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UNLABELLED: Several lines of evidence suggest that imatinib may affect skeletal tissue. We show that inhibition by imatinib of PDGFR signaling in osteoblasts activates osteoblast differentiation and inhibits osteoblast proliferation and that imatinib inhibits osteoclastogenesis by both stromal cell-dependent and direct effects on osteoclast precursors. INTRODUCTION: Imatinib mesylate,an orally active inhibitor of the c-abl,c-kit,and platelet-derived growth factor receptor (PDGFR) tyrosine kinases,is in clinical use for the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal cell tumors. Interruption of both c-kit and c-abl signaling in mice induces osteopenia,suggesting that imatinib might have adverse effects on the skeleton. However,biochemical markers of bone formation increase in patients with CML starting imatinib therapy,whereas bone resorption is unchanged,despite secondary hyperparathyroidism. We assessed the actions of imatinib on bone cells in vitro to study the cellular and molecular mechanism(s) underlying the skeletal effects we observed in imatinib-treated patients. MATERIALS AND METHODS: Osteoblast differentiation was assessed using a mineralization assay,proliferation by [(3)H]thymidine incorporation,and apoptosis by a TUNEL assay. Osteoclastogenesis was assessed using murine bone marrow cultures and RAW 264.7 cells. RT and multiplex PCR were performed on RNA prepared from human bone marrow samples,osteoblastic cells,and murine bone marrow cultures. Osteoprotegerin was measured by ELISA. RESULTS: The molecular targets of imatinib are expressed in bone cells. In vitro,imatinib increases osteoblast differentiation and prevents PDGF-induced inhibition of this process. Imatinib inhibits proliferation of osteoblast-like cells induced by serum and PDGF. In murine bone marrow cultures,imatinib inhibits osteoclastogenesis stimulated by 1,25-dihydroxyvitamin D(3) and partially inhibits osteoclastogenesis induced by RANKL and macrophage-colony stimulating factor. Imatinib partially inhibited osteoclastogenesis in RANKL-stimulated RAW-264.7 cells. Treatment with imatinib increases the expression of osteoprotegerin in bone marrow from patients with CML and osteoblastic cells. CONCLUSIONS: Taken together with recent in vivo data,these results suggest a role for the molecular targets of imatinib in bone cell function,that inhibition by imatinib of PDGFR signaling in osteoblasts activates bone formation,and that the antiresorptive actions of imatinib are mediated by both stromal cell-dependent and direct effects on osteoclast precursors. View Publication

Pluripotent embryonic stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here,we delineate the role of autocrine production of fibroblast growth factor 4 (Fgf4) and associated activation of the Erk1/2 (Mapk3/1) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead,such manipulations restrict the ability of ES cells to commit to differentiation. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction,and are refractory to BMP-induced non-neural differentiation. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus,FGF enables rather than antagonises the differentiation activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells,which fail to undergo either neural or mesodermal differentiation in adherent culture,and retain expression of pluripotency markers Oct4,Nanog and Rex1. These findings establish that Fgf4 stimulation of Erk1/2 is an autoinductive stimulus for naïve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos. View Publication

Neural tissue formation is induced by growth factors that activate networks of signal transduction cascades that ultimately lead to the expression of early neural genes,including transcription factors of the SoxB family. Here,we report that fibroblast growth factor (FGF)-induced Erk1/2 (Mapk3 and Mapk1,respectively) mitogen-activated protein kinase (MAPK),but not phosphatidylinositol 3'-OH kinase (PI3K,Pik3r1),signalling is required for neural specification in mouse embryonic stem (ES) cells and in the chick embryo. Further,blocking Erk1/2 inhibits the onset of key SoxB genes in both mouse ES cells (Sox1) and chick embryos (Sox2 and Sox3) and,in both contexts,Erk1/2 signalling is required during only a narrow time window,as neural specification takes place. In the absence of Erk1/2 signalling,differentiation of ES cells stalls following Fgf5 upregulation. Using differentiating ES cells as a model for neural specification,we demonstrate that sustained Erk1/2 activation controls the transition from an Fgf5-positive,primitive ectoderm-like cell state to a neural progenitor cell state without attenuating bone morphogenetic protein (BMP) signalling and we also define the minimum period of Erk1/2 activity required to mediate this key developmental step. Together,these findings identify a conserved,specific and stage-dependent requirement for Erk1/2 signalling downstream of FGF-induced neural specification in higher vertebrates and provide insight into the signalling dynamics governing this process. View Publication

It is well known that c-kit is related to pigmentation as well as to the oncology target protein. The objective of this study was to discover a skin-whitening agent that regulates c-kit activity. We have developed a high-throughput screening system using recombinant human c-kit protein. Approximately 10,000 synthetic compounds were screened for their effect on c-kit activity. Phenyl-imidazole sulfonamide derivatives showed inhibitory activity on c-kit phosphorylation in vitro. The effects of one derivative,[4-t-butylphenyl]-N-(4-imidazol-1-yl phenyl)sulfonamide (ISCK03),on stem-cell factor (SCF)/c-kit cellular signaling in 501mel human melanoma cells were examined further. Pretreatment of 501mel cells with ISCK03 inhibited SCF-induced c-kit phosphorylation dose dependently. ISCK03 also inhibited p44/42 ERK mitogen-activated protein kinase (MAPK) phosphorylation,which is known to be involved in SCF/c-kit downstream signaling. However ISCK03 did not inhibit hepatocyte growth factor (HGF)-induced phosphorylation of p44/42 ERK proteins. To determine the in vivo potency of ISCK03,it was orally administered to depilated C57BL/6 mice. Interestingly,oral administration of ISCK03 induced the dose-dependent depigmentation of newly regrown hair,and this was reversed with cessation of ISCK03 treatment. Finally,to investigate whether the inhibitory effect of ISCK03 on SCF/c-kit signaling abolished UV-induced pigmentation,ISCK03 was applied to UV-induced pigmented spots on brownish guinea pig skin. The topical application of ISCK03 promoted the depigmentation of UV-induced hyperpigmented spots. Fontana-Masson staining analysis showed epidermal melanin was diminished in spots treated with ISCK03. These results indicate that phenyl-imidazole sulfonamide derivatives are potent c-kit inhibitors and might be used as skin-whitening agents. View Publication

OBJECTIVE: In murine hematopoietic tissue,direct repopulation experiments have demonstrated that the side population (SP) represents a remarkable enrichment of hematopoietic stem cells. Human SP has been phenotyped as negative for lineage antigens as well as CD34. However,in the 9 years since the original publication,no long-term hematopoietic reconstitution has been reported for the adult human SP/CD34(-) subset. Elevated levels of aldehyde dehydrogenase (ALDH) have been demonstrated in murine and human progenitor cells when compared to other hematopoietic cells. METHODS: Here,we report the phenotype of human cord blood SP cells. We established the technique of simultaneous phenotyping,Hoechst exclusion,and ALDH labeling on murine tissues. We then performed the simultaneous analysis of phenotype,SP,and ALDH activity on human cord blood and bone marrow cells. Finally,we analyzed the phenotype and functional potential of human cord blood ALDH(+) cells to determine whether Lin(-)/CD34(-) cells are identified via this technique. RESULTS: We demonstrate that human Lin(-)/CD34(-)/ALDH(+) cells are capable of long-term repopulation. Although the SP technique identifies cells that overlap with the ALDH(+) cell population,this is restricted to the CD34(+) cell subset. CONCLUSION: Hoechst exclusion ability does not seem to be the method of choice for the isolation of human hematopoietic stem cells. View Publication

Cited2 (cAMP-responsive elementbinding protein [CBP]/p300-interacting transactivators with glutamic acid [E] and aspartic acid [D]-rich tail 2) is a newly identified transcriptional modulator. Knockout of the Cited2 gene results in embryonic lethality with embryos manifesting heart and neural tube defects. Cited2-/- fetal liver displayed significant reduction in the numbers of Lin(-)c-Kit+Sca-1+ cells,Lin(-)c-Kit+ cells,and progenitor cells of different lineages. Fetal liver cells from Cited2-/- embryos gave rise to markedly reduced number of colonies in the colony-forming unit assay. Primary and secondary transplantation studies showed significantly compromised reconstitution of T-lymphoid,B-lymphoid,and myeloid lineages in mice that received a transplant of Cited2-/- fetal liver cells. Competitive reconstitution experiments further showed that fetal liver hematopoietic stem cell (HSC) function is severely impaired due to Cited2 deficiency. Microarray analysis showed decreased expression of Wnt5a and a panel of myeloid molecular markers such as PRTN3,MPO,Neutrophil elastase,Cathepsin G,and Eosinophil peroxidase in Cited2-/- fetal livers. Decreased expression of Bmi-1,Notch1,LEF-1,Mcl-1,and GATA2 was also observed in Cited2-/- Lin(-)c-Kit+ cells. The present study uncovers for the first time a novel role of Cited2 in the maintenance of hematopoietic homeostasis during embryogenesis and thus provides new insights into the molecular regulation of hematopoietic development. View Publication

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) can modulate immune responses,but whether it directly affects B cell function is unknown. Patients with systemic lupus erythematosus,especially those with antinuclear Abs and increased disease activity,had decreased 1,25(OH)(2)D(3) levels,suggesting that vitamin D might play a role in regulating autoantibody production. To address this,we examined the effects of 1,25(OH)(2)D(3) on B cell responses and found that it inhibited the ongoing proliferation of activated B cells and induced their apoptosis,whereas initial cell division was unimpeded. The generation of plasma cells and postswitch memory B cells was significantly inhibited by 1,25(OH)(2)D(3),although the up-regulation of genetic programs involved in B cell differentiation was only modestly affected. B cells expressed mRNAs for proteins involved in vitamin D activity,including 1 alpha-hydroxylase,24-hydroxylase,and the vitamin D receptor,each of which was regulated by 1,25(OH)(2)D(3) and/or activation. Importantly,1,25(OH)(2)D(3) up-regulated the expression of p27,but not of p18 and p21,which may be important in regulating the proliferation of activated B cells and their subsequent differentiation. These results indicate that 1,25(OH)(2)D(3) may play an important role in the maintenance of B cell homeostasis and that the correction of vitamin D deficiency may be useful in the treatment of B cell-mediated autoimmune disorders. View Publication

Bone marrow mesenchymal stem cells (MSC) have potent immunosuppressive properties and have been advocated for therapeutic use in humans. The nature of their suppressive capacity is poorly understood but is said to be a primitive stem cell function. Demonstration that adult stromal cells such as fibroblasts (Fb) can modulate T cells would have important implications for immunoregulation and cellular therapy. In this report,we show that dermal Fb inhibit allogeneic T cell activation by autologously derived cutaneous APCs and other stimulators. Fb mediate suppression through soluble factors,but this is critically dependent on IFN-gamma from activated T cells. IFN-gamma induces IDO in Fb,and accelerated tryptophan metabolism is at least partly responsible for suppression of T cell proliferation. T cell suppression is reversible,and transient exposure to Fb during activation reprograms T cells,increasing IL-4 and IL-10 secretion upon restimulation. Increased Th2 polarization by stromal cells is associated with amelioration of pathological changes in a human model of graft-vs-host disease. Dermal Fb are highly clonogenic in vitro,suggesting that Fb-mediated immunosuppression is not due to outgrowth of rare MSC,although dermal Fb remain difficult to distinguish from MSC by phenotype or transdifferentiation capacity. These results suggest that immunosuppression is a general property of stromal cells and that dermal Fb may provide an alternative and accessible source of cellular therapy. View Publication

Brain tumors can arise following deregulation of signaling pathways normally activated during brain development and may derive from neural stem cells. Given the requirement for Hedgehog in non-neoplastic stem cells,we investigated whether Hedgehog blockade could target the stem-like population in glioblastoma multiforme (GBM). We found that Gli1,a key Hedgehog pathway target,was highly expressed in 5 of 19 primary GBM and in 4 of 7 GBM cell lines. Shh ligand was expressed in some primary tumors,and in GBM-derived neurospheres,suggesting a potential mechanism for pathway activation. Hedgehog pathway blockade by cyclopamine caused a 40%-60% reduction in growth of adherent glioma lines highly expressing Gli1 but not in those lacking evidence of pathway activity. When GBM-derived neurospheres were treated with cyclopamine and then dissociated and seeded in media lacking the inhibitor,no new neurospheres formed,suggesting that the clonogenic cancer stem cells had been depleted. Consistent with this hypothesis,the stem-like fraction in gliomas marked by both aldehyde dehydrogenase activity and Hoechst dye excretion (side population) was significantly reduced or eliminated by cyclopamine. In contrast,we found that radiation treatment of our GBM neurospheres increased the percentage of these stem-like cells,suggesting that this standard therapy preferentially targets better-differentiated neoplastic cells. Most importantly,viable GBM cells injected intracranially following Hedgehog blockade were no longer able to form tumors in athymic mice,indicating that a cancer stem cell population critical for ongoing growth had been removed. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

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The extended substrate specificity of granzyme B (GrB) was used to identify substrates among the chaperone superfamily. This approach identified Hsp90 and Bag1-L as novel GrB substrates,and an additional GrB cleavage site was identified in the Hsc70/Hsp70-Interacting Protein,Hip. Hsp90,Bag1L,and Hip were validated as GrB substrates in vitro,and mutational analysis confirmed the additional cleavage site in Hip. Because the role of Hip in apoptosis is unknown,its proteolysis by GrB was used as a basis to test whether it has anti-apoptotic activity. Previous work on Hip was limited to in vitro characterization; therefore,it was important to demonstrate Hip cleavage in a physiological context and to show its relevance to natural killer (NK) cell-mediated death. Hip is cleaved at both GrB cleavage sites during NK-mediated cell death in a caspase-independent manner,and its cleavage is due solely to GrB and not other granule components. Furthermore,Hip is not cleaved upon stimulation of the Fas receptor in the Jurkat T-cell line,suggesting that Hip is a substrate unique to GrB. RNA interference-mediated reduction of Hip within the K562 cell line rendered the cells more susceptible to NK cell-mediated lysis,indicating that proteolysis by GrB of Hip contributes to death induction. The small effect of RNA interference-mediated Hip deficiency on cytotoxicity is in agreement with the inherent redundancy of NK cell-mediated cell death. The identification of additional members of the chaperone superfamily as GrB substrates and the validation of Hip as an anti-apoptotic protein contribute to understanding the interplay between stress response and apoptosis. View Publication

TCR/CD28 engagement triggers the initiation of a variety of signal transduction pathways that lead to changes in gene transcription. Although reorganization of the actin cytoskeleton is required for T cell activation,the molecular pathways controlled by the actin cytoskeleton are ill defined. To this end,we analyzed TCR/CD28-stimulated signaling pathways in cytochalasin D-treated T cells to determine the cytoskeletal requirements for T cell activation. Cytochalasin D treatment impaired T cell activation by causing a reduction in TCR/CD28-mediated calcium flux,and blocked activation of two regulatory elements within the IL-2 promoter,NFAT/AP-1 and CD28RE/AP. Treatment had no effect on signaling leading to the activation of either AP-1 or NF-kappaB. Significantly,we found that NFAT1 is required for optimal c-rel up-regulation in response to TCR/CD28 stimulation. In fact,NFAT1 could be detected bound at the c-rel promoter in response to TCR/CD28 stimulation,and targeting of NFAT1 using RNA interference in human CD4(+) T cells abrogated c-rel transcription. Overall,these findings establish that disrupting actin cytoskeletal dynamics impairs TCR/CD28-mediated calcium flux required for NFAT1-mediated c-rel transcription and,thus,activation of the CD28RE/AP. View Publication