技术资料

UNLABELLED The effects of oleuropein on the processes of osteoblastogenesis and adipogenesis in mesenchymal stem cells (MSCs) from human bone marrow have been studied. We report that oleuropein,a polyphenol abundant in olive tree products,reduces the expression of peroxisome proliferator-activated receptor gamma (PPAR$\gamma$),inhibits adipocyte differentiation,and enhances differentiation into osteoblast. INTRODUCTION Age-related bone loss is associated with osteoblast insufficiency during continuous bone remodeling. It has been suggested that the formation of osteoblasts in bone marrow is closely associated with adipogenesis,and age-related changes in this relationship could be responsible for the progressive adiposity of bone marrow which occurs with osteoporosis. In addition,the consumption of oleuropein,a major polyphenol in olive leaves and olive oil,has been associated with a reduction in bone loss. METHODS We have analyzed the effects of oleuropein-at concentrations between 10(-6) and 10(-4) M-on the processes of osteoblastogenesis and adipogenesis in MSCs from human bone marrow. RESULTS The results show an increase in osteoblast differentiation and a decrease in adipocyte differentiation when there is oleuropein in the culture media. The gene expression of osteoblastogenesis markers,RUNXII,osterix,collagen type I,osteocalcin,or alkaline phosphatase (ALP),was higher in osteoblast-induced oleuropein-treated cells. Also,the ALP activity and extracellular matrix mineralization were higher when oleuropein was present in the media. Oleuropein in MSCs induced adipocytes to produce a decrease in the expression of the genes involved in adipogenesis,the PPAR$\gamma$,lipoprotein lipase,or fatty acid-binding protein 4,and minor fat accumulation. CONCLUSION Our data suggest that oleuropein,highly abundant in olive tree products included in the traditional Mediterranean diet,could prevent age-related bone loss and osteoporosis. View Publication

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown cause that is characterized by progressive fibrotic lung remodeling. An abnormal emergence of airway epithelial-like cells within the alveolar compartments of the lung,herein termed bronchiolization,is often observed in IPF. However,the origin of this dysfunctional distal lung epithelium remains unknown due to a lack of suitable human model systems. In this study,we established a human induced pluripotent stem cell (iPSC)-derived air-liquid interface (ALI) model of alveolar epithelial type II (ATII)-like cell differentiation that allows us to investigate alveolar epithelial progenitor cell differentiation in vitro. We treated this system with an IPF-relevant cocktail (IPF-RC) to mimic the pro-fibrotic cytokine milieu present in IPF lungs. Stimulation with IPF-RC during differentiation increases secretion of IPF biomarkers and RNA sequencing (RNA-seq) of these cultures reveals significant overlap with human IPF patient data. IPF-RC treatment further impairs ATII differentiation by driving a shift toward an airway epithelial-like expression signature,providing evidence that a pro-fibrotic cytokine environment can influence the proximo-distal differentiation pattern of human lung epithelial cells. In conclusion,we show for the first time,the establishment of a human model system that recapitulates aspects of IPF-associated bronchiolization of the lung epithelium in vitro. View Publication

BACKGROUND Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway starting from nicotinamide. Cancer cells have an increased demand for NAD due to their high proliferation and DNA repair rate. Consequently,NAMPT is considered as a putative target for anti-cancer therapies. There is evidence that AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) become dysregulated during the development of hepatocellular carcinoma (HCC). Here,we investigated the effects of NAMPT inhibition by its specific inhibitor FK866 on the viability of hepatocarcinoma cells and analyzed the effects of FK866 on the nutrient sensor AMPK and mTOR complex1 (mTORC1) signaling. RESULTS FK866 markedly decreased NAMPT activity and NAD content in hepatocarcinoma cells (Huh7 cells,Hep3B cells) and led to delayed ATP reduction which was associated with increased cell death. These effects could be abrogated by administration of nicotinamide mononucleotide (NMN),the enzyme product of NAMPT. Our results demonstrated a dysregulation of the AMPK/mTOR pathway in hepatocarcinoma cells compared to non-cancerous hepatocytes with a higher expression of mTOR and a lower AMPK$\alpha$ activation in hepatocarcinoma cells. We found that NAMPT inhibition by FK866 significantly activated AMPK$\alpha$ and inhibited the activation of mTOR and its downstream targets p70S6 kinase and 4E-BP1 in hepatocarcinoma cells. Non-cancerous hepatocytes were less sensitive to FK866 and did not show changes in AMPK/mTOR signaling after FK866 treatment. CONCLUSION Taken together,these findings reveal an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of hepatocarcinoma cells and suggest NAMPT inhibition as a potential treatment option for HCC. View Publication

AMPK is a master regulator of cellular metabolism that exerts either oncogenic or tumor suppressor activity depending on context. Here,we report that the specific AMPK agonist GSK621 selectively kills acute myeloid leukemia (AML) cells but spares normal hematopoietic progenitors. This differential sensitivity results from a unique synthetic lethal interaction involving concurrent activation of AMPK and mTORC1. Strikingly,the lethality of GSK621 in primary AML cells and AML cell lines is abrogated by chemical or genetic ablation of mTORC1 signaling. The same synthetic lethality between AMPK and mTORC1 activation is established in CD34-positive hematopoietic progenitors by constitutive activation of AKT or enhanced in AML cells by deletion of TSC2. Finally,cytotoxicity in AML cells from GSK621 involves the eIF2$\alpha$/ATF4 signaling pathway that specifically results from mTORC1 activation. AMPK activation may represent a therapeutic opportunity in mTORC1-overactivated cancers. View Publication

The caspase family represents a new class of intracellular cysteine proteases with known or suspected roles in cytokine maturation and apoptosis. These enzymes display a preference for Asp in the P1 position of substrates. To clarify differences in the biological roles of the interleukin-1beta converting enzyme (ICE) family proteases,we have examined in detail the specificities beyond the P1 position of caspase-1,-2,-3,-4,-6,and -7 toward minimal length peptide substrates in vitro. We find differences and similarities between the enzymes that suggest a functional subgrouping of the family different from that based on overall sequence alignment. The primary specificities of ICE homologs explain many observed enzyme preferences for macromolecular substrates and can be used to support predictions of their natural function(s). The results also suggest the design of optimal peptidic substrates and inhibitors. View Publication

Recent studies suggest the effect of radiation is observed not only in irradiated cells but also in adjacent non-irradiated cells (bystander effect),although the mechanism has not yet been fully revealed. This bystander effect may be caused by intercellular communication via a gap junction or by messengers released from irradiated cells,such as reactive oxygen species,nitric oxide,or cytokines. However,an unknown mechanism is also possible in the bystander effect. On the other hand,it is known that extracellular ATP,ADP,uridine 5'-triphosphate (UTP),and uridine 5'-diphosphate (UDP),which are released from cells,act as intercellular signaling molecules by activating purinergic P2X and P2Y receptors (purinergic signaling). Recently,I have suggested these extracellular nucleotides may be novel mediators of a radiation-induced bystander effect,because our recent studies indicated that purinergic signaling is involved in important cellular responses to radiation. Our data indicate that ionizing irradiation causes activation of the transient receptor potential melastatin type 2 (TRPM2) channel,and then ATP is released from cells through the anion channel or connexin43 hemichannel mediated by the activation of a P2X7 receptor. The released nucleotides activate P2Y6 and P2Y12 receptors,which are involved in the DNA damage response after irradiation. Activation of the P2Y6 receptor is also involved in radiation-induced activation of the epithelial growth factor receptor-extracellular signal regulated protein kinase (EGFR-ERK)1/2 pathway and subsequent nuclear translocation of EGFR,which plays a role in DNA repair. Further,the induction of an antioxidant after irradiation is also mediated by the activation of the P2Y receptor. In conclusion,purinergic signaling could play an important role in the protective cellular response to ionizing irradiation. View Publication

Lymphatic endothelial cells (LECs) chemoattract na{\{i}}ve T cells and promote their survival in the lymph nodes and can cross-present antigens to na{\"{i}}ve CD8+ T cells to drive their proliferation despite lacking key costimulatory molecules. However the functional consequence of LEC priming of CD8+ T cells is unknown. Here we show that while many proliferating LEC-educated T cells enter early apoptosis the remainders comprise a long-lived memory subset with transcriptional metabolic and phenotypic features of central memory and stem cell-like memory T cells. In vivo these memory cells preferentially home to lymph nodes and display rapid proliferation and effector differentiation following memory recall and can protect mice against a subsequent bacterial infection. These findings introduce a new immunomodulatory role for LECs in directly generating a memory-like subset of quiescent yet antigen-experienced CD8+ T cells that are long-lived and can rapidly differentiate into effector cells upon inflammatory antigenic challenge.""" View Publication

The function of a T cell depends on its subtype and activation state. Here,we show that imaging of the autofluorescence lifetime signals of quiescent and activated T cells can be used to classify the cells. T cells isolated from human peripheral blood and activated in culture using tetrameric antibodies against the surface ligands CD2,CD3 and CD28 showed specific activation-state-dependent patterns of autofluorescence lifetime. Logistic regression models and random forest models classified T cells according to activation state with 97-99{\%} accuracy,and according to activation state (quiescent or activated) and subtype (CD3+CD8+ or CD3+CD4+) with 97{\%} accuracy. Autofluorescence lifetime imaging can be used to non-destructively determine T-cell function. View Publication

Use of cisplatin,a chemotherapeutic agent,is associated with toxicity as a significant number of patients develop a decline in renal function. The mechanisms by which cisplatin produces renal injury are not well understood. It has been suggested that free radical-catalyzed lipid peroxidation can induce apoptosis or necrosis leading to renal injury. This study examined whether low concentrations of cisplatin induce apoptosis in LLC-PK1 cells and whether caspases 1,2,3,8,and 9 are activated during this event. Our results show a dose- and time-dependent induction of apoptosis by micromolar concentrations of cisplatin. Expression of oncogenes c-myc and p53 was induced,and except for caspase 1,all the other caspases tested were activated. Z-VAD,the broad-spectrum inhibitor of caspases,prevented caspase activation and apoptosis,but not c-myc and p53 induction. On the other hand,N-acetylcysteine prevented cisplatin-induced apoptosis as well as c-myc induction but not p53 induction. The antioxidant trolox also prevented cisplatin-induced apoptosis. The results suggest that antioxidants and caspase inhibitors may alleviate cisplatin-associated nephrotoxicity. View Publication

Cyst expansion in polycystic kidney disease (PKD) involves progressive fluid accumulation,which is believed to require chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Herein is reported that small-molecule CFTR inhibitors of the thiazolidinone and glycine hydrazide classes slow cyst expansion in in vitro and in vivo models of PKD. More than 30 CFTR inhibitor analogs were screened in an MDCK cell model,and near-complete suppression of cyst growth was found by tetrazolo-CFTR(inh)-172,a tetrazolo-derived thiazolidinone,and Ph-GlyH-101,a phenyl-derived glycine hydrazide,without an effect on cell proliferation. These compounds also inhibited cyst number and growth by {\textgreater}80{\%} in an embryonic kidney cyst model involving 4-d organ culture of embryonic day 13.5 mouse kidneys in 8-Br-cAMP-containing medium. Subcutaneous delivery of tetrazolo-CFTR(inh)-172 and Ph-GlyH-101 to neonatal,kidney-specific PKD1 knockout mice produced stable,therapeutic inhibitor concentrations of {\textgreater}3 microM in urine and kidney tissue. Treatment of mice for up to 7 d remarkably slowed kidney enlargement and cyst expansion and preserved renal function. These results implicate CFTR in renal cyst growth and suggest that CFTR inhibitors may hold therapeutic potential to reduce cyst growth in PKD. View Publication

Human embryonic stem cell-derived beta cells offer a promising cell-based therapy for diabetes. However,efficient stem cell to beta cell differentiation has proven difficult,possibly due to the lack of cross-talk with the appropriate mesenchymal niche. To define organ-specific niche signals,we isolated pancreatic and gastrointestinal stromal cells,and analyzed their gene expression during development. Our genetic studies reveal the importance of tightly regulated Hedgehog signaling in the pancreatic mesenchyme: inactivation of mesenchymal signaling leads to annular pancreas,whereas stroma-specific activation of signaling via loss of Hedgehog regulators,Sufu and Spop,impairs pancreatic growth and beta cell genesis. Genetic rescue and transcriptome analyses show that these Sufu and Spop knockout defects occur through Gli2-mediated activation of gastrointestinal stromal signals such as Wnt ligands. Importantly,inhibition of Wnt signaling in organoid and human stem cell cultures significantly promotes insulin-producing cell generation,altogether revealing the requirement for organ-specific regulation of stromal niche signals. View Publication

Peroxynitrite toxicity is a major cause of neuronal injury in stroke and neurodegenerative disorders. The mechanisms underlying the neurotoxicity induced by peroxynitrite are still unclear. In this study,we observed that TPEN [N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine],a zinc chelator,protected against neurotoxicity induced by exogenous as well as endogenous (coadministration of NMDA and a nitric oxide donor,diethylenetriamine NONOate) peroxynitrite. Two different approaches to detecting intracellular zinc release demonstrated the liberation of zinc from intracellular stores by peroxynitrite. In addition,we found that peroxynitrite toxicity was blocked by inhibitors of 12-lipoxygenase (12-LOX),p38 mitogen-activated protein kinase (MAPK),and caspase-3 and was associated with mitochondrial membrane depolarization. Inhibition of 12-LOX blocked the activation of p38 MAPK and caspase-3. Zinc itself induced the activation of 12-LOX,generation of reactive oxygen species (ROS),and activation of p38 MAPK and caspase-3. These data suggest a cell death pathway triggered by peroxynitrite in which intracellular zinc release leads to activation of 12-LOX,ROS accumulation,p38 activation,and caspase-3 activation. Therefore,therapies aimed at maintaining intracellular zinc homeostasis or blocking activation of 12-LOX may provide a novel avenue for the treatment of inflammation,stroke,and neurodegenerative diseases in which the formation of peroxynitrite is thought to be one of the important causes of cell death. View Publication