技术资料

Overexpression of fibroblast growth factor receptor (FGFR) tyrosine kinases has been found in many human breast cancers and has been associated with poor patient prognosis. In order to understand the mechanism by which FGFR mediates breast cancer cell proliferation,we used a low molecular weight compound,PD173074,that selectively inhibits FGFR tyrosine kinase activity and autophosphorylation. This potential anticancer agent caused a G1 growth arrest of MDA-MB-415,MDA-MB-453 and SUM 52 breast cancer cells. Our analyses revealed that FGFR signaling links to the cell cycle machinery via D-type cyclins. PD173074-mediated inhibition of FGFR activity caused downregulation of cyclin D1 and cyclin D2 expression,inhibition of cyclin D/cdk4 activity and,as a consequence,reduction of pRB phosphorylation. Retroviral-mediated ectopic expression of cyclin D1 prevented pRB hypophosphorylation and the cell cycle G1 block in PD173074-treated cells,suggesting a central role for D cyclins in proliferation of FGFR-driven breast cancer cells. The repression of FGFR activity caused downregulation of MAPK in MDA-MB-415 and MDA-MB-453 cells. In SUM 52 cells,both MAPK and PI3K signaling pathways were suppressed. In conclusion,results shown here describe a mechanism by which FGFR promotes proliferation of breast cancer cells. View Publication

RATIONALE: Excessive recruitment of polymorphonuclear leukocytes (PMNs) to the lung promotes acute lung injury (ALI). Chemokine receptors and adhesion molecules initiate leukocyte-endothelial interactions,but mediators of PMN migration through the alveolo-capillary membrane remain to be identified. p21-Activated kinase (PAK) is an effector of small GTPases and has been implicated in cell migration. OBJECTIVES: To test the role of PAK in ALI. METHODS: An inhibitory PAK peptide was used to determine the role of PAK in cytoskeletal actin polymerization,cell adhesion,and oxidative burst. PMN migration was investigated in vitro and in a murine model of lipopolysaccharide-induced lung injury. MEASUREMENTS AND MAIN RESULTS: PMN migration into lung interstitium and alveolar space was suppressed by an inhibitory PAK peptide. Neutrophils that had taken up the inhibitory PAK peptide were unable to enter the alveolar space. CXCL2/3,an important PMN chemoattractant in murine lung injury,induced PAK phosphorylation in PMNs. Blocking PAK function inhibited chemotaxis,chemokine-induced cytoskeletal actin polymerization,and adhesion-induced oxidative burst. CONCLUSIONS: We conclude that neutrophil PAK is a critical mediator of PMN migration and may be an attractive target in ALI. View Publication

Antagonists to alpha4 integrin show promise for several autoimmune and inflammatory diseases but may exhibit mechanism-based toxicities. We tested the capacity of blockade of alpha4 integrin signaling to perturb functions involved in inflammation,while limiting potential adverse effects. We generated and characterized mice bearing a Y991A mutation in alpha4 integrin [alpha4(Y991A) mice],which blocks paxillin binding and inhibits alpha4 integrin signals that support leukocyte migration. In contrast to the embryonic-lethal phenotype of alpha4 integrin-null mice,mice bearing the alpha4(Y991A) mutation were viable and fertile; however,they exhibited defective recruitment of mononuclear leukocytes into thioglycollate-induced peritonitis. Alpha4 integrins are essential for definitive hematopoiesis; however,the alpha4(Y991A) mice had intact lymphohematopoiesis and,with the exception of reduced Peyer's patches,normal architecture and cellularity of secondary lymphoid tissues. We conclude that interference with alpha4 integrin signaling can selectively impair mononuclear leukocyte recruitment to sites of inflammation while sparing vital functions of alpha4 integrins in development and hematopoiesis. View Publication

Increasing amounts of pathogen replication usually lead to a proportionate increase in size and effector differentiation of the CD8(+) T cell response,which is attributed to increased Ag and inflammation. Using a murine CMV that is highly sensitive to the antiviral drug famciclovir to modulate virus replication,we found that increased virus replication drove increased effector CD8(+) T cell differentiation,as expected. Paradoxically,however,increased virus replication dramatically decreased the size of the CD8(+) T cell response to two immunodominant epitopes. The decreased response was due to type I IFN-dependent depletion of conventional dendritic cells and could be reproduced by specific depletion of dendritic cells from day 2 postinfection or by sterile induction of type I IFN. Increased virus replication and type I IFN specifically inhibited the response to two immunodominant epitopes that are known to be dependent on Ag cross-presented by DCs,but they did not inhibit the response to inflationary" epitopes whose responses can be sustained by infected nonhematopoietic cells. Our results show that type I IFN can suppress CD8(+) T cell responses to cross-presented Ag by depleting cross-presenting conventional dendritic cells." View Publication

Characterizing the HIV-1 reservoir in blood and tissues is crucial for the development of curative strategies. Using an HIV Tat mRNA-containing lipid nanoparticle (Tat-LNP) in combination with panobinostat,we show that p24+ cells from blood and lymph nodes exhibit distinct phenotypes. Blood p24+ cells are found in both central/transitional (TCM/TTM) and effector memory subsets,mostly lack CXCR5 expression and are enriched in GZMA+ cells. In contrast,most lymph node p24+ cells display a TCM/TTM phenotype,with approximately 50% expressing CXCR5 and nearly all lacking GZMA expression. Furthermore,germinal center T follicular helper cells do not appear to harbor the translation-competent reservoir in long-term suppressed individuals. Near full-length HIV-1 sequencing in longitudinal samples from matched blood,lymph nodes,and gut indicates that clones of infected cells,including those carrying an inducible provirus,persist and spread across various anatomical compartments. Finally,uniform genetic diversity across sites suggests the absence of ongoing replication in tissues under treatment. Here,Pardons and Lambrechts et al show that HIV-1 reservoirs in blood and lymph nodes differ phenotypically. Furthermore, germinal center T follicular helper cells do not harbor the inducible reservoir in long-term suppressed individuals. Infected clones can spread across tissues and persist without active replication. View Publication

The generation of endothelial progenitor cells (EPCs) from blood monocytes has been propagated as a novel approach in the diagnosis and treatment of cardiovascular diseases. Low-density lipoprotein (LDL) uptake and lectin binding together with endothelial marker expression are commonly used to define these EPCs. Considerable controversy exists regarding their nature,in particular,because myelomonocytic cells share several properties with endothelial cells (ECs). This study was performed to elucidate whether the commonly used endothelial marker determination is sufficient to distinguish supposed EPCs from monocytes. We measured endothelial,hematopoietic,and progenitor cell marker expression of monocytes before and after angiogenic culture by fluorescence microscopy,flow cytometry,and real-time reverse transcription-polymerase chain reaction. The function of primary monocytes and monocyte-derived supposed EPCs was investigated during vascular network formation and EC colony-forming unit (CFU-EC) development. Monocytes cultured for 4 to 6 days under angiogenic conditions lost CD14/CD45 and displayed a commonly accepted EPC phenotype,including LDL uptake and lectin binding,CD31/CD105/CD144 reactivity,and formation of cord-like structures. Strikingly,primary monocytes already expressed most tested endothelial genes and proteins at even higher levels than their supposed EPC progeny. Neither fresh nor cultured monocytes formed vascular networks,but CFU-EC formation was strictly dependent on monocyte presence. LDL uptake,lectin binding,and CD31/CD105/CD144 expression are inherent features of monocytes,making them phenotypically indistinguishable from putative EPCs. Consequently,monocytes and their progeny can phenotypically mimic EPCs in various experimental models. View Publication

Increasing evidence supports the importance of cell extrinsic regulation in stem cell fate control. Hematopoietic stem cells (HSC) are responsive to local signals from their niche and to systemic feedback from progenitors and mature cells. The Notch ligand Delta-1 (DL1),a key component of the stem cell niche,regulates human hematopoietic lineage development in a dose-dependent manner and has been used clinically for primitive progenitor expansion. How DL1 acts to regulate HSC fate and whether these actions are related to its lineage skewing effects are poorly understood. Here we demonstrate that,although DL1 activates signal transducer and activator of transcription 3 signaling similarly to the gp130-activating cytokine interleukin-6 (IL-6),it has opposite effects on myeloid cell production. Mechanistically,these different outcomes are attributable to a DL1-mediated reduction in membrane (m)-bound IL-6 receptor (R) expression,converting progenitor cells from being directly IL-6 responsive to requiring both IL-6 and soluble (s) IL-6R for activation. Concomitant reduction of both mIL-6R (by DL1 supplementation) and sIL-6R (using dynamically fed cultures) reduced myeloid cell production and led to enhanced outputs of human HSCs. This work describes a new mode of cytokine action in which DL1 changes cytokine receptor distributions on hematopoietic cells,altering feedback networks and their impact on stem cell fate. View Publication

The limited proliferative capacity of erythroid precursors is a major obstacle to generate sufficient in vitro-derived red blood cells for clinical purposes. While BMI1,a Polycomb Repressive Complex 1 member,is both necessary and sufficient to drive extensive proliferation of self-renewing erythroblasts,its mechanism of action remains poorly understood. Here we report that BMI1 overexpression leads to 10 billion-fold increase in self-renewal of human erythroblasts,which can terminally mature and agglutinate with typing reagent monoclonal antibodies. BMI1 and RING1B occupancy,along with repressive histone marks,are present at known BMI1 target genes,including the INK-ARF locus,consistent with altered cell cycle kinetics following BMI1 inhibition. Upregulation of BMI1 target genes with low repressive histone modifications,including key regulators of cholesterol homeostasis,along with functional studies,suggest that both cholesterol import and synthesis are essential for BMI1-associated self-renewal. We conclude that BMI1 regulates erythroid self-renewal not only through gene repression but also through gene activation and offer a strategy to expand immature erythroid precursors for eventual clinical uses. Subject terms: Self-renewal,Cell growth,Stem-cell research View Publication

Despite advances in therapy,glioblastoma remains an incurable disease with a dismal prognosis. Recent studies have implicated cancer stem cells within glioblastoma (glioma stem cells,GSCs) as mediators of therapeutic resistance and tumor progression. In this study,we investigated the role of the transforming growth factor-$\beta$ (TGF-$\beta$) superfamily,which has been found to play an integral role in the maintenance of stem cell homeostasis within multiple stem cell systems,as a mediator of stem-like cells in glioblastoma. We find that BMP and TGF-$\beta$ signaling define divergent molecular and functional identities in glioblastoma,and mark relatively quiescent and proliferative GSCs,respectively. Treatment of GSCs with BMP inhibits cell proliferation,but does not abrogate their stem-ness,as measured by self-renewal and tumorigencity. Further,BMP pathway activation confers relative resistance to radiation and temozolomide chemotherapy. Our findings define a quiescent cancer stem cell population in glioblastoma that may be a cellular reservoir for tumor recurrence following cytotoxic therapy. View Publication

In contrast to mammals,adult zebrafish achieve complete heart regeneration via proliferation of cardiomyocytes. Surprisingly,we found that regenerating cardiomyocytes experience DNA replication stress,which represents one reason for declining tissue regeneration during aging in mammals. Pharmacological inhibition of ATM and ATR kinases revealed that DNA damage response signaling is essential for zebrafish heart regeneration. Manipulation of Bone Morphogenetic Protein (BMP)-Smad signaling using transgenics and mutants showed that BMP signaling alleviates cardiomyocyte replication stress. BMP signaling also rescues neonatal mouse cardiomyocytes,human fibroblasts and human hematopoietic stem and progenitor cells (HSPCs) from replication stress. DNA fiber spreading assays indicate that BMP signaling facilitates re-start of replication forks after replication stress-induced stalling. Our results identify the ability to overcome replication stress as key factor for the elevated zebrafish heart regeneration capacity and reveal a conserved role for BMP signaling in promotion of stress-free DNA replication. Subject terms: Cardiac regeneration,DNA damage and repair,Ageing View Publication

Bone Morphogenetic Protein (BMP) signaling pathways are involved in differentiation of stem cells into diverse cell types,and thus BMPs can be used as main guidance molecules for in vitro differentiation of human stem cells. View Publication

Adipogenesis plays a key role in the pathogenesis of obesity. It begins with the commitment of mesenchymal stem cells (MSCs) to the adipocyte lineage,followed by terminal differentiation of preadipocytes to mature adipocytes. A critical,but poorly understood,component of adipogenesis involves proliferation of MSCs and preadipocytes. The present study was undertaken to examine the hypothesis that bone morphogenetic protein-3 (BMP-3) promotes adipogenesis using C3H10T1/2 MSCs and 3T3-L1 preadipocytes as in vitro model systems. We demonstrated that although it did not promote the commitment of MSCs to the adipocyte lineage or the differentiation of preadipocytes to adipocytes,BMP-3-stimulated proliferation by threefold in both cell types. Owing to a lack of information on MSC proliferation,we then delineated the molecular mechanisms underlying BMP-3-stimulated MSC proliferation. We showed that BMP-3 activated the transforming growth factor-beta (TGF-beta)/activin but not ERK1/2,p38 MAPK,or JNK signaling pathways in C3H10T1/2 cells. Furthermore,the TGF-beta/activin receptor kinase inhibitor SB-431542 blocked BMP-3-stimulated proliferation. Importantly,siRNA-mediated knockdown of the key TGF-beta/activin signaling pathway components,ActRIIB,ALK4,or Smad2,abrogated the mitogenic effects of BMP-3 on MSCs. Together,these results demonstrate that BMP-3 stimulates MSC proliferation via the TGF-beta/activin signaling pathway,thus revealing a novel role for this divergent and poorly understood member of the TGF-beta superfamily in regulating MSC proliferation. View Publication