技术资料

NK cells from individuals with X-linked lymphoproliferative (XLP) disease exhibit functional defects when stimulated through the NK receptor,2B4 (CD244). These defects are likely a consequence of aberrant intracellular signaling initiated by mutations of the adaptor molecule SLAM-associated protein. In this report,we show that NK cells from individuals with XLP but not healthy individuals fail to phosphorylate and thereby inactivate glycogen synthase kinase-3 (GSK-3) following 2B4 stimulation. Lack of GSK-3 phosphorylation prevented the accumulation of the transcriptional coactivator beta-catenin in the cytoplasm and its subsequent translocation to the nucleus. Potential signaling pathways leading from 2B4 stimulation to GSK-3 phosphorylation were also investigated. Ligation of 2B4 resulted in the phosphorylation of the guanine nucleotide exchange factor,Vav-1,and subsequent activation of the GTP-binding protein Rac-1 (but not Ras) and the serine-threonine kinase Raf-1 in healthy but not XLP-derived NK cells. In addition,the activity of MEK-2 (but not MEK-1) was up-regulated,and Erk1/2 was phosphorylated in normal NK cells but not those from an individual with XLP suggesting that these proteins relay SLAM-associated protein-dependent signals from 2B4. Finally,inactivation of GSK-3 using a specific inhibitor of GSK-3beta increased the cytotoxicity and cytokine secretion of both healthy and XLP NK cells. These data indicate that the signaling of 2B4 in NK cells is mediated by GSK-3 and beta-catenin,possibly through a signal transduction pathway that involves Vav-1,Rac-1,Raf-1,MEK-2,and Erk1/2 and that this pathway is aberrant in individuals with XLP. View Publication

Histone deacetylase inhibitors have attracted considerable attention because of their ability to overcome the differentiation block in leukemic blasts,an effect achieved either alone or in combination with differentiating agents,such as all-trans retinoic acid. We have previously reported favorable effects of the potent histone deacetylase inhibitor valproic acid in combination with all-trans retinoic acid in patients with advanced acute myeloid leukemia leading to blast cell reduction and improvement of hemoglobin. These effects were accompanied by hypergranulocytosis most likely due to an enhancement of nonleukemic myelopoiesis and the suppression of malignant hematopoiesis rather than enforced differentiation of the leukemic cells. These data prompted us to investigate the effect of valproic acid on normal hematopoietic stem cells (HSC). Here we show that valproic acid increases both proliferation and self-renewal of HSC. It accelerates cell cycle progression of HSC accompanied by a down-regulation of p21(cip-1/waf-1). Furthermore,valproic acid inhibits GSK3beta by phosphorylation on Ser9 accompanied by an activation of the Wnt signaling pathway as well as by an up-regulation of HoxB4,a target gene of Wnt signaling. Both are known to directly stimulate the proliferation of HSC and to expand the HSC pool. In summary,we here show that valproic acid,known to induce differentiation or apoptosis in leukemic blasts,stimulates the proliferation of normal HSC,an effect with a potential effect on its future role in the treatment of acute myeloid leukemia. View Publication

The application of genome-wide expression analysis to a large-scale,multicentered program in critically ill patients poses a number of theoretical and technical challenges. We describe here an analytical and organizational approach to a systematic evaluation of the variance associated with genome-wide expression analysis specifically tailored to study human disease. We analyzed sources of variance in genome-wide expression analyses performed with commercial oligonucleotide arrays. In addition,variance in gene expression in human blood leukocytes caused by repeated sampling in the same subject,among different healthy subjects,among different leukocyte subpopulations,and the effect of traumatic injury,were also explored. We report that analytical variance caused by sample processing was acceptably small. Blood leukocyte gene expression in the same individual over a 24-h period was remarkably constant. In contrast,genome-wide expression varied significantly among different subjects and leukocyte subpopulations. Expectedly,traumatic injury induced dramatic changes in apparent gene expression that were greater in magnitude than the analytical noise and interindividual variance. We demonstrate that the development of a nation-wide program for gene expression analysis with careful attention to analytical details can reduce the variance in the clinical setting to a level where patterns of gene expression are informative among different healthy human subjects,and can be studied with confidence in human disease. View Publication

The compound BIRB796 inhibits the stress-activated protein kinases p38alpha and p38beta and is undergoing clinical trials for the treatment of inflammatory diseases. Here we report that BIRB796 also inhibits the activity and the activation of SAPK3/p38gamma. This occurs at higher concentrations of BIRB796 than those that inhibit p38alpha and p38beta and at lower concentrations than those that inhibit the activation of JNK isoforms. We also show that at these concentrations,BIRB796 blocks the stress-induced phosphorylation of the scaffold protein SAP97,further establishing that this is a physiological substrate of SAPK3/p38gamma. Our results demonstrate that BIRB796,in combination with SB203580,a compound that inhibits p38alpha and p38beta,but not the other p38 isoforms,can be used to identify physiological substrates of SAPK3/p38gamma as well as those of p38alpha and p38beta. View Publication

BACKGROUND Transplantation of endothelial progenitor cells (EPCs) improves neovascularization after ischemia,but patients with coronary artery disease (CAD) or diabetes mellitus show a reduced number of EPCs and impaired functional activity. Therefore,we investigated the effects of risk factors,such as glucose and TNF-alpha,on the number of EPCs in vitro to elucidate the underlying mechanisms. METHODS AND RESULTS EPCs of patients or healthy subjects were isolated from peripheral blood. Incubation with glucose or TNF-alpha dose-dependently reduced the number of EPCs (79.9+/-1.3% and 74.3+/-8.1% of control; Ptextless0.05,respectively). This reduction was not caused by apoptosis. TNF-alpha and glucose induced a dose- and time-dependent activation of the p38 MAP kinase,the downstream kinase mitogen- and stress-activated kinase 1,and the transcription factor cAMP-responsive element-binding protein (CREB),in EPCs. Moreover,EPCs from CAD patients had significantly higher basal p38-phosphorylation levels (1.83+/-0.2-fold increase; Ptextless0.05) compared with healthy subjects. The inhibition of the p38-kinase by SB203580 or infection with a dominant negative p38 kinase adenovirus significantly increased basal number of EPCs (136.7+/-6.3% and 142.9+/-18% versus control,respectively). Likewise,ex vivo cultivation of EPCs from patients with CAD with SB203580 significantly increased the number of EPCs and partially reversed the impaired capacity for neovascularization of EPCs in vivo (relative blood flow: 0.40+/-0.03 versus 0.64+/-0.08,Ptextless0.05). The increased numbers of EPCs by SB203580 were associated with an augmentation of EPC proliferation and a reduction of cells expressing the monocytic marker proteins CD14 and CD64,suggesting that p38 regulates proliferation and differentiation events. CONCLUSIONS These results demonstrate that p38 MAP kinase plays a pivotal role in the signal transduction pathways regulating the number of EPCs ex vivo. SB203580 can prevent the negative effects of TNF-alpha and glucose on the number of EPCs and may be useful to improve the number of EPCs for potential cell therapy. View Publication

Stem cell-based tissue engineering is a promising technology in the effort to create functional tissues of choice. To establish an efficient approach for generating hematopoietic cell lineages directly from embryonic stem (ES) cells and to study the effects of three-dimensional (3D) biomaterials on ES cell differentiation,we cultured mouse ES cells on 3D,highly porous,biomimetic scaffolds. Cell differentiation was evaluated by microscopy and flow cytometry analysis with a variety of hematopoiesis- specific markers. Our data indicate that ES cells differentiated on porous 3D scaffold structures developed embryoid bodies (EBs) similar to those in traditional two-dimensional (2D) cultures; however,unlike 2D differentiation,these EBs integrated with the scaffold and appeared embedded in a network of extracellular matrix. Most significantly,the efficiency of hematopoietic precursor cell (HPC) generation on 3D,as indicated by the expression of various HPC-specific surface markers (CD34,Sca-1,Flk-1,and c-Kit) and colony-forming cell (CFC) assays,was reproducibly increased (about 2-fold) over their 2D counterparts. Comparison of static and dynamic 3D cultures demonstrated that spinner flask technology also contributed to the higher hematopoietic differentiation efficiency of ES cells seeded on scaffolds. Continued differentiation of 3D-derived HPCs into the myeloid lineage demonstrated increased efficiency (2-fold) of generating myeloid compared with differentiation from 2D-derived HPCs. View Publication

Ex vivo amplification of human hematopoietic stem cells (HSC) without loss of their self-renewing potential represents an important target for transplantation,gene and cellular therapies. Valproic acid is a safe and widely used neurologic agent that acts as a potent inhibitor of histone deacetylase activities. Here,we show that valproic acid addition to liquid cultures of human CD34+ cells isolated from cord blood,mobilized peripheral blood,and bone marrow strongly enhances the ex vivo expansion potential of different cytokine cocktails as shown by morphologic,cytochemical,immunophenotypical,clonogenic,and gene expression analyses. Notably,valproic acid highly preserves the CD34 positivity after 1 week (range,40-89%) or 3 weeks (range,21-52%) amplification cultures with two (Flt3L + thrombopoietin) or four cytokines (Flt3L + thrombopoietin + stem cell factor + interleukin 3). Moreover,valproic acid treatment increases histone H4 acetylation levels at specific regulatory sites on HOXB4,a transcription factor gene with a key role in the regulation of HSC self-renewal and AC133,a recognized marker gene for stem cell populations. Overall,our results relate the changes induced by valproic acid on chromatin accessibility with the enhancement of the cytokine effect on the maintenance and expansion of a primitive hematopoietic stem cell population. These findings underscore the potentiality of novel epigenetic approaches to modify HSC fate in vitro. View Publication

The synthetic molecule sirtinol was shown previously to activate the auxin signal transduction pathway. Here we present a combination of genetic and chemical approaches to elucidate the action mechanisms of sirtinol in Arabidopsis. Analysis of sirtinol derivatives indicated that the active moiety" of sirtinol is 2-hydroxy-1-naphthaldehyde (HNA) View Publication

Transforming growth factor beta (TGF-beta) signaling pathways regulate a wide variety of cellular processes including cell proliferation,differentiation,extracellular matrix deposition,development,and apoptosis. TGF-beta type-I receptor (TbetaRI) is the major receptor that triggers several signaling events by activating downstream targets such as the Smad proteins. The intracellular kinase domain of TbetaRI is essential for its function. In this study,we have identified a short phospho-Smad peptide,pSmad3(-3),KVLTQMGSPSIRCSS(PO4)VS as a substrate of TbetaRI kinase for in vitro kinase assays. This peptide is uniquely phosphorylated by TbetaRI kinase at the C-terminal serine residue,the phosphorylation site of its parent Smad protein in vivo. Specificity analysis demonstrated that the peptide is phosphorylated by only TbetaRI and not TGF-beta type-II receptor kinase,indicating that the peptide is a physiologically relevant substrate suitable for kinetic analysis and screening of TbetaRI kinase inhibitors. Utilizing pSmad3(-3) as a substrate,we have shown that novel pyrazole compounds are potent inhibitors of TbetaRI kinase with K(i) value as low as 15 nM. Kinetic analysis revealed that these pyrazoles act through the ATP-binding site and are typical ATP competitive inhibitors with tight binding kinetics. More importantly,these compounds were shown to inhibit TGF-beta-induced Smad2 phosphorylation in vivo in NMuMg mammary epithelial cells with potency equivalent to the inhibitory activity in the in vitro kinase assay. Cellular selectivity analysis demonstrated that these pyrazoles are capable of inhibiting activin signaling but not bone morphogenic protein or platelet-derived growth factor signal transduction pathways. Further functional analysis revealed that pyrazoles are capable of blocking the TGF-beta-induced epithelial-mesenchymal transition in NMuMg cells,a process involved in the progression of cancer,fibrosis,and other human diseases. These pyrazoles provide a foundation for future development of potent and selective TbetaRI kinase inhibitors to treat human disease. View Publication

Experimental evidence indicates that shear stress (SS) exerts a morphogenetic function during cardiac development of mouse and zebrafish embryos. However,the molecular basis for this effect is still elusive. Our previous work described that in adult endothelial cells,SS regulates gene expression by inducing epigenetic modification of histones and activation of transcription complexes bearing acetyltransferase activity. In this study,we evaluated whether SS treatment could epigenetically modify histones and influence cell differentiation in mouse embryonic stem (ES) cells. Cells were exposed to a laminar SS of 10 dyne per cm2/s(-1),or kept in static conditions in the presence or absence of the histone deacetylase inhibitor trichostatin A (TSA). These experiments revealed that SS enhanced lysine acetylation of histone H3 at position 14 (K14),as well as serine phosphorylation at position 10 (S10) and lysine methylation at position 79 (K79),and cooperated with TSA,inducing acetylation of histone H4 and phosphoacetylation of S10 and K14 of histone H3. In addition,ES cells exposed to SS strongly activated transcription from the vascular endothelial growth factor (VEGF) receptor 2 promoter. This effect was paralleled by an early induction of cardiovascular markers,including smooth muscle actin,smooth muscle protein 22-alpha,platelet-endothelial cell adhesion molecule-1,VEGF receptor 2,myocyte enhancer factor-2C (MEF2C),and alpha-sarcomeric actin. In this condition,transcription factors MEF2C and Sma/MAD homolog protein 4 could be isolated from SS-treated ES cells complexed with the cAMP response element-binding protein acetyltransferase. These results provide molecular basis for the SS-dependent cardiovascular commitment of mouse ES cells and suggest that laminar flow may be successfully applied for the in vitro production of cardiovascular precursors. View Publication

Human embryonic stem cells (hESCs) self-renew indefinitely and give rise to derivatives of all three primary germ layers,yet little is known about the signaling cascades that govern their pluripotent character. Because it plays a prominent role in the early cell fate decisions of embryonic development,we have examined the role of TGFbeta superfamily signaling in hESCs. We found that,in undifferentiated cells,the TGFbeta/activin/nodal branch is activated (through the signal transducer SMAD2/3) while the BMP/GDF branch (SMAD1/5) is only active in isolated mitotic cells. Upon early differentiation,SMAD2/3 signaling is decreased while SMAD1/5 signaling is activated. We next tested the functional role of TGFbeta/activin/nodal signaling in hESCs and found that it is required for the maintenance of markers of the undifferentiated state. We extend these findings to show that SMAD2/3 activation is required downstream of WNT signaling,which we have previously shown to be sufficient to maintain the undifferentiated state of hESCs. Strikingly,we show that in ex vivo mouse blastocyst cultures,SMAD2/3 signaling is also required to maintain the inner cell mass (from which stem cells are derived). These data reveal a crucial role for TGFbeta signaling in the earliest stages of cell fate determination and demonstrate an interconnection between TGFbeta and WNT signaling in these contexts. View Publication

Ex vivo expansion of hematopoietic stem cells (HSCs) is important for many clinical applications,and knowledge of the surface phenotype of ex vivo-expanded HSCs will be critical to their purification and analysis. Here,we developed a simple culture system for bone marrow (BM) HSCs using low levels of stem cell factor (SCF),thrombopoietin (TPO),insulin-like growth factor 2 (IGF-2),and fibroblast growth factor-1 (FGF-1) in serum-free medium. As measured by competitive repopulation analyses,there was a more than 20-fold increase in numbers of long-term (LT)-HSCs after a 10-day culture of total BM cells. Culture of BM side population" (SP) cells View Publication