The IL-17F signaling pathway is involved in the induction of IFN-gamma-inducible protein 10 in bronchial epithelial cells

BACKGROUND: IL-17F is involved in airway inflammation, but its biologic activity and signaling pathway remain incompletely defined. Interferon-gamma-inducible protein 10 (IP-10) is widely expressed and plays a role in airway inflammatory diseases. OBJECTIVE: We sought to investigate the functional linkage between IL-17F and IP-10 expression in bronchial epithelial cells. METHODS: Bronchial epithelial cells were cultured in the presence or absence of IL-17F, and/or a T(H)1 cytokine, T(H)2 cytokines, proinflammatory cytokines, various kinase inhibitors, or a Raf1 dominant-negative mutant to analyze the expression of IP-10. Moreover, the involvement of p90 ribosomal S6 kinase (p90RSK) and cyclic AMP response element-binding protein (CREB) in IL-17F-induced IP-10 expression were investigated. RESULTS: IL-17F induces the gene and protein expression of IP-10. The addition of IFN-gamma, IL-1beta, and TNF-alpha augmented IL-17F-induced IP-10 expression. The mitogen-activated protein kinase kinase (MEK) inhibitors PD98059, U0126, and Raf1 kinase inhibitor I significantly inhibited its production. In contrast, a p38 inhibitor, a JNK inhibitor, protein kinase C inhibitors, and a phosphatidylinositol 3-kinase inhibitor, showed no inhibitory effect. Furthermore, overexpression of a Raf1 dominant-negative mutant inhibited its expression. Of interest, IL-17F phosphorylated p90RSK and CREB, and transfection of the cells with a short interfering RNA for p90RSK or CREB inhibited its expression, suggesting p90RSK and CREB as novel signaling molecules of IL-17F. CONCLUSION: IL-17F is a potent inducer of IP-10 in bronchial epithelial cells through the activation of the Raf1-MEK1/2-extracellular signal-regulated kinase 1/2-p90RSK-CREB pathway, supporting its regulatory role in airway inflammation. CLINICAL IMPLICATIONS: The IL-17F-IP-10 axis might be a novel and critical therapeutic target for airway inflammatory diseases.     

肺炎克雷伯杆菌荚膜多糖经EGFR途径诱导支气管上皮细胞分泌细胞因子

目的:探讨表皮生长因子受体(EGFR)在肺炎克雷伯杆菌(KP)荚膜多糖(CPS)诱导人正常支气管上皮BEAS-2B细胞分泌炎性细胞因子中的作用。方法:体外培养KP并提取CPS,用不同浓度的CPS刺激BEAS-2B细胞,通过ELISA检测细胞上清中肿瘤坏死因子α(TNF-α)和白细胞介素8(IL-8)的水平,并于刺激后不同时点通过Western blot检测EGFR的磷酸化水平;EGFR抑制剂AG1478预处理BEAS-2B细胞后,Western blot检测ERK磷酸化水平,间接免疫荧光染色检测p65的核转位,并观察细胞上清中TNF-α和IL-8的变化情况;最后经ERK抑制剂PD98059和NF-κB抑制剂PDTC分别预处理后用CPS刺激细胞,ELISA检测细胞上清中TNF-α和IL-8的水平。结果:10 mg/L CPS刺激BEAS-2B细胞12 h能够显著诱导其分泌TNF-α和IL-8;Western blot和间接免疫荧光染色检测结果显示,CPS刺激可显著诱导BEAS-2B细胞中EGFR和ERK的磷酸化及p65的核转位。经EGFR抑制剂AG1478预处理细胞后,ERK的磷酸化水平...     

Possible novel receptor for PGD2 on human bronchial epithelial cells

Prostaglandin D(2) (PGD(2)), a major prostanoid produced by activated mast cells, has long been implicated in allergic diseases. Recent studies have shown that PGD(2) exerts its effects through two different G-protein-coupled receptors (GPCRs), the D-prostanoid receptor (DP) and the chemoattractant receptor-homologous molecule expressed on T helper type-2 cells (CRTH2), expressed in various human tissues. The PGD(2)/CRTH2 system mediates the chemotaxis of eosinophils, basophils, and Th2 cells, which are involved in the induction of allergic inflammation. We have reported that normal human bronchial epithelial cells (NHBE) and epithelial cell lines (NCI-H(292)) expressed CRTH2, and PGD(2) induces production of IL-8 and GM-CSF. This review discusses the role of CRTH2/DP on epithelial cells and mentions a possible novel receptor for PGD(2).     

细胞因子诱导支气管上皮细胞表达嗜酸粒细胞趋化因子

Eotaxin和新近发现的Eotaxin-2在支气管上皮细胞中的表达以及Th2型细胞因子的调节作用。以支气管上皮细胞株BEAS-2B细胞为研究对象,通过RT-PCR的方法测定Th2型细胞因子IL-4、IL-13以及促炎症因子TNF-α单独和协同刺激下BEAS-2B细胞Eotaxin和Eotaxin-2的基因表达,通过ELISA方法测定细胞培养上清液中Eotaxin蛋白的表达。EotaxinmRNA在TNF-α刺激12h后表达最高,Th2型细胞因子IL-4和IL-13与TNF-α协同刺激后表达进一步增强,Eotaxin蛋白的表达在协同刺激下也呈剂量和时间依赖性增高(P<0.01)。Eotaxin-2mRNA在TNF-α的刺激下于8h表达最高,IL-4或IL-13与TNF-α的协同刺激也使表达进一步增强,Eotaxin和Eotaxin-2两者基因表达具有相关性(P<0.05)。Th2型细胞因子可与促炎症因子TNF-α协同刺激支气管上皮细胞增强表达嗜酸粒细胞趋化因子Eotaxin和Eotaxin-2,从而吸引嗜酸粒细胞浸润至气道参与哮喘炎症过程。     

TGF-beta suppresses EGF-induced MAPK signaling and proliferation in asthmatic epithelial cells

Epithelial damage is an important pathophysiologic feature of asthma. Bronchial epithelium damage results in release of growth factors such as transforming growth factor (TGF)-beta(1) that may affect epithelial cell proliferation. The objective of our study is to evaluate the importance of TGF-beta(1) in regulating epithelial cell repair in asthma. We evaluated the effect of TGF-beta(1) on epidermal growth factor (EGF)-induced proliferation and downstream signaling in epithelial cells obtained from subjects with asthma compared with cells from healthy subjects. Cell proliferation was evaluated by bromodeoxyuridine incorporation. EGF receptor (EGFR), mitogen-activated protein kinase, TGF-beta receptors, Smads, Smad anchor for receptor activation (SARA), and cyclin-dependant kinase inhibitors were evaluated by Western blot. TGF-beta(1) and receptor expression were measured by RT-PCR and by enzyme-linked immunosorbent assay. Proliferation of epithelial cells at baseline and after EGF stimulation was significantly reduced in cells derived from subjects with asthma compared with cells obtained from healthy control subjects. EGF-induced ERK1/2 phosphorylation was reduced in epithelial cells from subjects with asthma compared with cells from healthy control subjects. This was paralleled with a reduced EGFR phosphorylation. Addition of TGF-beta(1) significantly decreased EGF-induced cell proliferation. TGF-beta(1) production was higher in asthmatic epithelial cells compared with normal cells. This was supported by a high expression of pSmad 3 and SARA in cells derived from individuals with asthma compared with normal subjects. Cycline-dependent kinase inhibitors were highly expressed in asthmatic compared with normal cells. Inhibition of TGF-beta(1) signaling in asthmatic epithelial cells restored EGFR, ERK1/2 phosphorylation, and cell proliferation induced by EGF. Our results suggest that TGF-beta restrains EGFR phosphorylation and downstream signaling in bronchial epithelial cells.     

Protein kinase C activation is required for cigarette smoke-enhanced C5a-mediated release of interleukin-8 in human bronchial epithelial cells.

Complement-derived anaphylatoxin C5a is a glycopolypeptide important in the regulation of inflammation. Previously, we have shown that C5a receptors (C5aR) are constitutively expressed on human bronchial epithelial cells (HBECs) grown in culture. We have also shown that the expression of C5aR is increased upon exposure of HBECs to 5% cigarette smoke extract (CSE), and that this subtoxic dose of CSE significantly enhances C5a-stimulated interleukin (IL)-8 release. To determine the intracellular signaling pathway of CSE + C5a-mediated IL-8 release, we assayed protein kinase C (PKC) activity of HBECs after exposing the cells to CSE and/or C5a. No increase in PKC activity was observed when HBECs were treated with 50 nM C5a for various times. However, PKC activity was increased by 2- to 3-fold in HBECs stimulated with 5% CSE for 1 h, as compared with cells incubated with medium only. No additional increase in PKC was observed when HBECs were treated with CSE and C5a together. When HBECs were pretreated with the PKC-specific inhibitor calphostin C (1 microM), no CSE-mediated PKC activation was observed. We then correlated PKC activation with IL-8 release in the same cells. Although HBECs required stimulation by both CSE and C5a to release maximal levels of IL-8, preincubation of CSE-stimulated HBECs with calphostin C inhibited IL-8 release by CSE + C5a. These results suggest that PKC activation by CSE alone does not result in IL-8 release, but that CSE-stimulated PKC activation is required for C5a-mediated IL-8 release from HBECs.     

Suppression of cytokine production by glucocorticoids is mediated by MKP-1 in human lung epithelial cells

Mitogen-activated protein kinase phosphatase 1 (MKP-1) expression is induced by inflammatory factors and serves as an endogenous p38 MAPK suppressor to limit inflammatory response. Glucocorticoids are very effective anti-inflammatory drugs and they are used for the treatment of many inflammatory diseases, such as asthma and COPD. We investigated the role of MKP-1 in the inhibition of cytokine production by dexamethasone in human A549 bronchial epithelial cells. We found that dexamethasone increased MKP-1 expression, inhibited p38 MAPK phosphorylation, and suppressed TNF and MIP-3α production in A549 cells. Interestingly, the suppression of p38 MAPK phosphorylation and the inhibition of TNF expression by dexamethasone were attenuated in cells, where MKP-1 expression was silenced by siRNA. In conclusion, these data suggest that dexamethasone increases MKP-1 expression and this results in the suppression of p38 MAPK signaling leading to the inhibition of cytokine production in human bronchial epithelial cells. These results point to the role of MKP-1 as an important factor in the therapeutic effects of glucocorticoids in the treatment of inflammatory lung diseases.     

人支气管上皮细胞的体外培养

背景：人支气管上皮细胞培养在呼吸系统疾病研究中应用越来越广泛。 目的：探索美国典型培养物保藏中心人支气管上皮细胞体外培养方法的可行性。 方法：对人支气管上皮细胞培养条件进行反复摸索,最终确定应用含体积分数20%胎牛血清的DMEM/F12培养基进行培养。 结果与结论：实验培养的人支气管上皮细胞扁平,呈多边形,长满后呈“铺路石”样分布,经免疫组化检测发现培养的细胞表达上皮细胞标志物细胞角蛋白。在支气管扩张症患者痰液上清刺激下,细胞表达的核因子κB、肿瘤坏死因子α和白细胞介素8明显增强,证实培养的人支气管上皮细胞获得成功。提示不必拘泥于美国典型培养物保藏中心推荐的培养条件,改进的培养方法简便易行,有应用价值。     

Erk1/2 signaling is required for Tgf-beta 2-induced suture closure.

Transforming growth factors beta (Tgf-betas) act by means of Smad signaling pathways and may also interact with the mitogen-activated protein kinase pathway. The hypothesis was tested that Erk1/2 signaling is required for Tgf-beta2-induced suture closure, by culturing embryonic mouse calvariae in the presence of Tgf-beta2 with or without Erk1/2 inhibitor PD98059 (PD). Suture widths were measured daily, and microdissected sutures and bones were homogenized and protein analyzed by Western blots. Tgf-beta2 induced narrowing of the sutures after 72 hr, an effect inhibited by treatment with PD. Erk1/2 and Egf but not Smad2/3 protein expression was up-regulated by Tgf-beta2 calvarial tissues at 72 hr. PD inhibited endogenous and Tgf-beta2-stimulated Erk1/2 protein as well as Tgf-beta2-stimulated Egf, but increased Smad2/3 protein expression. In tissues harvested 0, 15, and 30 min after exposure to Tgf-beta2, Erk1/2 phosphorylation was up-regulated after 15 min, an effect abrogated by the simultaneous addition of PD. In summary, Tgf-beta2 stimulated Erk1/2 phosphorylation and induced Egf and Erk1/2 expression, associated with suture closure after 72 hr. Blocking Erk1/2 activity with PD inhibited these effects but increased Smad2/3 expression. We postulate that Tgf-beta2 regulates suture closure directly by means of phosphorylation of Erk1/2 and indirectly by up-regulating Erk1/2, a substrate for Fgf receptor signaling required for Fgf induction of premature suture obliteration.     

Dectin-1 is inducible and plays a crucial role in Aspergillus-induced innate immune responses in human bronchial epithelial cells

Airway epithelial cells are the first cells to be challenged upon contact with the conidia of Aspergillus. In response, they express pattern-recognition receptors that play fundamental roles as sentinels and mediators of pulmonary innate immunity. The C-type lectin Dectin-1 is expressed predominantly on the surface of myeloid lineage cells. We examined the induction, regulation, and functions of Dectin-1 in pulmonary epithelial cells by challenging human bronchial epithelial (HBE) cells with A. fumigatus. Inflammatory, antimicrobial peptide genes and reactive oxygen species (ROS) were quantified, with and without knockdown of Dectin-1. We found that A. fumigatus induced the expression of Dectin-1 mRNA and protein in HBE cells in a toll-like receptor (TLR) 2-dependent manner. In addition, A. fumigatus-mediated generation of ROS was dependent on the upregulation of Dectin-1. Moreover, A. fumigatus actively induced the expression of TNFα, GM-CSF, IL8, HBD2, and HBD9. Knockdown of Dectin-1 inhibited TNFα, IL8, HBD2, and HBD9 expression. Hence, Dectin-1 was required for the upregulation of pro-inflammatory cytokines and antimicrobial peptides. Finally, knockdown of TLR2 significantly inhibited Dectin-1 upregulation. Our results demonstrate the novel induction of Dectin-1 in human bronchial epithelial cells and its critical role in the innate immune response against A. fumigatus in non-phagocytic cells.     

Smad3-dependent nuclear translocation of beta-catenin is required for TGF-beta1-induced proliferation of bone marrow-derived adult human mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) derived from bone marrow contribute to the regeneration of multiple types of mesenchymal tissues. Here we describe the functional role of a novel form of cross-talk between the transforming growth factor beta1 (TGF-beta1) and Wnt signaling pathways in regulating the activities of human MSCs. We show that TGF-beta1 induces rapid nuclear translocation of beta-catenin in MSCs in a Smad3-dependent manner. Functionally, this pathway is required for the stimulation of MSC proliferation and the inhibition of MSC osteogenic differentiation by TGF-beta1, likely through the regulation of specific downstream target genes. These results provide evidence for a new mode of cooperation between the TGF-beta and Wnt signaling pathways in this specific cellular context and suggest a potentially important role for this distinct signaling pathway in the control of self-renewal and differentiation of a specific type of MSCs.     

CD40 expression by human bronchial epithelial cells

CD40 is a 50-kDa protein expressed on B cells, dendritic cells, monocytes and epithelial cells, but the distribution of CD40 expression in humans is not completely known. It binds to a ligand (CD40L) which is expressed essentially on activated T cells. The interaction between CD40 and CD40L plays important roles in immune responses. CD40 expression was investigated on bronchial tissues and human bronchial cell lines using immunohistochemistry, immunofluorescence staining and analysis with a cytometer, respectively. Constitutive CD40 expression, but not that of CD40L, was slightly detectable on normal human bronchial epithelial cells (HBEC) in situ and on an adult lung adenocarcinoma (SKLU1) cell line, while another cell line, a bronchial transformed SV40 cell line (WI26VA4), was negative for CD40. Among the various cytokines tested, only interferon (IFN)-gamma was found to induce CD40 expression on WI26VA4. Tumour necrosis factor (TNF)-alpha was the best cytokine able to up-regulate CD40 in SKLU1 cells. A combination of IFN-gamma and TNF-alpha was slightly more effective than the cytokine alone at up-regulating CD40 expression on both cell lines. We further investigated the functional consequences of CD40 ligation on both cell lines. These bronchial cells expressed CD40, HLADR and CD54 under basal conditions or when stimulated by cytokines. Stimulation through CD40 did not affect cell-surface-antigen expression on either cell line. The production of cytokines such as interleukin (IL)-6 and granulocyte macrophage-colony stimulating factor (GM-CSF) by HBEC has been described. SKLU1 and WI26VA4 cells released IL-6 and GM-CSF spontaneously. Whatever the case, CD40 engagement did not modulate spontaneous or TNF-alpha-induced production of these two cytokines. These data indicate for the first time that normal HBEC express CD40 in situ. Further investigations are required in order to determine the role of CD40 on normal HBEC.     

TGF-beta signaling in neuronal stem cells

Transforming growth factor beta (TGF-beta) signaling has diverse and complex roles in various biological phenomena such as cell growth, differentiation, embryogenesis and morphogenesis. ES cells provide an essential model for understanding the role of TGF-beta signaling in lineage specification and differentiation. Recent studies have suggested significant role of TGF-beta in stem/progenitor cell biology. Here in this review, we focus on the role of the TGF-beta superfamily in neuronal development.     

SIRT1 is required for long-term growth of human mesenchymal stem cells

Human mesenchymal stem cells (MSCs) have therapeutic potential because of their ability to self-renew and differentiate into multiple tissues. However, senescence often occurs in MSCs when they are cultured in vitro and the molecular mechanisms underlying this effect remain unclear. In this study, we found that NAD-dependent protein deacetylase SIRT1 is differentially expressed in both human bone marrow-derived MSCs (B-MSCs) and adipose tissue-derived MSCs after increasing passages of cell culture. Using lentiviral shRNA we demonstrated that selective knockdown of SIRT1 in human MSCs at early passage slows down cell growth and accelerates cellular senescence. Conversely, overexpression of SIRT1 delays senescence in B-MSCs that have undergone prolonged in vitro culturing and the cells do not lose adipogenic and osteogenic potential. In addition, we found that the delayed accumulation of the protein p16 is involved in the effect of SIRT1. However, resveratrol, which has been used as an activator of SIRT1 deacetylase activity, only transiently promotes proliferation of B-MSCs. Our findings will help us understand the role of SIRT1 in the aging of normal diploid cells and may contribute to the prevention of human MSCs senescence thus benefiting MSCs-based tissue engineering and therapies.     

Low-affinity receptor for IgE on human bronchial epithelial cells in asthma.

Bronchial epithelial cells are activated in asthma but the mechanisms underlying this activation are poorly understood. We tested the possibility that bronchial epithelial cells recovered by brushing from 15 asthmatic and 11 control subjects may be activated by an IgE-dependent mechanism. The expression of the low-affinity IgE receptor (CD23) was studied by immunocytochemistry using the alkaline phosphatase anti-alkaline phosphatase technique and immunofluorescence using confocal microscopy. Four of eight allergic asthmatic patients and none of the seven non-allergic asthmatic or control subjects had a positive expression of CD23. The functional activity of CD23 was examined in the cells recovered from these subjects by stimulating them with IgE/anti-IgE. 15-HETE was not released but endothelin was released in the three or four asthmatic patients who had a positive expression of CD23. None of the other subjects released any endothelin. This study suggests that bronchial epithelial cells of asthmatic patients may be directly activated by an IgE-mediated mechanism.     

MUC1 mucin mRNA expression in cultured human nasal and bronchial epithelial cells.

The MUC1 mucin mRNA, for which the cDNA was previously cloned from human breast and pancreatic tissues, was found to be expressed in nasal and bronchial epithelial cell primary cultures from cystic fibrotic, atopic, and normal individuals. Sequence analysis of cDNA clones from the CF/T43 cystic fibrosis nasal epithelial cell line revealed only insignificant differences in the 3' untranslated region of the mRNA when compared with the pancreas and breast mucin cDNAs.