Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells

A human epithelial cell line, WISH, and a mouse cell line, LB6-uPAR, transfected with the human urokinase receptor (uPAR), both expressed high affinity uPAR but undetectable levels of urokinase (uPA). In two independent assays, binding of exogenous pro-uPA produced an up to threefold enhancement of migration. The migration was time and concentration dependent and did not involve extracellular proteolysis. This biologic response suggested that uPAR can trigger an intracellular signal. Since this receptor is a glycosyl-phosphatidylinositol-linked protein, we postulated that it must do so by interacting with other proteins, among which, by analogy to other systems, would be a kinase. To test this hypothesis, we carried out a solid phase capture of uPAR from WISH cell lysates using either antibodies against uPAR or pro-uPA adsorbed to plastic wells, followed by in vitro phosphorylation of the immobilized proteins. SDS-PAGE and autoradiography revealed two phosphorylated protein bands of 47 and 55 kD. Both proteins were phosphorylated on serine residues. Partial sequence of the two proteins showed a 100% homology to cytokeratin 18 (CK18) and 8 (CK8), respectively. A similar pattern of phosphorylation was obtained with lysates from A459 cells, a lung carcinoma, but not HL60, LB6-uPAR or HEp3 cell lysates, suggesting that the identified multiprotein uPAR- complex may be specific for simple epithelia. Moreover, immunocapture with antibody to another glycosyl-phosphatidylinositol-linked protein, CD55, which is highly expressed in WISH cells, was ineffective. The kinase was tentatively identified as protein kinase C, because it was inhibited by an analogue of staurosporine more specific for PKC and not by a PKA or tyrosine kinase inhibitors. The kinase was tentatively identified as PKC epsilon because of its resistance to PMA down- modulation, independence of Ca2+ for activity, and reaction with a specific anti-PKC epsilon antibody in Western blots. Cell fractionation into cytosolic and particulate fractions revealed that all four proteins, the kinase, uPAR, CK18, and CK8, were present in the particulate fraction. In vivo, CK8, and to a lesser degree CK18, were found to be phosphorylated on serine residues. Occupation of uPAR elicited a time-dependent increase in the phosphorylation intensity of CK8, a cell shape change and a redistribution of the cytokeratin filaments. These results strongly suggest that uPAR serves not only as an anchor for uPA but participates in a signal transduction pathway resulting in a pronounced biological response.     

Cytokeratins in normal lung and lung carcinomas. I. Adenocarcinomas, squamous cell carcinomas and cultured cell lines

The various epithelial cells of the lower respiratory tract and the carcinomas derived from them differ markedly in their differentiation characteristics. Using immunofluorescence microscopy and two-dimensional gel electrophoresis of cytoskeletal proteins from microdissected tissues we have considered whether cytokeratin polypeptides can serve as markers of cell differentiation in epithelia from various parts of the human and bovine lower respiratory tract. In addition , we have compared these protein patterns with those found in the two commonest types of human lung carcinoma and in several cultured lung carcinoma cell lines. By immunofluorescence microscopy, broad spectrum antibodies to cytokeratins stain all epithelial cells of the respiratory tract, including basal, ciliated, goblet, and alveolar cells as well as all tumor cells of adenocarcinomas and squamous cell carcinomas. However, in contrast, selective cytokeratin antibodies reveal cell type-related differences. Basal cells of the bronchial epithelium react with antibodies raised against a specific epidermal keratin polypeptide but not with antibodies derived from cytokeratins characteristic of simple epithelia. When examined by two-dimensional gel electrophoresis, the alveolar cells of human lung show cytokeratin polypeptides typical of simple epithelia (nos. 7, 8, 18 and 19) whereas the bronchial epithelium expresses, in addition, basic cytokeratins (no. 5, small amounts of no. 6) as well as the acidic polypeptides nos. 15 and 17. Bovine alveolar cells also differ from cells of the tracheal epithelium by the absence of a basic cytokeratin polypeptide. All adenocarcinomas of the lung reveal a "simple-epithelium-type" cytokeratin pattern (nos. 7, 8, 18 and 19). In contrast, squamous cell carcinomas of the lung contain an unusual complexity of cytokeratins. We have consistently found polypeptides nos. 5, 6, 8, 13, 17, 18 and 19 and, in some cases, variable amounts of cytokeratins nos. 4, 14 and 15. Several established cell lines derived from human lung carcinomas (SK-LU-1, Calu -1, SK-MES-1 and A-549) show a uniform pattern of cytokeratin polypeptides (nos. 7, 8, 18 and 19), similar to that found in adenocarcinomas. In addition, vimentin filaments are produced in all the cell lines examined, except for SK-LU-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct and monocyte-induced innate immune response of human lung epithelial cells to Brucella abortus infection

Although Brucella frequently infects humans through inhalation, its interaction with pulmonary cells has been overlooked. We examined whether human lung epithelial cells produce proinflammatory mediators in response to Brucella infection. Infection with smooth or rough strains of Brucella abortus induced the secretion of IL-8 and GM-CSF by the bronchial epithelial cell lines Calu-6 and 16HBE14o-, but not by the alveolar epithelial cell line A549. Infected Calu-6 cells also produced low levels of MCP-1. Since monocyte-derived cytokines may induce chemokine secretion in epithelial cells, cocultures of human monocytes (THP-1 cell line) and respiratory epithelial cells were used to study such interaction. IL-8 and MCP-1 levels in B. abortus-infected THP-1:A549 and THP-1:Calu-6 cocultures, and MCP-1 levels in THP-1:16HBE14o- cocultures, were higher than those detected in infected epithelial monocultures. Conditioned medium from infected monocytes induced the secretion of IL-8 and/or MCP-1 by A549 and Calu-6 cells, and these effects were mainly mediated by IL-1 (in A549 cells) or TNF-α (in Calu-6 cells). Conversely, culture supernatants from Brucella-infected bronchial epithelial cells induced MCP-1 production by monocytes, an effect largely mediated by GM-CSF. This study shows that human lung epithelial cells mount a proinflammatory response to Brucella, either directly or after interaction with Brucella-infected monocytes.     

Monoclonal cytokeratin antibody recognizing a heterotypic complex: immunological probing of conformational states of cytoskeletal proteins in filaments and in solution

A novel type of monoclonal murine antibody (Ks18.18) directed against an epitope depending on human cytokeratin (CK) 18, a member of the acidic (type I) CK subfamily, is described. We show by SDS-PAGE immunoblots and dot-blot assays that this antibody is unreactive with both the denatured and the renatured individual polypeptides but binds strongly to heterotypic coiled-coil complexes of CK 18 with several members of the complementary basic (type II) CK subfamily, notably with CK 8; i.e., its most frequent natural partner. We also show that specific interactions between complementary CK polypeptides take place during the incubation steps of immunoblotting procedures as polypeptides, or fragments thereof, that detach from the substrate can bind to complementary polypeptides attached to the substratum, which may result in false assignments of antibody reactivities. The conformation-specific, CK 18-dependent epitope of Ks18.18 was detected in intermediate filaments (IFs) of cultured cells, simple epithelia, and many carcinomas and, surprisingly, also in the basal cells of some stratified epithelia. Ks18.18 also reacts with altered CK configurations as present in the spheroidal bodies of mitotic cells and in the Mallory bodies of hepatocytes intoxicated with certain drugs, thus indicating that the heterotypic CK complexes are maintained in these structures. We have also used antibody Ks18.18 to demonstrate the existence of heterotypic CK 8 and 18 complexes in a distinct soluble form among supernatant proteins from cell homogenates which is indistinguishable from the heterotypic tetramer obtained after experimental disintegration of IFs. The potential value of such IF conformation-specific antibodies in cell biological research and pathology is discussed.     

Expression of functional TRPA1 receptor on human lung fibroblast and epithelial cells

The transient receptor potential subfamily A member 1 (TRPA1) is a non-selective cation channel implicated in the pathogenesis of several airway diseases like asthma and chronic obstructive pulmonary disease (COPD). Most of the research on TRPA1 focuses on its expression and function in neuronal context; studies investigating non-neuronal expression of TRPA1 are lacking. In the present study, we show functional expression of TRPA1 in human lung fibroblast cells (CCD19-Lu) and human pulmonary alveolar epithelial cell line (A549). We demonstrate TRPA1 expression at both mRNA and protein levels in these cell types. TRPA1 selective agonists like allyl isothiocyanate (AITC), 4-hydroxynonenal (4-HNE), crotonaldehyde and zinc, induced a concentration-dependent increase in Ca⁺² influx in CCD19-Lu and A549 cells. AITC-induced Ca⁺² influx was inhibited by Ruthenium red (RR), a TRP channel pore blocker, and by GRC 17536, a TRPA1 specific antagonist. Furthermore, we also provide evidence that activation of the TRPA1 receptor by TRPA1 selective agonists promotes release of the chemokine IL-8 in CCD19-Lu and A549 cells. The IL-8 release in response to TRPA1 agonists was attenuated by TRPA1 selective antagonists. In conclusion, we demonstrate here for the first time that TRPA1 is functionally expressed in cultured human lung fibroblast cells (CCD19-Lu) and human alveolar epithelial cell line (A549) and may have a potential role in modulating release of this important chemokine in inflamed airways.     

Phorbol acetate enhances the phosphorylation of cytokeratins 8 and 18 in human colonic epithelial cells.

The phosphorylation of epithelial-specific cytokeratin (CK) 8 and 18 was studied in the human colonic cell line HT29. Metabolic labelling of cells with orthophosphate resulted in phosphorylation of cytokeratins 8/18 on serine residues. When phorbol acetate was added to labelled cells, a 2.2-fold increase in CK8/18 phosphate labelling was noted, whereas increasing intracellular cAMP levels using forskolin or 8-Br-cAMP showed no significant change in CK phosphorylation. CKs8/18 were also phosphorylated by added PKC in the presence of [gamma-32P]ATP. Tryptic peptide map analysis of the phosphorylated CK8 species showed that treatment of cells with 8-Br-cAMP or phorbol acetate generated a phosphopeptide not seen in control cells. In contrast, tryptic peptide maps of phosphorylated CK18 showed no discernable differences. Our results support a role for PKC in the phosphorylation of epithelial cytokeratins, with some phosphorylation sites being modulated by cAMP dependent protein kinase.     

Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae

A 40-kD protein kinase C (PKC)epsilon related activity was found to associate with human epithelial specific cytokeratin (CK) polypeptides 8 and 18. The kinase activity coimmunoprecipitated with CK8 and 18 and phosphorylated immunoprecipitates of the CK. Immunoblot analysis of CK8/18 immunoprecipitates using an anti-PKC epsilon specific antibody showed that the 40-kD species, and not native PKC epsilon (90 kD) associated with the cytokeratins. Reconstitution experiments demonstrated that purified CK8 or CK18 associated with a 40-kD tryptic fragment of purified PKC epsilon, or with a similar species obtained from cells that express the fragment constitutively but do not express CK8/18. A peptide pseudosubstrate specific for PKC epsilon inhibited phosphorylation of CK8/18 in intact cells or in a kinase assay with CK8/18 immunoprecipitates. Tryptic peptide map analysis of the cytokeratins that were phosphorylated by purified rat brain PKC epsilon or as immunoprecipitates by the associated kinase showed similar phosphopeptides. Furthermore, PKC epsilon immunoreactive species and CK8/18 colocalized using immunofluorescent double staining. We propose that a kinase related to the catalytic fragment of PKC epsilon physically associates with and phosphorylates cytokeratins 8 and 18.     

Dendritic cells efficiently acquire and present antigen derived from lung cancer cells and induce antigen-specific T-cell responses

Active immunotherapy of cancer requires the availability of a source of tumor antigens. To date, no such antigen associated with lung cancer has been identified. We have therefore investigated the ability of dendritic cells (DC) to capture whole irradiated human lung tumor cells and to present a defined surrogate antigen derived from the ingested tumor cells. We also describe an in vitro system using a modified human adenocarcinoma cell line (A549-M1) that expresses the well-characterized, immunogenic influenza M1 matrix protein as a surrogate tumor antigen. Peripheral blood monocyte-derived DC, when co-cultured with sub-lethally irradiated A549 cells or primary lung tumor cells derived from surgical resection of non-small cell carcinoma (NSCLC), efficiently ingested the tumor cells as determined by flow cytometry analysis and confocal microscopic examination. More importantly, DC loaded with irradiated A549-M1 cells efficiently processed and presented tumor cell-derived M1 antigen to T cells and elicited antigen-specific immune responses that included IFNgamma release from an M1-specific T-cell line, expansion of M1 peptide-specific Vbeta17+ and CD8+ peripheral T cells and generation of M1-specific cytotoxic T lymphocytes (CTL). We also compared DC loaded with irradiated tumor cells to those loaded with tumor cell lysate or killed tumor cells and found that irradiated lung tumor cells as a source of tumor antigen for DC loading is superior to tumor cell lysate or killed tumor cells in efficient induction of antigen-specific T-cell responses. Our results demonstrate the feasibility of using lung tumor cell-loaded DC to induce immune responses against lung cancer-associated antigens and support ongoing efforts to develop a DC-based lung cancer vaccine.     

Comparative proteomics of pulmonary tumors with neuroendocrine differentiation

We aimed to evaluate neuroendocrine pulmonary tumors (NEPT) by a novel method involving map tree construction by comparing all of the protein spots. We performed a proteomics analysis to assess the similarities in protein expression between neuroendocrine pulmonary tumors (NEPT), including typical carcinoids (TC), atypical carcinoids (AC), large cell neuroendocrine carcinomas (LCNEC) and small cell carcinomas (SCLC). Total protein lysates were obtained from seven histologically confirmed frozen NEPT tissues, including 1TC, 2 SCLC, and 4 cases ranging from AC to LCNEC. 2-DE demonstrated that TC was similar to normal lung. AC, LCNEC, and SCLC were similar to each other, forming a group separate from TC, however, SCLC at an early stage showed a similarity to TC. MALDI analysis detected 9 surrogate endpoint biomarkers, including eIF5A1, GST M3, cytokeratin 18 (CK 18), FK506-binding protein p59, p63, MAGE-D2, mitochondrial short-chain enoyl-coenzyme A hydratase 1, tranferrin and poly (rC) binding protein 1. Immunohistochemical staining revealed a gradual decrease in expression rate of p63 and CK 18 with poor differentiation of NEPT. Our results demonstrate that (1) the comparative proteomics of NEPT match the WHO classification except for AC and LCNEC; (2) SCLC show differences in their proteomics according to tumor stage; and (3) CK 18 and p63 may be useful as diagnostically and prognostically available markers.     

Expression of functional TRPA1 receptor on human lung fibroblast and epithelial cells

The transient receptor potential subfamily A member 1 (TRPA1) is a non-selective cation channel implicated in the pathogenesis of several airway diseases like asthma and chronic obstructive pulmonary disease (COPD). Most of the research on TRPA1 focuses on its expression and function in neuronal context; studies investigating non-neuronal expression of TRPA1 are lacking. In the present study, we show functional expression of TRPA1 in human lung fibroblast cells (CCD19-Lu) and human pulmonary alveolar epithelial cell line (A549). We demonstrate TRPA1 expression at both mRNA and protein levels in these cell types. TRPA1 selective agonists like allyl isothiocyanate (AITC), 4-hydroxynonenal (4-HNE), crotonaldehyde and zinc, induced a concentration-dependent increase in Ca+2 influx in CCD19-Lu and A549 cells. AITC-induced Ca+2 influx was inhibited by Ruthenium red (RR), a TRP channel pore blocker, and by GRC 17536, a TRPA1 specific antagonist. Furthermore, we also provide evidence that activation of the TRPA1 receptor by TRPA1 selective agonists promotes release of the chemokine IL-8 in CCD19-Lu and A549 cells. The IL-8 release in response to TRPA1 agonists was attenuated by TRPA1 selective antagonists. In conclusion, we demonstrate here for the first time that TRPA1 is functionally expressed in cultured human lung fibroblast cells (CCD19-Lu) and human alveolar epithelial cell line (A549) and may have a potential role in modulating release of this important chemokine in inflamed airways.     

Cytokeratin 8-MHC class I interactions: A potential novel immune escape phenotype by a lymph node metastatic carcinoma cell line

Defective human leukocyte antigen (HLA) class I expression in malignant cells facilitates their escape from destruction by CD8⁺ cytotoxic T lymphocytes. In this study, a post-translational mechanism of HLA class I abnormality that does not involve defects in the HLA subunits and antigen processing machinery components was identified and characterized. The marked HLA class I downregulation phenotype of a metastatic carcinoma cell line can be readily reversed by trypsin, suggesting a masking effect by serine protease-sensitive HLA class I-interacting factors. Co-immunoprecipitation, combined with LC–tandem mass spectrometry and immunoblotting identified these factors as cytokeratin (CK) 8 and its heterodimeric partners CK18 and CK19. Ectopic CK8/18 or CK8/19 expression in HEK293 cells resulted in surface CK8 expression with an HLA class I downregulation phenotype, while redirecting CK8/18 and CK8/19 to the endoplasmic reticulum (ER) had no such effect. This observation and the failure to constrain CK8/18 and CK8/19 membrane trafficking by an ER-Golgi transport inhibitor suggested an ER-independent route for CK8 access to HLA class I molecules. Monoclonal antibody mapping revealed a potential CK8 blockade of HLA class I-CD8 and -TCR contacts. These findings, along with the emerging role of cell surface CK8 in cancer metastasis, may imply a dual strategy for tumor cell survival in the host.     

CK2 inhibitor CX4945 induces sequential inactivation of proteins in the signaling pathways related with cell migration and suppresses metastasis of A549 human lung cancer cells

Casein kinase 2 (CK2) is known to be involved in various cellular processes such as cell cycle, apoptosis and proliferation. It has been reported that the inhibition of CK2 induced by recently developed small molecule CX4945 shows anti-cancer effects including anti-proliferation and anti-angiogenesis in several different cancers including prostate cancer. Here we report that migration and invasion of A549 human lung cancer cells are suppressed by the inhibition of CK2 induced by CX4945. We found that CX4945 sequentially attenuates the proteins in PI3K/Akt and MAPK pathways, two signaling pathways related with cell migration. This sequential control of signal pathways inhibits the expression of membrane type 1-matrix metalloproteinase and this leads to the selective attenuation of one of the gelatinases, MMP-2, which can degrade components of extracellular matrix, and metastasis of A549 human lung cancer cell.     

稳定表达p120ctn的人肺腺癌A549细胞株的构建

目的:构建稳定表达p120ctn的A549细胞株,以研究p120ctn蛋白在肺癌发生和转移过程中的作用。方法:通过分子克隆,将pc DNA3.1多克隆位点插入Flag标签的编码序列,得到pc DNA.Flag表达载体。然后PCR扩增p120ctn的编码序列,插入Flag标签下游,构建pc DNA.Flag-p120ctn质粒,筛选阳性克隆并进行酶切及测序鉴定。利用脂质体Lipofectamine 2000将pc DNA.Flag-p120ctn质粒转染到肺癌细胞A549中,通过G418筛选得到稳定转染细胞株,免疫印迹法检测p120ctn的表达。结果:本文构建了融合有Flag标签的p120ctn真核表达载体并转染到A549中,免疫印迹结果表明p120ctn蛋白在A549细胞中高效的表达。结论:本文成功构建了稳定高表达p120ctn的A549细胞模型,为深入研究p120ctn在肺癌的发生和转移过程中的作用奠定了基础。     

DEDD association with cytokeratin filaments correlates with sensitivity to apoptosis

The cytokeratin 8/18 (CK8/18) cytoskeleton network is an early target for caspase cleavage during apoptosis. Recent reports suggest that the highly conserved and ubiquitous death effector domain containing DNA binding protein (DEDD) plays a role in the recruitment of procaspase-9 and -3 at this CK8/18 scaffold. DEDD interacts with both the CK8/18 intermediate filament network and procaspase-3 and –9. It is suggested that the CK8/18 fibrils may provide a scaffold for the proximity-induced autocleavage and activation of procaspase-9 in close association with caspase-3. We addressed this issue by investigating DEDD staining patterns in various cell lines and by correlating these expression patterns with the sensitivity of these cell lines for roscovitine-induced apoptosis. We showed that in some cell lines DEDD revealed a bright filamentous staining pattern in others DEDD staining was weak and diffusely distributed in the cytoplasm of the cells. The difference in staining patterns was irrespective of the phosphorylation status of the cytokeratin filaments. In cells showing a filamentous staining pattern, DEDD was strongly associated with the CK8/18 cytokeratin filaments as evidenced by double immunofluorescence and its resistance to extraction with Triton X-100. Subcellular fractionation indicates that DEDD co-purifies with CK18, which corroborates a strong association of DEDD and the cytokeratin network. DEDD was either mono- or diubiquinated. Cells showing a filamentous DEDD distribution are more apoptosis-prone as evidenced by the rapid appearance of M30 CytoDeath-positive cells after induction of apoptosis. The sensitivity towards apoptosis is irrespective of the procaspase-3 content of the cells. Our data support the notion that DEDD-mediated accumulation of procaspases at the cytokeratin scaffold leads to an increase in the local concentration, which renders cells more apoptosis-prone.     

Subtractive screening of genes involved in cellular senescence

We attempted to identify the genes involved in cellularsenescence, telomere maintenance and telomerase regulationthrough subtractive screening of cDNA libraries prepared froma human lung adenocarcinoma cell line A549 and its sublinesnamed A5DC7, CK and AST-9. Cell phenotypes of A5DC7, CK andAST-9 are normal cell-like, cancer cell-like and intermediate,respectively. These cell lines have different phenotypes interms of telomerase activity and telomere maintenance, andthus are thought to be useful for identifying the genesinvolved in cellular senescence and telomerase regulation. In this study, we identified 86 independent cDNA clones bysubtractive screening. Among these cDNA clones, subtractingA5DC7 cDNAs from A549 cDNAs and CK cDNAs gave 7 and 3 cDNAclones which highly and specifically expressed in tester celllines. Genes corresponding to these 10 cDNA clones mightparticipate in maintaining cancer-cell phenotypes. As aresult of database searching, each four of A549 specific cDNAclones are found to correspond to known cDNAs. Each two ofA549 specific and two of CK specific cDNA clones have highhomology to independent ESTs. Sequences having homology toeach one of A549 specific and one of CK specific cDNA cloneshave not been deposited in the Genbank database, indicatingthat these two cDNA clones are part of novel genes. Weanticipate that their involvement in telomerase regulationand/or senescence program can be clarified by functionalanalysis using each full-length cDNA.     

Isolation and characterization of a human hepatic epithelial-like cell line (AKN-1) from a normal liver

Summary The isolation and characterization of human liver cell lines are rather difficult due to limited material and poor growth in cell culture. In this report, we present the isolation, culture and characterization of a new epithelial-like liver cell line (AKN-1) with a heterogeneous cell population and many characteristics of the biliary epithelium. The AKN-1 cell line stained positively with antibodies to epithelial cytokeratin polypetides CK 8, 18, and 19. In addition, the cell line expressed the anti-human epithelial-related antigen (MOC-31), the human epithelial antigen (HEA), and the gamma-glutamyl transpeptidase, the hematopoietic growth factor, stem cell factor, and also its receptor, c-kit. The cell line failed to express albumin and factor 8 by immunohistochemistry. It did show, however, a twofold increase in 7-ethoxyresorufin-O-deethylase activity. Cytogenetic characterization revealed rare breakpoints in chromosome 2, which to our knowledge, have not yet been reported in liver cells.     

Effect of proinflammatory cytokines on interleukin-8 mRNA expression and protein production by isolated human alveolar epithelial cells type II in primary culture

Alveolar epithelial cells type II (AEC-II) are ideally situated to regulate the recruitment and activation of different types of cells through the production of chemokines in response to inflammatory stimulation from the alveolar space. We hypothesized that these cells are important producers of interleukin-8 (IL-8) in the lung. This lead us to investigate the capacity of isolated human AEC-II cells to release IL-8 and whether this IL-8 release is regulated by proinflammatory cytokines, i.e. IL-1 beta, TNF-alpha and IFN-gamma. We isolated AEC-II from tumor-free sections of human lungs obtained by pneumectomy and purified the cells by magnetic activated cell sorting. For control experiments the AEC-II-like cell line A549 was used. IL-8 concentration was measured by ELISA in supernatants of unstimulated and LPS-, IL-1 beta-, TNF-alpha- and IFN-gamma- stimulated cells. IL-8 mRNA expression was evaluated by RT-PCR. Spontaneous IL-8 mRNA expression and protein secretion by AEC-II were significantly higher in comparison with A549 cells. TNF-alpha increased both IL-8 mRNA expression and protein production, whereas IL-1 beta slightly increased IL-8 release but did not change mRNA expression in AEC-II. LPS and IFN-gamma did not influence IL-8 expression in AEC-II and A549 cells. These results show considerable differences between A549 cell and AEC-II. The latter are capable of producing IL-8 under the control of proinflammatory cytokines. Our findings demonstrate that the modulation of IL-8 release in AEC-II may have an important impact on the immunoreactivity of these cells during pulmonary inflammation in vivo.     

Characterization and dynamics of O-linked glycosylation of human cytokeratin 8 and 18.

The glycosylation of human cytokeratin (CK) 8 and 18 was studied after metabolic labeling of HT29 colonic cells with [3H]glucosamine. Labeling of CK8/18 was not inhibited by tunicamycin, suggesting that glycosylation was not N-linked. Acid hydrolysis of CK8 and CK18, purified from [3H]glucosamine-labeled cells, generated free glucosamine. In the presence of UDP-[3H]galactose, galactosyltransferase catalyzed the labeling of cytokeratin 8 and 18. beta-Elimination of the [3H]galactose- labeled CK8/18 generated the disaccharide N-acetyllactosaminitol, indicating that cytokeratin 8 and 18 contain single O-linked N-acetylglucosamine residues. Using chemical analysis, the stoichiometry of glycosylation was found to be 1.5 and 2 molecules of N-acetylglucosamine/protein molecule of CK8 and CK18, respectively. Peptide maps of [3H]glucosamine-labeled CK8/18 showed that multiple peptides were labeled with the amino sugar. The biosynthetic and degradation rates of the carbohydrate moiety were faster than the protein core as determined by metabolic radiolabeling or pulse-chase experiments, respectively. Our results show that CK8 and 18 are glycosylated at multiple sites with a single O-linked N-acetylglucosamine. Furthermore, CK8/18 glycosylation is a dynamic process which is likely to have functional relevance.