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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

Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity,yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here,we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation,which revealed distinct gene networks individually regulated by FOXP3 and PRDM1,in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2,to our knowledge not previously implicated in Treg cell function,coregulates another gene network with SATB1 and is important for Treg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling,we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies. 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

Manganese ions block apoptosis of phagocytes induced by various agents. The prevention of apoptosis was attributed to the activation of manganous superoxide dismutase (Mn-SOD) and to the antioxidant function of free Mn2+ cations. However,the effect of Mn2+ on B cell apoptosis is not documented. In this study,we investigated the effects of Mn2+ on the apoptotic process in human B cells. We observed that Mn2+ but not Mg2+ or Ca2+,inhibited cell growth and induced apoptosis of activated tonsilar B cells,Epstein Barr virus (EBV)-negative Burkitt's lymphoma cell lines (BL-CL) and EBV-transformed B cell lines (EBV-BCL). In the same conditions,no apoptosis was observed in U937,a monoblastic cell line. Induction of B cell apoptosis by Mn2+ was time- and dose-dependent. The cell permeable tripeptide inhibitor of ICE family cysteine proteases,zVAD-fmk,suppressed Mn2+-induced apoptosis. Furthermore,Mn2+ triggered the activation of interleukin-1beta converting enzyme (ICE/caspase 1),followed by the activation of CPP32/Yama/Apopain/caspase-3. In addition,poly-(ADP-ribose) polymerase (PARP),a cellular substrate for CPP32 protease was degraded to generate apoptotic fragments in Mn2+-treated B cell lines. The inhibitor,zVAD-fmk suppressed Mn2+-triggered CPP32 activation and PARP cleavage and apoptosis. These results indicate that the activation of caspase family proteases is required for the apoptotic process induced by Mn2+ treatment of B cells. While the caspase-1 inhibitor YVAD was unable to block apoptosis,the caspase-3 specific inhibitor DEVD-cmk,partially inhibited Mn2+-induced CPP32 activation,PARP cleavage and apoptosis of cells. Moreover,Bcl-2 overexpression in BL-CL effectively protected cells from apoptosis and cell death induced by manganese. This is the first report showing the involvement of Mn2+ in the regulation of B lymphocyte death presumably via a caspase-dependent process with a death-protective effect of Bcl-2. View Publication

Pygopus 2 (Pygo2) is a coactivator of Wnt/$\beta$-catenin signaling that can bind bi- or trimethylated lysine 4 of histone-3 (H3K4me2/3) and participate in chromatin reading and writing. It remains unknown whether the Pygo2-H3K4me2/3 association has a functional relevance in breast cancer progression in vivo. To investigate the functional relevance of histone-binding activity of Pygo2 in malignant progression of breast cancer,we generated a knock-in mouse model where binding of Pygo2 to H3K4me2/3 was rendered ineffective. Loss of Pygo2-histone interaction resulted in smaller,differentiated,and less metastatic tumors,due,in part,to decreased canonical Wnt/$\beta$-catenin signaling. RNA- and ATAC-sequencing analyses of tumor-derived cell lines revealed downregulation of TGF$\beta$ signaling and upregulation of differentiation pathways such as PDGFR signaling. Increased differentiation correlated with a luminal cell fate that could be reversed by inhibition of PDGFR activity. Mechanistically,the Pygo2-histone interaction potentiated Wnt/$\beta$-catenin signaling,in part,by repressing the expression of Wnt signaling antagonists. Furthermore,Pygo2 and $\beta$-catenin regulated the expression of miR-29 family members,which,in turn,repressed PDGFR expression to promote dedifferentiation of wild-type Pygo2 mammary epithelial tumor cells. Collectively,these results demonstrate that the histone binding function of Pygo2 is important for driving dedifferentiation and malignancy of breast tumors,and loss of this binding activates various differentiation pathways that attenuate primary tumor growth and metastasis formation. Interfering with the Pygo2-H3K4me2/3 interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer. SIGNIFICANCE: Pygo2 represents a potential therapeutic target in metastatic breast cancer,as its histone-binding capability promotes $\beta$-catenin-mediated Wnt signaling and transcriptional control in breast cancer cell dedifferentiation,EMT,and metastasis. View Publication

Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase) is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC50 values against the purified kinase. Although XL413 is highly selective for DDK,its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491,whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly,their activities in cell lines were highly divergent. Unlike PHA-767491,XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines,this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors,we also tested the cross-reactivity of ∼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA). We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491,including Chk1 and PKR kinase inhibitors,but had divergent chemical scaffolds from known DDK inhibitors. Taken together,these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific,biologically active DDK inhibitors for use as chemotherapeutic agents. View Publication

We examined the potential role of myosin and actin in the release of human immunodeficiency virus type 1 (HIV-1) from infected cells. Wortmannin (100 nM to 5 microM),an effective inhibitor of myosin light chain kinase,blocked the release of HIV-1 from infected T-lymphoblastoid and monocytoid cells in a concentration-dependent manner. Cytochalasin D,a reagent that disrupts the equilibrium between monomeric and polymeric actin,also partially inhibited the release of HIV-1 from the infected cells. At the budding stage,myosin and HIV-1 protein were detected in the same areas on the plasma membrane by using dual-label immunofluorescence microscopy and immunoelectron microscopy. In the presence of 5 microM wortmannin,viral components were observed on the plasma membrane by using immunofluorescence microscopy and electron microscopy,implying that wortmannin did not disturb the transport of viral proteins to the plasma membrane but rather inhibited budding. View Publication

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

Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs alpha and beta) that bind triiodothyronine (T(3),3,5,3'-triiodo-l-thyronine) with high affinity,and its precursor thyroxine (T(4),3,5,3',5'-tetraiodo-l-thyronine) with lower affinity. T(4) contains a bulky 5' iodine group absent from T(3). Because T(3) is buried in the core of the ligand binding domain (LBD),we have predicted that TH analogues with 5' substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T(4) affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics,migration on hydrophobic interaction columns,and non-denaturing gels) that TR-T(4) complexes adopt a conformation that differs from TR-T(3) complexes in solution. Nonetheless,T(4) behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells,when conversion to T(3) does not contribute to agonist activity. We determined x-ray crystal structures of the TRbeta LBD in complex with T(3) and T(4) at 2.5-A and 3.1-A resolution. Comparison of the structures reveals that TRbeta accommodates T(4) through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket,which,together,enlarge a niche that permits helix 12 to pack over the 5' iodine and complete the coactivator binding surface. While T(3) is the major active TH,our results suggest that T(4) could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5' extension should be considered in TR ligand design. View Publication

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2,an emerging virus that utilizes host proteins ACE2 and TMPRSS2 as entry factors. Understanding the factors affecting the pattern and levels of expression of these genes is important for deeper understanding of SARS-CoV-2 tropism and pathogenesis. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway,through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci for both ACE2 and TMPRSS2,that vary in frequency across world populations. We find TMPRSS2 is part of a mucus secretory network,highly upregulated by type 2 (T2) inflammation through the action of interleukin-13,and that the interferon response to respiratory viruses highly upregulates ACE2 expression. IL-13 and virus infection mediated effects on ACE2 expression were also observed at the protein level in the airway epithelium. Finally,we define airway responses to common coronavirus infections in children,finding that these infections generate host responses similar to other viral species,including upregulation of IL6 and ACE2. Our results reveal possible mechanisms influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes. View Publication

In vitro cultures of primary human airway epithelial cells (hAECs) grown at air-liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions,and for drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number of different differentiation media,are now available,making comparison of data between studies difficult. Here,we investigated the impact of two common differentiation media on phenotypic,transcriptomic,and physiological features of CF and non-CF epithelia. Cellular architecture and density were strongly impacted by the choice of medium. RNA-sequencing revealed a shift in airway cell lineage; one medium promoting differentiation into club and goblet cells whilst the other enriched the growth of ionocytes and multiciliated cells. Pathway analysis identified differential expression of genes involved in ion and fluid transport. Physiological assays (intracellular/extracellular pH,Ussing chamber) specifically showed that ATP12A and CFTR function were altered,impacting pH and transepithelial ion transport in CF hAECs. Importantly,the two media differentially affected functional responses to CFTR modulators. We argue that the effect of growth conditions should be appropriately determined depending on the scientific question and that our study can act as a guide for choosing the optimal growth medium for specific applications. View Publication

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