Expression and Activity of Phosphodiesterase Isoforms during Epithelial Mesenchymal Transition: The Role of Phosphodiesterase 4

Epithelial–mesenchymal transition (EMT) has emerged as a critical event in the pathogenesis of organ fibrosis and cancer and is typically induced by the multifunctional cytokine transforming growth factor (TGF)-β1. The present study was undertaken to evaluate the potential role of phosphodiesterases (PDEs) in TGF-β1-induced EMT in the human alveolar epithelial type II cell line A549. Stimulation of A549 with TGF-β1 induced EMT by morphological alterations and by expression changes of the epithelial phenotype markers E-cadherin, cytokeratin-18, zona occludens-1, and the mesenchymal phenotype markers, collagen I, fibronectin, and α-smooth muscle actin. Interestingly, TGF-β1 stimulation caused twofold increase in total cAMP-PDE activity, contributed mostly by PDE4. Furthermore, mRNA and protein expression demonstrated up-regulation of PDE4A and PDE4D isoforms in TGF-β1-stimulated cells. Most importantly, treatment of TGF-β1 stimulated epithelial cells with the PDE4-selective inhibitor rolipram or PDE4 small interfering RNA potently inhibited EMT changes in a Smad-independent manner by decreasing reactive oxygen species, p38, and extracellular signal-regulated kinase phosphorylation. In contrast, the ectopic overexpression of PDE4A and/or PDE4D resulted in a significant loss of epithelial marker E-cadherin but did not result in changes of mesenchymal markers. In addition, Rho kinase signaling activated by TGF-β1 during EMT demonstrated to be a positive regulator of PDE4. Collectively, the findings presented herein suggest that TGF-β1 mediated up-regulation of PDE4 promotes EMT in alveolar epithelial cells. Thus, targeting PDE4 isoforms may be a novel approach to attenuate EMT-associated lung diseases such as pulmonary fibrosis and lung cancer.     

机械拉伸下人肺上皮细胞增殖及整联蛋白再分布

应用体外周期性拉伸装置研究机械拉伸对人肺上皮细胞A5 4 9增殖及其膜表面受体———整联蛋白α5、β1再分布的调控作用。结果表明 :在应变为 15 % ,频率为 2 0次 /min、4 0次 /min的拉伸刺激下 ,4 8h后 ,应用流式细胞技术检测细胞的增殖活性指数明显降低 ,A5 4 9细胞的DNA合成受到显著抑制。在 4 0次 /min的拉伸频率下 ,整联蛋白α5、β1的分布发生重组并向基底层转移 ,形成局部粘附连接。研究表明 :整联蛋白α5、β1可能在肺上皮细胞感应机械应力过程中起了重要的作用。     

Cyclic AMP-dependent Cl Secretion Is Regulated by Multiple Phosphodiesterase Subtypes in Human Colonic Epithelial Cells

The role of phosphodiesterase (PDE) isoforms in regulation of transepithelial Cl secretion was investigated using cultured monolayers of T84 cells grown on membrane filters. Identification of the major PDE isoforms present in these cells was determined using ion exchange chromatography in combination with biochemical assays for cGMP and cAMP hydrolysis. The most abundant PDE isoform in these cells was PDE4 accounting for 70-80% of the total cAMP hydrolysis within the cytosolic and membrane fractions from these cells. The PDE3 isoform was also identified in both cytosolic and membrane fractions accounting for 20% of the total cAMP hydrolysis in the cytosolic fraction and 15-30% of the total cAMP hydrolysis observed in the membrane fraction. A large portion of the total cGMP hydrolysis detected in cytosolic and membrane fractions of T84 cells was mediated by PDE5 (50-75%). Treatment of confluent monolayers of T84 cells with various PDE inhibitors produced significant increases in short-circuit current (Isc). The PDE3-selective inhibitors terqinsin, milrinone and cilostamide produced increases in Isc with EC50 values of 0.6 nM, 8.0 nM and 0.5 microM respectively. These values were in close agreement with the IC50 values for cAMP hydrolysis. The effects of the PDE1-(8-MM-IBMX) and PDE4-(RP-73401) selective inhibitors on Isc were significantly less potent than PDE3 inhibitors with EC50 values of >7 microM and >50 microM respectively. However, the effects of 8-MM-IBMX and terqinsin on Cl secretion were additive, suggesting that inhibition of PDE1 also increases Cl secretion. The effect of PDE inhibitors on Isc were significantly blocked by apical treatment with glibenclamide (an inhibitor of the CFTR Cl channel) and by basolateral bumetanide, an inhibitor of Na-K-2Cl cotransport activity. These results indicate that inhibition of PDE activity in T84 cells stimulates transepithelial Cl secretion and that PDE1 and PDE3 are involved in regulating the rate of secretion.     

大气细颗粒污染物 PM2.5 浓度及对肺上皮细胞炎性因子的影响

目的:研究大气细颗粒污染物(PM2.5)浓度及对肺上皮细胞(A549细胞)炎性因子的影响。方法:测定2013年1月至2013年12月北京市某城区PM2.5浓度,比较不同PM2.5浓度对A549细胞炎性因子IL-6、TNF-α表达水平的影响。结果:北京市细颗粒污染物PM2.5日均值春季、夏季、秋季、冬季分别为174.3μg/m3、143.5μg/m3、166.7μg/m3、189.6μg/m3,四季超标率差异无统计学意义(P>0.05);大气细颗粒污染物PM2.5对肺上皮细胞IL-6、TNF-α的影响,春季、夏季、秋季、冬季四季之间差异无统计学意义(P>0.05);随着PM2.5浓度升高IL-6、TNF-α表达水平升高,差异有统计学意义(P<0.05);随着染毒时间延长IL-6、TNF-α表达水平升高,差异有统计学意义(P<0.05)。结论:大气细颗粒污染物浓度升高会使肺上皮细胞炎性因子表达增强。     

周期性应变诱导人肺上皮细胞整联蛋白再分布的研究

为了探讨机械拉伸应变对人肺上皮细胞表面整联蛋白α₃ 、α₅和β₁分布的调控作用,建立了体外周期性拉伸应变装置,并应用胞外基质蛋白——纤连蛋白（Fn）、胶原蛋白Ⅳ（Col Ⅳ）裱衬基底膜,激光共聚焦显微镜分析了在应变为15％,频率为40次／min的拉伸刺激下,正常人肺上皮细胞H727表面α₃、α₅和β₁整联蛋白的再分布.结果表明：在人肺上皮细胞H727中,α₃、α₅和β₁整联蛋白是对拉伸应变敏感的膜受体,周期性拉伸刺激可诱导其激活,使之发生分布的重组,并向基底层转移,形成局部粘附连接,增强了整联蛋白受体与其特异性配体的结合能力.结果提示：一个有效的局部粘附连接的形成是受体聚集和配体占据共同启动的一个协调反应,该反应可能参与了细胞响应机械应力的起始过程,可能进一步通过力-化学信号的耦合或张力整合的形式,最终对细胞的生物学行为产生影响.     

Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells

The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells). Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS) and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI) via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates.     

Immune Modulatory Effects of IL-22 on Allergen-Induced Pulmonary Inflammation

IL-22 is a Th17/Th22 cytokine that is increased in asthma. However, recent animal studies showed controversial findings in the effects of IL-22 in allergic asthma. To determine the role of IL-22 in ovalbumin-induced allergic inflammation we generated inducible lung-specific IL-22 transgenic mice. Transgenic IL-22 expression and signaling activity in the lung were determined. Ovalbumin (OVA)-induced pulmonary inflammation, immune responses, and airway hyperresponsiveness (AHR) were examined and compared between IL-22 transgenic mice and wild type controls. Following doxycycline (Dox) induction, IL-22 protein was readily detected in the large (CC10 promoter) and small (SPC promoter) airway epithelial cells. IL-22 signaling was evidenced by phosphorylated STAT3. After OVA sensitization and challenge, compared to wild type littermates, IL-22 transgenic mice showed decreased eosinophils in the bronchoalveolar lavage (BAL), and in lung tissue, decreased mucus metaplasia in the airways, and reduced AHR. Among the cytokines and chemokines examined, IL-13 levels were reduced in the BAL fluid as well as in lymphocytes from local draining lymph nodes of IL-22 transgenic mice. No effect was seen on the levels of serum total or OVA-specific IgE or IgG. These findings indicate that IL-22 has immune modulatory effects on pulmonary inflammatory responses in allergen-induced asthma.     

Targeting Immunoliposomes to Pulmonary Type II and Tracheobroncheal Epithelial Cells

Abstract

Specific agents are necessary to target therapies to certain cell types, both to maximize benefit and to minimize damage to healthy cells. Immunoliposomes (IL) may help to target the delivery of therapeutic drugs and DNA molecules to specific cells or organs. We describe here the characterization in vitro of an immunoliposome incorporating A2R, a monoclonal antibody that recognizes a receptor for surfactant protein-A (SP-A) present on both type II pneumocytes and the epithelium of the conducting airways. A2R-immunoliposomes (A2R-IL) containing β-galactosidase as a marker were prepared by coupling derivatized A2R to liposomes made of phosphatidylcholine and cholesterol. Control IL were prepared similarly, but incorporated normal IgG instead of A2R (IgG-IL), or no Ig (Null-IL). Type II cells in primary explant cell culture were treated with either A2R-IL, IgG-IL, or null-IL. We found highly significant differences in binding between specific and nonspecific IL: 31.44% of type II cells incubated with A2R-IL showed transfer of the β-gal. Pretreatment of the type II cells with SP-A blocked SP-A receptors and reduced the uptake of A2R-IL almost to background levels. As SP-A receptor is present on the cells of the conducting airways, we tested tracheal explants for their ability to take up A2R-IL, and found that these cells, similarly, could be targeted by A2R-IL. Thus, this immunoliposome could facilitate future study and therapies directed at type II alveolar cells and/or tracheobroncheal epithelium.     

Innate Immune Responses of Pulmonary Epithelial Cells to Burkholderia pseudomallei Infection

Background

Burkholderia pseudomallei, a facultative intracellular pathogen, causes systemic infection in humans with high mortality especially when infection occurs through an infectious aerosol. Previous studies indicated that the epithelial cells in the lung are an active participant in host immunity. In this study, we aimed to investigate the innate immune responses of lung epithelial cells against B. pseudomallei.

Methodology and Principal Findings

Using a murine lung epithelial cell line, primary lung epithelial cells and an inhalational murine infection model, we characterized the types of innate immunity proteins and peptides produced upon B. pseudomallei infection. Among a wide panel of immune components studied, increased levels of major pro-inflammatory cytokines IL-6 and TNFα, chemokine MCP-1, and up-regulation of secretory leukocyte protease inhibitor (SLPI) and chemokine (C-C motif) ligand 20 (CCL20) were observed. Inhibition assays using specific inhibitors suggested that NF-κB and p38 MAPK pathways were responsible for these B. pseudomallei-induced antimicrobial peptides.

Conclusions

Our findings indicate that the respiratory epithelial cells, which form the majority of the cells lining the epithelial tract and the lung, have important roles in the innate immune response against B. pseudomallei infection.     

Electronic Cigarette Liquid Increases Inflammation and Virus Infection in Primary Human Airway Epithelial Cells

Background/Objective

The use of electronic cigarettes (e-cigarettes) is rapidly increasing in the United States, especially among young people since e-cigarettes have been perceived as a safer alternative to conventional tobacco cigarettes. However, the scientific evidence regarding the human health effects of e-cigarettes on the lung is extremely limited. The major goal of our current study is to determine if e-cigarette use alters human young subject airway epithelial functions such as inflammatory response and innate immune defense against respiratory viral (i.e., human rhinovirus, HRV) infection.

Methodology/Main Results

We examined the effects of e-cigarette liquid (e-liquid) on pro-inflammatory cytokine (e.g., IL-6) production, HRV infection and host defense molecules (e.g., short palate, lung, and nasal epithelium clone 1, SPLUNC1) in primary human airway epithelial cells from young healthy non-smokers. Additionally, we examined the role of SPLUNC1 in lung defense against HRV infection using a SPLUNC1 knockout mouse model. We found that nicotine-free e-liquid promoted IL-6 production and HRV infection. Addition of nicotine into e-liquid further amplified the effects of nicotine-free e-liquid. Moreover, SPLUNC1 deficiency in mice significantly increased lung HRV loads. E-liquid inhibited SPLUNC1 expression in primary human airway epithelial cells. These findings strongly suggest the deleterious health effects of e-cigarettes in the airways of young people. Our data will guide future studies to evaluate the impact of e-cigarettes on lung health in human populations, and help inform the public about potential health risks of e-cigarettes.     

EGFR 对博莱霉素诱导小鼠肺纤维化中上皮- 间质转分化的影响

目的:探讨表皮生长因子受体(EGFR)在肺内的表达对博莱霉素(BLM)诱导小鼠肺纤维化中上皮-间质转分化的影响。方法:将40只4～6周龄C57BLB/c雄性小鼠随机分为正常对照组(气管滴入PBS),纤维化组(气管滴入BLM 3 mg/kg),EGFRRNAi组(气管滴入BLM 3 mg/kg+气管滴入siRNA 20μl)和RNAi阴性对照组(气管滴入BLM 3 mg/kg+气管滴入siRNA阴性对照20μl)。实验第10天处死小鼠,收获肺组织,检测羟脯氨酸含量;采用逆转录-聚合酶链反应(RT-PCR)法检测EGFR和α平滑肌肌动蛋白(α-SMA)mRNA的表达;肺组织切片行HE染色观察肺组织病理改变,免疫组化染色检测EGFR和α-SMA表达。结果:纤维化组EGFR和α-SMA两者的mRNA和蛋白表达均较正常对照组显著增加;RNAi组肺病理损伤较纤维化组减轻,气道上皮下胶原沉积及肺羟脯氨酸含量减少(P<0.05),肺组织EGFR和α-SMA两者的mRNA和蛋白表达均较纤维化组显著下降(P<0.05)。结论:在博来霉素诱导的肺纤维化中EGFR RNAi抑制EGFR活化,下调α-SMA的表达,减轻了博莱霉素诱导的肺纤维化病理改变。其抑制肺纤维化病理过程可能与其抑制上皮-间质转分化(EMT)有关。     

The Effects of Fulvic Acid on Copper Bioavailability to Porcine Oviductal Epithelial Cells

This study assessed the effect of dissolved organic matter on the copper (Cu) bioavailability to mammalian cells, porcine oviductal epithelial cells (POEC), in order to imply its effect onto humans. Cu toxicity was investigated in the presence of with and without fulvic acid (FA). Dissociation and exchange rate constants were calculated by using competing ligand Chelex-100, and optical parameters were employed to help explain the complexation of their aromatic and aliphatic structures. Their morphological change was observed using transmission electron microscope (TEM), and Cu species were calculated using MINTEQA2 program. The results showed that the dissociation rate constant of Cu²⁺–FA was equal to 9.08?×?10^?4?s^?1, which was slower than the exchange rate at 1.95?×?10^?3?s^?1. Although Cu–FA was significantly absorbed into the cells higher than Cu²⁺, it showed less damage than tested with Cu²⁺. TEM and optical studies showed many aggregations around nucleus suggesting the amphipathic character of FA helped binging to the nuclear surfaces of both Cu–FA and FA treatments. Even though the MINTEQA2 calculations showed that there was free Cu²⁺ in the mixed solutions around 39.2%, it could not bind with the cell surface. This suggested that the effect of FA was strong and had a lot of influence on the living surface of POEC, modifying the effect of Cu toxicity.     

Effects of Weaning on Intestinal Upper Villus Epithelial Cells of Piglets

The intestinal upper villus epithelial cells represent the differentiated epithelial cells and play key role in digesting and absorbing lumenal nutrients. Weaning stress commonly results in a decrease in villus height and intestinal dysfunction in piglets. However, no study have been conducted to test the effects of weaning on the physiology and functions of upper villus epithelial cells. A total of 40 piglets from 8 litters were weaned at 14 days of age and one piglet from each litter was killed at 0 d (w0d), 1 d (w1d), 3 d (w3d), 5 d (w5d), and 7 d (w7d) after weaning, respectively. The upper villus epithelial cells in mid-jejunum were isolated using the distended intestinal sac method. The expression of proteins in upper villus epithelial cells was analyzed using the isobaric tags for relative and absolute quantification or Western blotting. The expression of proteins involved in energy metabolism, Golgi vesicle transport, protein amino acid glycosylation, secretion by cell, transmembrane transport, ion transport, nucleotide catabolic process, translational initiation, and epithelial cell differentiation and apoptosis, was mainly reduced during the post-weaning period, and these processes may be regulated by mTOR signaling pathway. These results indicated that weaning inhibited various cellular processes in jejunal upper villus epithelial cells, and provided potential new directions for exploring the effects of weaning on the functions of intestine and improving intestinal functions in weaning piglets.     

藤黄新酸抑制肝癌细胞生长的机制研究

在肿瘤细胞中蛋白酶体活性的抑制可以导致细胞凋亡和周期阻滞.藤黄新酸(TH2)是从中药藤黄中提取的一个新的(口山)酮类衍生物.研究结果显示,TH2可以抑制人肝癌Bel-7402细胞的增殖,诱导细胞产生凋亡,且呈浓度和时间依赖性.同时,10μmol/L的TH2与细胞作用24h后,可以导致早期凋亡标志性蛋白PARP发生裂解.在体外采用特异性荧光底物检测TH2对蛋白酶体活性的影响,发现该化合物能够抑制蛋白酶体的糜乳蛋白酶样、胰蛋白酶样活性和谷氨酰后水解活性.抑癌基因p53是细胞内的蛋白酶体降解底物,TH2可使p53蛋白的降解受到阻滞,表达增加.由此可见,TH2具有抑制人肝癌Bel-7402细胞增殖、诱导细胞凋亡的作用,可能的分子机制与其抑制细胞内蛋白酶体活性、导致p53蛋白降解受阻有关.     

兔机械性角膜上皮损伤对结膜杯状细胞及结膜上皮细胞的作用研究

目的探讨机械性角膜上皮损伤对结膜杯状细胞及结膜上皮细胞的作用。方法选取雄性新西兰大白兔12只,建立机械性角膜上皮损伤模型（角膜中央直径8 mm上皮刮除）,建模后使用盐酸林可霉素滴眼,用法为3次/日,1滴/次,观察时间为7 d。在模型建立后第1、4、7天共3个时间点进行结膜印迹细胞学检查、结膜组织透射电镜检查,对结膜上皮细胞及杯状细胞数量及形态进行分析。结果成功建立机械性角膜上皮损伤模型。结膜印迹细胞学检查显示,造模前结膜杯状细胞数量平均值为66.367±2.466（个/每200μm×150μm面积）,Nelson 0级;造模后第1天,结膜杯状细胞数量明显下降,平均值为2.933±0.242（个/每200μm×150μm面积）,Nelson 3级;造模后第4天,结膜杯状细胞数量开始恢复,平均值为17.350±0.991（个/每200μm×150μm面积）,Nelson 2级;造模后第7天,结膜杯状细胞数量已明显恢复,平均值为32.467±2.244（个/每200μm×150μm面积）,Nelson 1级。结膜组织透射电镜检查可见到造模后结膜杯状细胞大量减少,分泌颗粒排空,细胞凋亡,结膜上皮细胞脱落坏死,胞核固缩,胞质中可见溶酶体,上皮下及上皮细胞间炎症细胞浸润;随时间推移,结膜杯状细胞数量及形态逐渐恢复,初期细胞形态欠规则,结膜上皮细胞胞间隙大,连接松散;后期杯状细胞数量明显恢复,形态饱满,分泌功能开始恢复。结膜上皮细胞分化好,细胞连接较为紧密。结论机械性角膜上皮损伤可造成结膜杯状细胞的数量下降及分泌增加,同时可造成结膜上皮细胞凋亡增加,炎症细胞浸润。结膜杯状细胞的数量、功能以及结膜上皮细胞正常结构可在一定时间内自行修复。     

Cytotoxic Effects of Curcumin in Human Retinal Pigment Epithelial Cells

Backround

Curcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE) cells in vitro.

Methodology/Principal Findings

Cellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM) and delayed apoptosis (above 1 µM). The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein.

Conclusion

It is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (∼10 µM) has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as concomitant therapy of cancer or in the treatment of eye diseases, retinal function should be monitored carefully.     

Lung Epithelial Cells Coordinate Innate Lymphocytes and Immunity against Pulmonary Fungal Infection

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气道上皮细胞在哮喘中的作用

随着现代医学的发展,人们对支气管哮喘发病机制的研究有了进一步发展.支气管哮喘(简称哮喘)是一种由多种细胞,多种细胞因子参与形成的慢性气道炎症性疾病.支气管上皮细胞是气道结构细胞,它是抵抗外界损伤因素的第一道防线,当吸人性刺激物质时,首先激化支气管上皮细胞并破坏支气管上皮细胞的正常结构和生理功能,在应激状态下的上皮细胞通过分泌炎性介质与自身细胞或其他气道结构细胞、炎性细胞、抗原递呈细胞等相互作用,积极参与哮喘的气道慢性炎症发生与发展进程.因此气道上皮损伤是影响哮喘发生发展的重要因素,阐明维持气道上皮正常结构和功能的分子机制是目前防治哮喘的重要课题.本文综述气道上皮在哮喘发生发展中的作用及相关机制研究进展.