技术资料

Recent studies suggest that forced activation of AMP-activated protein kinase (AMPK) could inhibit melanoma cell proliferation. In this report,we evaluated the anti-melanoma cell activity by a novel small-molecular AMPK activator,GSK621. Treatment of GSK621 decreased survival and proliferation of human melanoma cells (A375,WM-115 and SK-Mel-2 lines),which was accompanied by activation of caspase-3/-9 and apoptosis. Reversely,caspase inhibitors attenuated GSK621-induced cytotoxicity against melanoma cells. Significantly,GSK621 was more potent than other AMPK activators (A769662,Compound 13 and AICAR) in inhibiting melanoma cells. Intriguingly,same GSK621 treatment was non-cytotoxic or pro-apoptotic against human melanocytes. Molecularly,we showed that activation of AMPK mediated GSK621's activity against melanoma cells. AMPK$\alpha$1 shRNA knockdown or dominant negative mutation (T172A) dramatically attenuated GSK621-induced melanoma cell lethality. Further studies revealed that MEK-ERK activation might be the primary resistance factor of GSK621. MEK-ERK inhibition,either genetically or pharmacologically,significantly sensitized melanoma cells to GSK-621. Remarkably,intraperitoneal (i.p.) injection of GSK621 inhibited A375 tumor growth in SCID mice. Co-administration of MEK-ERK inhibitor MEK162 further sensitized GSK621-induced anti-A375 tumor activity in vivo. Together,the results imply that targeted activation of AMPK by GSK621 inhibits melanoma cell survival and proliferation. MEK-ERK inhibition may further sensitize GSK621's anti-melanoma cell activity in vitro and in vivo. 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

Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However,CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR,suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein,we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production,obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air-liquid interface and organoid cultures,the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium. 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

In human cytomegalovirus (HCMV)-seropositive patients,CD34+ hematopoietic progenitor cells (HPCs) provide an important source of latent virus that reactivates following cellular differentiation into tissue macrophages. Multiple groups have used primary CD34+ HPCs to investigate mechanisms of viral latency. However,analyses of mechanisms of HCMV latency have been hampered by the genetic variability of CD34+ HPCs from different donors,availability of cells,and low frequency of reactivation. In addition,multiple progenitor cell types express surface CD34,and the frequencies of these populations differ depending on the tissue source of the cells and culture conditions in vitro In this study,we generated CD34+ progenitor cells from two different embryonic stem cell (ESC) lines,WA01 and WA09,to circumvent limitations associated with primary CD34+ HPCs. HCMV infection of CD34+ HPCs derived from either WA01 or WA09 ESCs supported HCMV latency and induced myelosuppression similar to infection of primary CD34+ HPCs. Analysis of HCMV-infected primary or ESC-derived CD34+ HPC subpopulations indicated that HCMV was able to establish latency and reactivate in CD38+ CD90+ and CD38+/low CD90- HPCs but persistently infected CD38- CD90+ cells to produce infectious virus. These results indicate that ESC-derived CD34+ HPCs can be used as a model for HCMV latency and that the virus either latently or persistently infects specific subpopulations of CD34+ cells.IMPORTANCE Human cytomegalovirus infection is associated with severe disease in transplant patients and understanding how latency and reactivation occur in stem cell populations is essential to understand disease. CD34+ hematopoietic progenitor cells (HPCs) are a critical viral reservoir; however,these cells are a heterogeneous pool with donor-to-donor variation in functional,genetic,and phenotypic characteristics. We generated a novel system using embryonic stem cell lines to model HCMV latency and reactivation in HPCs with a consistent cellular background. Our study defined three key stem cell subsets with differentially regulated latent and replicative states,which provide cellular candidates for isolation and treatment of transplant-mediated disease. This work provides a direction toward developing strategies to control the switch between latency and reactivation. View Publication

Cystic fibrosis (CF),a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene,is characterized by chronic bacterial infections and inflammation in the lung. Having previously shown that deletion of CFTR is associated with lower expression of the endogenous anti-inflammatory protein Annexin A1 (AnxA1),we investigated further this possible functional connection using a validated CFTR inhibitor. Treatment of mice with the CFTR inhibitor-172 (CFTR(172)) augmented the acute peritonitis promoted by zymosan,an effect associated with lower AnxA1 levels in peritoneal cells. Similar results were obtained with another,chemically distinct,CFTR inhibitor. The pro-inflammatory effect of CFTR(172) was lost in AnxA1(-/-),as well as CFTR(-/-) mice. Importantly,administration of hrAnxA1 and its peptido-mimetic to CFTR(-/-) animals or to animals treated with CFTR(172) corrected the exaggerated leukocyte migration seen in these animals. In vitro assays with human Polymorphonuclear leukocyte (PMN) demonstrated that CFTR(172) reduced cell-associated AnxA1 by promoting release of the protein in microparticles. We propose that the reduced impact of the counterregulatory properties of AnxA1 in CF cells contributes to the inflammatory phenotype characteristic of this disease. Thus,these findings provide an important insight into the mechanism underlying the inflammatory disease associated with CFTR inhibition while,at the same time,providing a novel pharmacological target for controlling the inflammatory phenotype of CF. View Publication

Inhibition of the metabolic regulator AMP-activated protein kinase (AMPK) is increasingly being investigated for its therapeutic potential in diseases where AMPK hyperactivity results in poor prognoses,as in established cancers and neurodegeneration. However,AMPK-inhibitory tool compounds are largely limited to compound C,which has a poor selectivity profile. Here we identify the pyrimidine derivative SBI-0206965 as a direct AMPK inhibitor. SBI-0206965 inhibits AMPK with 40-fold greater potency and markedly lower kinase promiscuity than compound C and inhibits cellular AMPK signaling. Biochemical characterization reveals that SBI-0206965 is a mixed-type inhibitor. A co-crystal structure of the AMPK kinase domain/SBI-0206965 complex shows that the drug occupies a pocket that partially overlaps the ATP active site in a type IIb inhibitor manner. SBI-0206965 has utility as a tool compound for investigating physiological roles for AMPK and provides fresh impetus to small-molecule AMPK inhibitor therapeutic development. View Publication