技术资料

We report transgenic mouse models in which three or four reprogramming factors are expressed from a single genomic locus using a drug-inducible transgene. Multiple somatic cell types can be directly reprogrammed to generate induced pluripotent stem cells (iPSCs) by culture in doxycycline. Because reprogramming factors are carried on a single polycistronic construct,the mice can be easily maintained,and the transgene can be easily transferred into other genetic backgrounds. View Publication

To define novel human NK cell markers,we generated two mAbs specific for G-protein-coupled receptor 56 (GPR56),a surface glycoprotein that appears to be involved in cell-to-cell and cell-to-matrix interactions. GPR56 has been described in selected normal tissues,and in certain tumors,while,as yet,its expression on leukocytes is unknown. In this study,we show that anti-GPR56 mAbs,among leukocytes,prevalently recognize NK cells. In particular,these mAbs brightly stain CD56(dull) CD16(+) NK cells while react poorly with CD56(bright) CD16(+/-) NK cells. Consistently,we found that GPR56 was expressed on NK cells populating inflamed peripheral tissues while it was absent in lymph node-derived NK cells. We also show that activating stimuli,such as cytokines or exposure to monocyte-derived dendritic cell,down-regulate NK cell expression of GPR56 both at the protein and at the transcriptional level. Interestingly,IL-18,known to induce de novo expression of CCR7 on CD56(dull) CD16(+) NK cells,displayed the highest capability of modulating GPR56. Thus,together with the identification of GPR56 as a novel marker capable of discriminating different NK cells subsets,our data suggest that GPR56 may take part to the mechanisms regulating NK cell migration through the blood stream,peripheral tissues and lymph nodes. View Publication

The discovery of an activating tyrosine kinase mutation JAK2V617F in myeloproliferative neoplasms (MPNs),polycythemia vera (PV),essential thrombocythemia (ET) and primary myelofibrosis (PMF) has resulted in the development of JAK2 inhibitors,of which several are being evaluated in phase I/II clinical studies. It is important to recognize that because the V617F mutation is localized in a region outside the adenosine triphosphate (ATP)-binding pocket of JAK2 enzyme,ATP-competitive inhibitors of JAK2 kinase (like the current JAK2 inhibitors in the clinic) are not likely to discriminate between wild-type and mutant JAK2 enzymes. Therefore,JAK2 inhibitors,by virtue of their near equipotent activity against wild-type JAK2 that is important for normal hematopoiesis,may have adverse myelosuppression as an expected side effect,if administered at doses that aim to completely inhibit the mutant JAK2 enzyme. While they may prove to be effective in controlling hyperproliferation of hematopoietic cells in PV and ET,they may not be able to eliminate mutant clones. On the other hand,JAK inhibitors may have great therapeutic benefit by controlling the disease for patients with MPNs who suffer from debilitating signs (eg,splenomegaly) or constitutional symptoms (which presumably result from high levels of circulating cytokines that signal through JAK enzymes). Indeed,the primary clinical benefits observed so far in MF patients have been significant reduction is splenomegaly,elimination of debilitating disease-related symptoms,and weight gain. Most importantly,patients with and without the JAK2V617F mutation appear to benefit to the same extent. In this review we summarize current clinical experience with JAK2 inhibitors in MPNs. View Publication

Neonatal hyperoxia impairs vascular and alveolar growth in mice and decreases endothelial progenitor cells. To determine the role of bone marrow-derived cells in restoration of neonatal lung structure after injury,we studied a novel bone marrow myeloid progenitor cell population from Tie2-green fluorescent protein (GFP) transgenic mice (bone marrow-derived angiogenic cells; BMDAC). We hypothesized that treatment with BMDAC would restore normal lung structure in infant mice during recovery from neonatal hyperoxia. Neonatal mice (1-day-old) were exposed to 80% oxygen for 10 days. BMDACs (1 x 10(5)),embryonic endothelial progenitor cells,mouse embryonic fibroblasts (control),or saline were then injected into the pulmonary circulation. At 21 days of age,saline-treated mice had enlarged alveoli,reduced septation,and a reduction in vascular density. In contrast,mice treated with BMDAC had complete restoration of lung structure that was indistinguishable from room air controls. BMDAC comprised 12% of distal lung cells localized to pulmonary vessels or alveolar type II (AT2) cells and persist (8.8%) for 8 wk postinjection. Coculture of AT2 cells or lung endothelial cells (luEC) with BMDAC augmented AT2 and luEC cell growth in vitro. We conclude that treatment with BMDAC after neonatal hyperoxia restores lung structure in this model of bronchopulmonary dysplasia. View Publication

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS,we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3),including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also,GSK3 phosphorylated PAX3-FKHR in vitro,suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies. View Publication

Blood of both humans and mice contains 2 main monocyte subsets. Here,we investigated the extent of their similarity using a microarray approach. Approximately 270 genes in humans and 550 genes in mice were differentially expressed between subsets by 2-fold or more. More than 130 of these gene expression differences were conserved between mouse and human monocyte subsets. We confirmed numerous of these differences at the cell surface protein level. Despite overall conservation,some molecules were conversely expressed between the 2 species' subsets,including CD36,CD9,and TREM-1. Other differences included a prominent peroxisome proliferator-activated receptor gamma (PPARgamma) signature in mouse monocytes,which is absent in humans,and strikingly opposed patterns of receptors involved in uptake of apoptotic cells and other phagocytic cargo between human and mouse monocyte subsets. Thus,whereas human and mouse monocyte subsets are far more broadly conserved than currently recognized,important differences between the species deserve consideration when models of human disease are studied in mice. View Publication

Gfi-1B is a transcriptional repressor that is crucial for erythroid differentiation: inactivation of the GFI1B gene in mice leads to embryonic death due to failure to produce differentiated red cells. Accordingly,GFI1B expression is tightly regulated during erythropoiesis,but the mechanisms involved in such regulation remain partially understood. We here identify HMGB2,a high-mobility group HMG protein,as a key regulator of GFI1B transcription. HMGB2 binds to the GFI1B promoter in vivo and up-regulates its trans-activation most likely by enhancing the binding of Oct-1 and,to a lesser extent,of GATA-1 and NF-Y to the GFI1B promoter. HMGB2 expression increases during erythroid differentiation concomitantly to the increase of GfI1B transcription. Importantly,knockdown of HMGB2 in immature hematopoietic progenitor cells leads to decreased Gfi-1B expression and impairs their erythroid differentiation. We propose that HMGB2 potentiates GATA-1-dependent transcription of GFI1B by Oct-1 and thereby controls erythroid differentiation. View Publication

Since the discovery of rapamycin,considerable progress has been made in unraveling the details of the mammalian target of rapamycin (mTOR) signaling network,including the upstream mechanisms that modulate mTOR signaling functions,and the roles of mTOR in the regulation of mRNA translation and other cell growth-related responses. mTOR is found in two different complexes within the cell,mTORC1 and mTORC2,but only mTORC1 is sensitive to inhibition by rapamycin. mTORC1 is a master controller of protein synthesis,integrating signals from growth factors within the context of the energy and nutritional conditions of the cell. Activated mTORC1 regulates protein synthesis by directly phosphorylating 4E-binding protein 1 (4E-BP1) and p70S6K (S6K),translation initiation factors that are important to cap-dependent mRNA translation,which increases the level of many proteins that are needed for cell cycle progression,proliferation,angiogenesis,and survival pathways. In normal physiology,the roles of mTOR in both glucose and lipid catabolism underscore the importance of the mTOR pathway in the production of metabolic energy in quantities sufficient to fuel cell growth and mitotic cell division. Several oncogenes and tumor-suppressor genes that activate mTORC1,often through the phosphatidylinositol 3-kinase (PI3K)/AKT pathway,are frequently dysregulated in cancer. Novel analogs of rapamycin (temsirolimus,everolimus,and deforolimus),which have improved pharmaceutical properties,were designed for oncology indications. Clinical trials of these analogs have already validated the importance of mTOR inhibition as a novel treatment strategy for several malignancies. Inhibition of mTOR now represents an attractive anti-tumor target,either alone or in combination with strategies to target other pathways that may overcome resistance. The far-reaching downstream consequences of mTOR inhibition make defining the critical molecular effector mechanisms that mediate the anti-tumor response and associated biomarkers that predict responsiveness to mTOR inhibitors a challenge and priority for the field. View Publication

p53 can induce apoptosis through mitochondrial membrane permeabilization by interaction of its DNA binding region with the anti-apoptotic proteins BclxL and Bcl2. However,little is known about the action of p53 at the mitochondria in molecular detail. By using NMR spectroscopy and fluorescence polarization we characterized the binding of wild-type and mutant p53 DNA binding domains to BclxL and show that the wild-type p53 DNA binding domain leads to structural changes in the BH3 binding region of BclxL,whereas mutants fail to induce such effects due to reduced affinity. This was probed by induced chemical shift and residual dipolar coupling data. These data imply that p53 partly achieves its pro-apoptotic function at the mitochondria by facilitating interaction between BclxL and BH3-only proteins in an allosteric mode of action. Furthermore,we characterize for the first time the binding behavior of Pifithrin-mu,a specific small molecule inhibitor of the p53-BclxL interaction,and present a structural model of the protein-ligand complex. A rather unusual behavior is revealed whereby Pifithrin-mu binds to both sides of the protein-protein complex. These data should facilitate the rational design of more potent specific BclxL-p53 inhibitors. View Publication

The regulatory pathways necessary for the maintenance of adult hematopoietic stem cells (HSCs) remain poorly defined. By using loss-of-function approaches,we report a selective and cell-autonomous requirement for the p300/CBP-binding transcriptional coactivator Cited2 in adult HSC maintenance. Conditional deletion of Cited2 in the adult mouse results in loss of HSCs causing multilineage bone marrow failure and increased lethality. In contrast,conditional ablation of Cited2 after lineage specification in lymphoid and myeloid lineages has no impact on the maintenance of these lineages. Additional deletion of Ink4a/Arf (encoding p16(Ink4a) and p19(Arf)) or Trp53 (encoding p53,a downstream target of p19(Arf)) in a Cited2-deficient background restores HSC functionality and rescues mice from bone marrow failure. Furthermore,we show that the critical role of Cited2 in primitive hematopoietic cells is conserved in humans. Taken together,our studies provide genetic evidence that Cited2 selectively maintains adult HSC functions,at least in part,via Ink4a/Arf and Trp53. View Publication

The aim of this study was to determine if treatment with reversine,a purine analog,promoted generation of skeletal progenitor cells from lineage-committed annulus fibrosus cells. Reversine modulated cell growth,morphology,and the actin cytoskeleton of annulus fibrosus cells. Microarray profiling coupled with Ingenuity Pathway Analysis revealed that reversine treatment resulted in a significant expression change in many genes including those required for cell-cell interaction,cell movement,cell growth,and development. Further analysis revealed that there was involvement of gene networks concerned with cellular assembly and organization,DNA replication and repair,tissue morphology,and cell-to-cell signaling. The gene expression profile was dependent on reversine concentration. In osteogenic media,cells pretreated with 300 nM reversine exhibited an increased induction in alkaline phosphatase activity and enhanced expression of alkaline phosphatase,bone sialoprotein,osteocalcin,and collagen type I mRNA. Maintained in adipogenic media,the reversine-pretreated annulus cells displayed evidence of adipogenic differentiation: accumulation of cytosolic lipid droplets and increased expression of PPAR-gamma2,LPL,and Fabp mRNA. In chondrogenic media,cells pretreated with reversine exhibited marked increase in the induction of aggrecan,collagen types II,IX,and XI,and versican. It is concluded that reversine treatment induced annulus fibrosus cell plasticity and promoted their differentiation along mesenchymal lineages. This agent could be used to generate skeletal progenitor cells to orchestrate the repair of the intervertebral disc. View Publication

Leucocyte function-associated antigen-1 (LFA-1) is known to be involved in immune reactions leading to allograft rejection. The role of deactivating LFA-1 in this context has not been investigated yet,although it is accepted that regulating LFA-1 activity is essential for T-cell function. Expressing LFA-1 locked in an active state in mice (LFA-1(d/d)) allowed us to investigate the in vivo function of LFA-1 deactivation for allograft rejection in a model of heterotopic cardiac transplantation. We provide in vivo evidence that regulating LFA-1 activity from an active to an inactive state controls antigen-specific priming and proliferation of T cells in response to allogeneic stimuli. Consequently,defective LFA-1 deactivation significantly prolonged cardiac allograft survival. Furthermore,reduced numbers of alloantigen-specific T cells and non-allo-specific innate immune cells within allografts of LFA-1(d/d) recipients indicate that expression of active LFA-1 impairs inflammatory responses involving all major leucocyte subpopulations. Taken together,our in vivo data suggest that LFA-1 deactivation is important for the formation of inflammatory lesions and rejection of cardiac allografts. Thus,the dynamic regulation of LFA-1 activity,rather than the mere presence of LFA-1,appears to contribute to the control of immune reactions inducing allogeneic transplant rejection. View Publication