be separated into the following phenotypically defined subsets of primitive hematopoietic cells: CD43(+)CD235a(+)CD41a(+/-) (erythro-megakaryopoietic),lin(-)CD34(+)CD43(+)CD45(-) (multipotent),and lin(-)CD34(+)CD43(+)CD45(+) (myeloid-skewed) cells. Although we observed some variations in the efficiency of hematopoietic differentiation between different hiPSCs,the pattern of differentiation was very similar in all seven tested lines obtained through reprogramming of human fetal,neonatal,or adult fibroblasts with three or four genes. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes,patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and for identification of molecules that can correct affected genetic networks. View Publication

The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway commonly occurs in cancers and is a crucial event in tumorigenesis. Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The PI3K/Akt pathway is activated by Bcr-Abl chimera protein and mediates the leukemogenesis in CML. However,the mechanism by which Bcr-Abl activates the PI3K/Akt pathway is not completely understood. In the present study,we found that pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1 and PHLPP2) were depleted in CML cells. We investigated the interaction between PHLPPs and Bcr-Abl in CML cell lines and Bcr-Abl+ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of PHLPP1 and PHLPP2,which dephosphorylated Ser-473 on Akt1,-2,and -3,resulting in inhibited proliferation of CML cells. The reduction of PHLPP1 and PHLPP2 expression by short interfering RNA in CML cells weakened the Abl kinase inhibitor-mediated inhibition of proliferation. In colony-forming unit-granulocyte,erythroid,macrophage,megakaryocyte; colony-forming unit-granulocyte,macrophage; and burst-forming unit-erythroid,treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced PHLPP1 and PHLPP2 expression and inhibited colony formation of Bcr-Abl+ progenitor cells,whereas depletion of PHLPP1 and PHLPP2 weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl+ progenitor cells. Thus,Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and continuously activates Akt1,-2,and -3 via phosphorylation on Ser-473,resulting in the proliferation of CML cells. View Publication

The number of neutrophils in the blood is tightly regulated to ensure adequate protection against microbial pathogens while minimizing damage to host tissue. Neutrophil homeostasis in the blood is achieved through a balance of neutrophil production,release from the bone marrow,and clearance from the circulation. Accumulating evidence suggests that signaling by CXCL12,through its major receptor CXCR4,plays a key role in maintaining neutrophil homeostasis. Herein,we generated mice with a myeloid lineage-restricted deletion of CXCR4 to define the mechanisms by which CXCR4 signals regulate this process. We show that CXCR4 negatively regulates neutrophil release from the bone marrow in a cell-autonomous fashion. However,CXCR4 is dispensable for neutrophil clearance from the circulation. Neutrophil mobilization responses to granulocyte colony-stimulating factor (G-CSF),CXCL2,or Listeria monocytogenes infection are absent or impaired,suggesting that disruption of CXCR4 signaling may be a common step mediating neutrophil release. Collectively,these data suggest that CXCR4 signaling maintains neutrophil homeostasis in the blood under both basal and stress granulopoiesis conditions primarily by regulating neutrophil release from the bone marrow. View Publication

T cell homeostasis is crucial for maintaining an efficient and balanced T cell immunity. The interaction between TCR and self peptide (sp) MHC ligands is known to be the key driving force in this process,and it is believed to be functionally and mechanistically different from that initiated by the antigenic TCR stimulation. Yet,very little is known about the downstream signaling events triggered by this TCR-spMHC interaction and how they differ from those triggered by antigenic TCR stimulation. In this study,we show that T cell conditional ablation of MEKK3,a Ser/Thr kinase in the MAPK cascade,causes a significant reduction in peripheral T cell numbers in the conditional knockout mice,but does not perturb thymic T cell development and maturation. Using an adoptive mixed transfer method,we show that MEKK3-deficient T cells are severely impaired in lymphopenia-induced cell proliferation and survival. Interestingly,the Ag-induced T cell proliferation proceeds normally in the absence of MEKK3. Finally,we found that the activity of ERK1/2,but not p38 MAPK,was attenuated during the lymphopenia-driven response in MEKK3-deficient T cells. Together,these data suggest that MEKK3 may play a crucial selective role for spMHC-mediated T cell homeostasis. View Publication

The receptor tyrosine kinase c-Kit plays a critical role in hematopoiesis,and gain-of-function mutations of the receptor are frequently seen in several malignancies,including acute myeloid leukemia,gastrointestinal stromal tumors,and testicular carcinoma. The most common mutation of c-Kit in these disorders is a substitution of the aspartic acid residue in position 816 to a valine (D816V),leading to constitutive activation of the receptor. In this study,we aimed to investigate the role of Src family kinases in c-Kit/D816V signaling. Src family kinases are necessary for the phosphorylation of wild-type c-Kit as well as of activation of downstream signaling pathways including receptor ubiquitination and the Ras/Mek/Erk pathway. Our data demonstrate that,unlike wild-type c-Kit,the phosphorylation of c-Kit/D816V is not dependent on Src family kinases. In addition,we found that neither receptor ubiquitination nor Erk activation by c-Kit/D816V required activation of Src family kinases. In vitro kinase assay using synthetic peptides revealed that c-Kit/D816V had an altered substrate specificity resembling Src and Abl tyrosine kinases. We further present evidence that,in contrast to wild-type c-Kit,Src family kinases are dispensable for c-Kit/D816V cell survival,proliferation,and colony formation. Taken together,we demonstrate that the signal transduction pathways mediated by c-Kit/D816V are markedly different from those activated by wild-type c-Kit and that altered substrate specificity of c-Kit circumvents a need for Src family kinases in signaling of growth and survival,thereby contributing to the transforming potential of c-Kit/D816V. View Publication

Prostaglandins,particularly prostaglandin E2 (PGE2),play an important role during inflammation. This is exemplified by the clinical use of cyclooxygenase 2 inhibitors,which interfere with PGE2 synthesis,as effective antiinflammatory drugs. Here,we show that PGE2 directly promotes differentiation and proinflammatory functions of human and murine IL-17-producing T helper (Th17) cells. In human purified naive T cells,PGE2 acts via prostaglandin receptor EP2- and EP4-mediated signaling and cyclic AMP pathways to up-regulate IL-23 and IL-1 receptor expression. Furthermore,PGE2 synergizes with IL-1beta and IL-23 to drive retinoic acid receptor-related orphan receptor (ROR)-gammat,IL-17,IL-17F,CCL20,and CCR6 expression,which is consistent with the reported Th17 phenotype. While enhancing Th17 cytokine expression mainly through EP2,PGE2 differentially regulates interferon (IFN)-gamma production and inhibits production of the antiinflammatory cytokine IL-10 in Th17 cells predominantly through EP4. Furthermore,PGE2 is required for IL-17 production in the presence of antigen-presenting cells. Hence,the combination of inflammatory cytokines and noncytokine immunomodulators,such as PGE2,during differentiation and activation determines the ultimate phenotype of Th17 cells. These findings,together with the altered IL-12/IL-23 balance induced by PGE2 in dendritic cells,further highlight the crucial role of the inflammatory microenvironment in Th17 cell development and regulation. View Publication

The zinc finger protein growth factor independent-1 (Gfi1) is a transcriptional repressor that is critically required for normal granulocytic differentiation. GFI1 loss-of-function mutations are found in some patients with severe congenital neutropenia (SCN). The SCN-associated GFI1-mutant proteins act as dominant negatives to block granulopoiesis through selective deregulation of a subset of GFI1 target genes. Here we show that Gfi1 is a master regulator of microRNAs,and that deregulated expression of these microRNAs recapitulates a Gfi1 loss-of-function block to granulocyte colony-stimulating factor (G-CSF)-stimulated granulopoiesis. Specifically,bone marrow cells from a GFI1-mutant SCN patient and Gfi1(-/-) mice display deregulated expression of miR-21 and miR-196B expression. Flow cytometric analysis and colony assays reveal that the overexpression or depletion of either miR induces changes in myeloid development. However,coexpression of miR-21 and miR-196b (as seen in Gfi1(-/-) mice and a GFI1N382S SCN patient) completely blocks G-CSF-induced granulopoiesis. Thus,our results not only identify microRNAs whose regulation is required during myelopoiesis,but also provide an example of synergy in microRNA biologic activity and illustrate potential mechanisms underlying SCN disease pathogenesis. View Publication

MicroRNAs (miRNAs) are a newly discovered endogenous class of small noncoding RNAs that play important posttranscriptional regulatory roles by targeting mRNAs for cleavage or translational repression. Accumulating evidence now supports the importance of miRNAs for human embryonic stem cell (hESC) self-renewal,pluripotency,and differentiation. However,with respect to induced pluripotent stem cells (iPSC),in which embryonic-like cells are reprogrammed from adult cells using defined factors,the role of miRNAs during reprogramming has not been well-characterized. Determining the miRNAs that are associated with reprogramming should yield significant insight into the specific miRNA expression patterns that are required for pluripotency. To address this lack of knowledge,we use miRNA microarrays to compare the microRNA-omes" of human iPSCs� View Publication