技术资料

Doxorubicin is an anthracycline chemotherapy agent effective in treating a wide range of malignancies,but it causes a dose-related cardiotoxicity that can lead to heart failure in a subset of patients. At present,it is not possible to predict which patients will be affected by doxorubicin-induced cardiotoxicity (DIC). Here we demonstrate that patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can recapitulate the predilection to DIC of individual patients at the cellular level. hiPSC-CMs derived from individuals with breast cancer who experienced DIC were consistently more sensitive to doxorubicin toxicity than hiPSC-CMs from patients who did not experience DIC,with decreased cell viability,impaired mitochondrial and metabolic function,impaired calcium handling,decreased antioxidant pathway activity,and increased reactive oxygen species production. Taken together,our data indicate that hiPSC-CMs are a suitable platform to identify and characterize the genetic basis and molecular mechanisms of DIC. View Publication

The adoption of Warburg metabolism is critical for the activation of macrophages in response to lipopolysaccharide. Macrophages stimulated with lipopolysaccharide increase their expression of nicotinamide phosphoribosyltransferase (NAMPT),a key enzyme in NAD+ salvage,and loss of NAMPT activity alters their inflammatory potential. However,the events that lead to the cells' becoming dependent on NAD+ salvage remain poorly defined. We found that depletion of NAD+ and increased expression of NAMPT occurred rapidly after inflammatory activation and coincided with DNA damage caused by reactive oxygen species (ROS). ROS produced by complex III of the mitochondrial electron-transport chain were required for macrophage activation. DNA damage was associated with activation of poly(ADP-ribose) polymerase,which led to consumption of NAD+. In this setting,increased NAMPT expression allowed the maintenance of NAD+ pools sufficient for glyceraldehyde-3-phosphate dehydrogenase activity and Warburg metabolism. Our findings provide an integrated explanation for the dependence of inflammatory macrophages on the NAD+ salvage pathway. View Publication

Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes,but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study,we demonstrated that stabilizing actin filaments by inhibiting gene expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs,enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro,as well as heterotopic bone formation in vivo. Similarly,treating hMSC with Phalloidin,which is known to stabilize polymerized actin filaments,increased hMSCs viability and OB differentiation. Conversely,Cytocholasin D,an inhibitor of actin polymerization,reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level,preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1 (LIMK1) decreased cell viability and impaired OB differentiation of hMSCs. Moreover,depolymerizing actin reduced FAK,p38 and JNK activation during OB differentiation of hMSCs,while polymerizing actin enhanced these signaling pathways. Our results demonstrate that the actin dynamic reassembly and Cofilin phosphorylation loop is involved in the control of hMSC proliferation and osteoblasts differentiation. View Publication

R428 (BGB324) is an anti-cancer drug candidate under clinical investigation. It inhibits the receptor tyrosine kinase Axl and induces apoptosis of many types of cancer cells,but the relationship between the two has not been well established. We investigated the molecular mechanisms of the R428-induced apoptosis and found that R428 induced extensive cytoplasmic vacuolization and caspase activation,independent of its inhibitory effects on Axl. Further analyses revealed that R428 blocked lysosomal acidification and recycling,accumulated autophagosomes and lysosomes,and induced cell apoptosis. Inhibition of autophagy by autophagy inhibitors or autophagic gene-knockout alleviated the R428-induced vacuoles formation and cell apoptosis. Our study uncovered a novel function and mechanism of R428 in addition to its ability to inhibit Axl. These data will help to better direct the application of R428 as an anti-cancer reagent. It also adds new knowledge to understand the regulation of autophagy and apoptosis. View Publication

BACKGROUND Cell-surface mucins are expressed in apical epithelial cells of the respiratory tract,and contribute a crucial part of the innate immune system. Despite anti-inflammatory or antiviral functions being revealed for certain cell-surface mucins such as MUC1,the roles of other mucins are still poorly understood,especially in viral infections. METHODS To further identify mucins significant in influenza infection,we screened the expression of mucins in human nasal epithelial cells infected by H3N2 influenza A virus. RESULTS We found that the expression of MUC15 was significantly upregulated upon infection,and specific only to active infection. While MUC15 did not interact with virus particles or reduce viral replication directly,positive correlations were observed between MUC15 and inflammatory factors in response to viral infection. Given that the upregulation of MUC15 was only triggered late into infection when immune factors (including cytokines,chemokines,EGFR and phosphorylated ERK) started to peak and plateau,MUC15 may potentially serve an immunomodulatory function later during influenza viral infection. CONCLUSIONS Our study revealed that MUC15 was one of the few cell-surface mucins induced during influenza infection. While MUC15 did not interact directly with influenza virus,we showed that its increase coincides with the peak of immune activation and thus MUC15 may serve an immunomodulatory role during influenza infection. View Publication

S-glutathionylation of reactive protein cysteines is a post-translational event that plays a critical role in transducing signals from oxidants into biological responses. S-glutathionylation can be reversed by the deglutathionylating enzyme glutaredoxin (GLRX). We have previously demonstrated that ablation of Glrx sensitizes mice to the development of parenchymal lung fibrosis(1). It remains unclear whether GLRX also controls airway fibrosis,a clinical feature relevant to asthma and chronic obstructive pulmonary disease,and whether GLRX controls the biology of airway epithelial cells,which have been implicated in the pathophysiology of these diseases. In the present study we utilized a house dust mite (HDM) model of allergic airway disease in wild type (WT) and Glrx-/- mice on a C57BL/6 background prone to develop airway fibrosis,and tracheal basal stem cells derived from WT mice,global Glrx-/- mice,or bi-transgenic mice allowing conditional ablation of the Glrx gene. Herein we show that absence of Glrx led to enhanced HDM-induced collagen deposition,elevated levels of transforming growth factor beta 1 (TGFB1) in the bronchoalveolar lavage,and resulted in increases in airway hyperresponsiveness. Airway epithelial cells isolated from Glrx-/- mice or following conditional ablation of Glrx showed spontaneous increases in secretion of TGFB1. Glrx-/- basal cells also showed spontaneous TGFB pathway activation,in association with increased expression of mesenchymal genes,including collagen 1a1 and fibronectin. Overall,these findings suggest that GLRX regulates airway fibrosis via a mechanism(s) that involve the plasticity of basal cells,the stem cells of the airways. View Publication

Cardiac differentiation of human pluripotent stem cells may be induced under chemically defined conditions,wherein the regulation of Wnt/$\beta$-catenin pathway is often desirable. Here,we examined the effect of trolox,a vitamin E analog,on the cardiac differentiation of human embryonic stem cells (hESCs). 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) significantly enhanced cardiac differentiation in a time- and dose-dependent manner after the mesodermal differentiation of hESCs. Trolox promoted hESC cardiac differentiation through its inhibitory activity against the Wnt/$\beta$-catenin pathway. This study demonstrates an efficient cardiac differentiation method and reveals a novel Wnt/$\beta$-catenin regulator. View Publication

Receptor tyrosine kinases (RTK) are therapeutic targets for the treatment of malignancy. However,tumor cells develop resistance to targeted therapies through the activation of parallel signaling cascades. Recent evidence has shown that redundant or compensatory survival signals responsible for resistance are initiated by nontargeted glycoprotein RTKs coexpressed by the cell. We hypothesized that disrupting specific functions of the posttranslational machinery of the secretory pathway would be an effective strategy to target both primary and redundant RTK signaling. Using the N-linked glycosylation inhibitor,tunicamycin,we show that expression levels of several RTKS (EGFR,ErbB2,ErbB3,and IGF-IR) are exquisitely sensitive to inhibition of N-linked glycosylation. Disrupting this synthetic process reduces both cellular protein levels and receptor activity in tumor cells through retention of the receptors in the endoplasmic reticulum/Golgi compartments. Using U251 glioma and BXPC3 pancreatic adenocarcinoma cell lines,two cell lines resistant to epidermal growth factor receptor-targeted therapies,we show that inhibiting N-linked glycosylation markedly reduces RTK signaling through Akt and radiosensitizes tumor cells. In comparison,experiments in nontransformed cells showed neither a reduction in RTK-dependent signaling nor an enhancement in radiosensitivity,suggesting the potential for a therapeutic ratio between tumors and normal tissues. This study provides evidence that enzymatic steps regulating N-linked glycosylation are novel targets for developing approaches to sensitize tumor cells to cytotoxic therapies. View Publication

Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs),and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo,with a consequent enhancement of MCC. To this end,camostat,a trypsin-like protease inhibitor,provided a potent (IC(50) approximately 50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells,a potency order of placental bikunin {\textgreater} camostat {\textgreater} 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester {\textgreater} aprotinin {\textgreater} soybean trypsin inhibitor = alpha1-antitrypsin,was largely consistent with that observed for inhibition of prostasin,a molecular candidate for the airway CAP. In vivo,topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED(50) = 3 microg/kg). When administered by aerosol inhalation in conscious sheep,camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary,camostat attenuates ENaC function and enhances MCC,providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically. View Publication

Latrunculin A,a toxin purified from the red sea sponge Latrunculia magnifica,was found previously to induce striking reversible changes in the morphology of mammalian cells in culture and to disrupt the organization of their microfilaments. We now provide evidence that latrunculin A affects the polymerization of pure actin in vitro in a manner consistent with the formation of a 1:1 molar complex between latrunculin A and G-actin. The equilibrium dissociation constant (Kd) for the reaction in vitro is about 0.2 microM whereas the effects of the drug on cultured cells are detectable at concentrations in the medium of 0.1-1 microM. View Publication