技术资料

MED1/TRAP220,a subunit of the transcriptional Mediator/TRAP complex,is crucial for various biological events through its interaction with distinct activators,such as nuclear receptors and GATA family activators. In hematopoiesis,MED1 plays a pivotal role in optimal nuclear receptor-mediated myelomonopoiesis and GATA-1-induced erythropoiesis. In this study,we present evidence that MED1 in stromal cells is involved in supporting hematopoietic stem and/or progenitor cells (HSPCs) through osteopontin (OPN) expression. We found that the proliferation of bone marrow (BM) cells cocultured with MED1 knockout (Med1(-/-)) mouse embryonic fibroblasts (MEFs) was significantly suppressed compared to the control. Furthermore,the number of long-term culture-initiating cells (LTC-ICs) was attenuated for BM cells cocultured with Med1(-/-) MEFs. The vitamin D receptor (VDR)- and Runx2-mediated expression of OPN,as well as Mediator recruitment to the Opn promoter,was specifically attenuated in the Med1(-/-) MEFs. Addition of OPN to these MEFs restored the growth of cocultured BM cells and the number of LTC-ICs,both of which were attenuated by the addition of the anti-OPN antibody to Med1(+/+) MEFs and to BM stromal cells. Consequently,MED1 in niche appears to play an important role in supporting HSPCs by upregulating VDR- and Runx2-mediated transcription on the Opn promoter. View Publication

Adhesion properties of hematopoietic stem cells (HSCs) in the bone marrow (BM) niches control their migration and affect their cell-cycle dynamics. The serum response factor (Srf) regulates growth factor-inducible genes and genes controlling cytoskeleton structures involved in cell spreading,adhesion,and migration. We identified a role for Srf in HSC adhesion and steady-state hematopoiesis. Conditional deletion of Srf in BM cells resulted in a 3-fold expansion of the long- and short-term HSCs and multipotent progenitors (MPPs),which occurs without long-term modification of cell-cycle dynamics. Early differentiation steps to myeloid and lymphoid lineages were normal,but Srf loss results in alterations in mature-cell production and severe thrombocytopenia. Srf-null BM cells also displayed compromised engraftment properties in transplantation assays. Gene expression analysis identified Srf target genes expressed in HSCs,including a network of genes associated with cell migration and adhesion. Srf-null stem cells and MPPs displayed impair expression of the integrin network and decreased adherence in vitro. In addition,Srf-null mice showed increase numbers of circulating stem and progenitor cells,which likely reflect their reduced retention in the BM. Altogether,our results demonstrate that Srf is an essential regulator of stem cells and MPP adhesion,and suggest that Srf acts mainly through cell-matrix interactions and integrin signaling. View Publication

The importance of cancer metabolism has been appreciated for many years,but the intricacies of how metabolic pathways interconnect with oncogenic signaling are not fully understood. With a clear understanding of how metabolism contributes to tumorigenesis,we will be better able to integrate the targeting of these fundamental biochemical pathways into patient care. The mevalonate (MVA) pathway,paced by its rate-limiting enzyme,hydroxymethylglutaryl coenzyme A reductase (HMGCR),is required for the generation of several fundamental end-products including cholesterol and isoprenoids. Despite years of extensive research from the perspective of cardiovascular disease,the contribution of a dysregulated MVA pathway to human cancer remains largely unexplored. We address this issue directly by showing that dysregulation of the MVA pathway,achieved by ectopic expression of either full-length HMGCR or its novel splice variant,promotes transformation. Ectopic HMGCR accentuates growth of transformed and nontransformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice and,importantly,cooperates with RAS to drive the transformation of primary mouse embryonic fibroblasts cells. We further explore whether the MVA pathway may play a role in the etiology of human cancers and show that high mRNA levels of HMGCR and additional MVA pathway genes correlate with poor prognosis in a meta-analysis of six microarray datasets of primary breast cancer. Taken together,our results suggest that HMGCR is a candidate metabolic oncogene and provide a molecular rationale for further exploring the statin family of HMGCR inhibitors as anticancer agents. View Publication

Although practiced clinically for more than 40 years,the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with primary human HSCs identified a purine derivative,StemRegenin 1 (SR1),that promotes the ex vivo expansion of CD34+ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy. View Publication

The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment,we silenced c-myb in human CD34(+) hematopoietic stem/progenitor cells. Noteworthy,c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages,whereas erythroid differentiation was impaired,as demonstrated by clonogenic assay,morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb-silenced CD34(+) cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as putative targets,which can account for c-myb knockdown effects. Indeed,chromatin immunoprecipitation and luciferase reporter assay demonstrated that c-myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently,the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing,whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34(+) cells. Our data collectively demonstrate that c-myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment,by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed,we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision. View Publication

Vinculin is a highly conserved actin-binding protein that is localized in integrin-mediated focal adhesion complexes. Although critical roles have been proposed for integrins in hematopoietic stem cell (HSC) function,little is known about the involvement of intracellular focal adhesion proteins in HSC functions. This study showed that the ability of c-Kit(+)Sca1(+)Lin(-) HSCs to support reconstitution of hematopoiesis after competitive transplantation was severely impaired by lentiviral transduction with short hairpin RNA sequences for vinculin. The potential of these HSCs to differentiate into granulocytic and monocytic lineages,to migrate toward stromal cell-derived factor 1α,and to home to the bone marrow in vivo were not inhibited by the loss of vinculin. However,the capacities to form long term culture-initiating cells and cobblestone-like areas were abolished in vinculin-silenced c-Kit(+)Sca1(+)Lin(-) HSCs. In contrast,adhesion to the extracellular matrix was inhibited by silencing of talin-1,but not of vinculin. Whole body in vivo luminescence analyses to detect transduced HSCs confirmed the role of vinculin in long term HSC reconstitution. Our results suggest that vinculin is an indispensable factor determining HSC repopulation capacity,independent of integrin functions. View Publication

Self-renewal and differentiation of hematopoietic stem cells (HSCs) are balanced by the concerted activities of the fibroblast growth factor (FGF),Wnt,and Notch pathways,which are tuned by enzyme-mediated remodeling of heparan sulfate proteoglycans (HSPGs). Sulfatase modifying factor 1 (SUMF1) activates the Sulf1 and Sulf2 sulfatases that remodel the HSPGs,and is mutated in patients with multiple sulfatase deficiency. Here,we show that the FGF signaling pathway is constitutively activated in Sumf1(-/-) HSCs and hematopoietic stem progenitor cells (HSPCs). These cells show increased p-extracellular signal-regulated kinase levels,which in turn promote beta-catenin accumulation. Constitutive activation of FGF signaling results in a block in erythroid differentiation at the chromatophilic erythroblast stage,and of B lymphocyte differentiation at the pro-B cell stage. A reduction in mature myeloid cells and an aberrant development of T lymphocytes are also seen. These defects are rescued in vivo by blocking the FGF pathway in Sumf1(-/-) mice. Transplantation of Sumf1(-/-) HSPCs into wild-type mice reconstituted the phenotype of the donors,suggesting a cell autonomous defect. These data indicate that Sumf1 controls HSPC differentiation and hematopoietic lineage development through FGF and Wnt signaling. View Publication

AIMS: Macrophage scavenger receptor A (SR-A) and class B scavenger receptor CD36 (CD36) contribute to foam cell formation and atherogenesis via uptake of modified lipoproteins. So far,the role of these scavenger receptors has been studied mainly using knockout models totally lacking these receptors. We studied the role of SR-A and CD36 in foam cell formation and atherogenesis by RNA interference (RNAi)-mediated silencing,which is a clinically feasible method to down-regulate the expression of these receptors. METHODS AND RESULTS: We constructed lentivirus vectors encoding short hairpin RNAs (shRNAs) against mouse SR-A and CD36. Decreased SR-A but not CD36 expression led to reduced foam cell formation caused by acetylated low-density lipoprotein (LDL) in mouse macrophages,whereas the uptake of oxidized LDL was not altered. More importantly,silencing of SR-A upregulates CD36 and vice versa. In LDL receptor-deficient apolipoprotein B100 (LDLR(-/-)ApoB(100/100)) mice kept on a western diet,silencing of either SR-A or CD36 in bone marrow cells led to a marked decrease (37.4 and 34.2%,respectively) in cross-sectional lesion area,whereas simultaneous silencing of both receptors was not effective. CONCLUSION: Our results suggest that silencing of either SR-A or CD36 alone reduces atherogenesis in mice. However,due to reciprocal upregulation,silencing of both SR-A and CD36 is not effective. View Publication

RATIONALE: Fibrocytes are progenitor cells characterized by the simultaneous expression of mesenchymal,monocyte,and hematopoietic stem cell markers. We previously documented their presence in lungs of patients with idiopathic pulmonary fibrosis. However,the mechanisms involved in their migration,subsequent homing,and local role remain unclear. Matrix metalloproteinases (MMPs) facilitate cell migration and have been implicated in the pathogenesis of pulmonary fibrosis. OBJECTIVES: To evaluate the expression and role of matrix metalloproteinases in human fibrocytes. METHODS: Fibrocytes were purified from CD14(+) monocytes and cultured for 8 days; purity of fibrocyte cultures was 95% or greater as determined by flow cytometry. Conditioned media and total RNA were collected and the expression of MMP-1,MMP-2,MMP-7,MMP-8,and MMP-9 was evaluated by real-time polymerase chain reaction. Protein synthesis was examined using a Multiplex assay,Western blot,fluorescent immunocytochemistry,and confocal microscopy. MMP-2 and MMP-9 enzymatic activities were evaluated by gelatin zymography. Migration was assessed using collagen I-coated Boyden chambers. Stromal cell-derived factor-1α and platelet-derived growth factor-B were used as chemoattractant with or without a specific MMP-8 inhibitor. MEASUREMENTS AND MAIN RESULTS: Fibrocytes showed gene and protein expression of MMP-2,MMP-9,MMP-8,and MMP-7. MMP-2 and MMP-9 enzymatic activities were also demonstrated by gelatin zymography. Likewise,we found colocalization of MMP-8 and MMP-7 with type I collagen in fibrocytes. Fibrocyte migration toward platelet-derived growth factor-B or Stromal cell-derived factor-1α in collagen I-coated Boyden chambers was significantly reduced by a specific MMP-8 inhibitor. CONCLUSIONS: Our findings reveal that fibrocytes express a variety of MMPs and that MMP-8 actively participates in the process of fibrocyte migration. View Publication

Retroviral overexpression of NF-Ya,the regulatory subunit of the transcription factor NF-Y,activates the transcription of multiple genes implicated in hematopoietic stem cell (HSC) self-renewal and differentiation and directs HSCs toward self-renewal. We asked whether TAT-NF-Ya fusion protein could be used to transduce human CD34(+) cells as a safer,more regulated alternative approach to gene therapy. Here we show that externally added recombinant protein was able to enter the cell nucleus and activate HOXB4,a target gene of NF-Ya,using real-time polymerase chain reaction RNA and luciferase-based protein assays. After TAT-NF-Ya transduction,the proliferation of human CD34(+) cells in the presence of myeloid cytokines was increased 4-fold. Moreover,TAT-NF-Ya-treated human primary bone marrow cells showed a 4-fold increase in the percentage of huCD45(+) cells recovered from the bone marrow of sublethally irradiated,transplanted NOD-Scid IL2Rγ(null) mice. These data demonstrate that TAT-peptide therapies are an alternative approach to retroviral stem cell therapies and suggest that NF-Ya peptide delivery should be further evaluated as a tool for HSC/progenitors ex vivo expansion and therapy. View Publication

p400/mDomino is an ATP-dependent chromatin-remodeling protein that catalyzes the deposition of histone variant H2A.Z into nucleosomes to regulate gene expression. We previously showed that p400/mDomino is essential for embryonic development and primitive hematopoiesis. Here we generated a conditional knock-out mouse for the p400/mDomino gene and investigated the role of p400/mDomino in adult bone marrow hematopoiesis and in the cell-cycle progression of embryonic fibroblasts. The Mx1-Cre- mediated deletion of p400/mDomino resulted in an acute loss of nucleated cells in the bone marrow,including committed myeloid and erythroid cells as well as hematopoietic progenitor and stem cells. A hematopoietic colony assay revealed a drastic reduction in colony-forming activity after the deletion of p400/mDomino. Moreover,the loss of p400/mDomino in mouse embryonic fibroblasts (MEFs) resulted in strong growth inhibition. Cell-cycle analysis revealed that the mDomino-deficient MEFs exhibited a pleiotropic cell-cycle defect at the S and G(2)/M phases,and polyploid and multi-nucleated cells with micronuclei emerged. DNA microarray analysis revealed that the p400/mDomino deletion from MEFs caused the impaired expression of many cell-cycle-regulatory genes,including G(2)/M-specific genes targeted by the transcription factors FoxM1 and c-Myc. These results indicate that p400/mDomino plays a key role in cellular proliferation by controlling the expression of cell-cycle-regulatory genes. View Publication

In up to one-third of patients with acute myeloid leukemia,a C-terminal frame-shift mutation results in abnormal and abundant cytoplasmic accumulation of the usually nucleoli-bound protein nucleophosmin (NPM),and this is thought to function in cancer pathogenesis. Here,we demonstrate a gain-of-function role for cytoplasmic NPM in the inhibition of caspase signaling. The NPM mutant specifically inhibits the activities of the cell-death proteases,caspase-6 and -8,through direct interaction with their cleaved,active forms,but not the immature procaspases. The cytoplasmic NPM mutant not only affords protection from death ligand-induced cell death but also suppresses caspase-6/-8-mediated myeloid differentiation. Our data hence provide a potential explanation for the myeloid-specific involvement of cytoplasmic NPM in the leukemogenesis of a large subset of acute myeloid leukemia. View Publication