TRX-ASK1-JNK signaling regulation of cell density-dependent cytotoxicity in cigarette smoke-exposed human bronchial epithelial cells

Cigarette smoke is a major environmental air pollutant that injures airway epithelium and incites subsequent diseases including chronic obstructive pulmonary disease. The lesion that smoke induces in airway epithelium is still incompletely understood. Using a LIVE/DEAD cytotoxicity assay, we observed that subconfluent cultures of bronchial epithelial cells derived from both human and monkey airway tissues and an immortalized normal human bronchial epithelial cell line (HBE1) were more susceptible to injury by cigarette smoke extract (CSE) and by direct cigarette smoke exposure than cells in confluent cultures. Scraping confluent cultures also caused an enhanced cell injury predominately in the leading edge of the scraped confluent cultures by CSE. Cellular ATP levels in both subconfluent and confluent cultures were drastically reduced after CSE exposure. In contrast, GSH levels were significantly reduced only in subconfluent cultures exposed to smoke and not in confluent cultures. Western blot analysis demonstrated ERK activation in both confluent and subconfluent cultures after CSE. However, activation of apoptosis signal-regulating kinase 1 (ASK1), JNK, and p38 were demonstrated only in subconfluent cultures and not in confluent cultures after CSE. Using short interfering RNA (siRNA) to JNK1 and JNK2 and a JNK inhibitor, we attenuated CSE-mediated cell death in subconfluent cultures but not with an inhibitor of the p38 pathway. Using the tetracycline (Tet)-on inducible approach, overexpression of thioredoxin (TRX) attenuated CSE-mediated cell death and JNK activation in subconfluent cultures. These results suggest that the TRX-ASK1-JNK pathway may play a critical role in mediating cell density-dependent CSE cytotoxicity.     

Control of glycoprotein synthesis. The use of oligosaccharide substrates and HPLC to study the sequential pathway for N-acetylglucosaminyltransferases I, II, III, IV, V, and VI in the biosynthesis of highly branched N-glycans by hen oviduct membranes

Glycoproteins isolated from hen oviduct contain highly branched asparagine-linked oligosaccharides (N-glycans). Six N-acetylglucosaminyltransferases (GlcNAc-T I, II, III, IV, V, and VI) are involved in initiating the synthesis of these branches, as indicated below: (formula; see text) where R is GlcNAc beta 1----4(+/-Fuc alpha 1----6)GlcNAcAsn-X. HPLC has been used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct. Oligosaccharides with free reducing GlcNAc termini were prepared from various glycoproteins by hydrazinolysis-re-N-acetylation and used as GlcNAc-T substrates and HPLC standards. Enzyme assay components were separated on AG1 x 8, followed by HPLC on amine-bonded silica columns eluted with acetonitrile-water mixtures. Absorbance at 195 nm and radioactivity of eluted compounds were monitored. Substrates and products were identified by comparison of their retention times with those of oligosaccharides with known structures. Enzyme assay by HPLC is more rapid and convenient than previous GlcNAc-T assays using lectin columns or electrophoresis. Since some substrates yielded multiple products, these could be used to assay more than one GlcNAc-T in the same incubation. GlcNAc-T VI was shown to act on both bisected and nonbisected GlcNAc-terminating tetraantennary oligosaccharide substrates; GlcNAc-T II, IV, and V acted poorly or not at all on bisected substrates. GlcNAc-T V was the only enzyme among the six transferases studied that could be assayed in the absence of Mn2+.     

Regulation of N-acetylglucosaminyltransferase V activity. Kinetic comparisons of parental, Rous sarcoma virus-transformed BHK, and L-phytohemagglutinin-resistant BHK cells using synthetic substrates and an inhibitory substrate analog

Baby hamster kidney (BHK) cells transformed with Rous sarcoma virus, RS-BHK cells, demonstrate a 2.5-fold increase in the activity of N-acetylglucosaminyltransferase V (GlcNAc-T V, EC 2.4.1.155), and this increase in activity appears to be specific for this enzyme. By contrast, a lectin-resistant BHK cell line selected for its ability to grow in high levels of L-phytohemagglutinin, LP3.3, is characterized by a specific decrease in its GlcNAc-T V activity. To test if these alterations in the apparent Vmax of GlcNAc-T V are due to changes in the efficiency of populations of enzymes in RS-BHK and LP3.3 cells compared to the parental BHK cells, we have compared the kinetic properties of the enzymes from these three sources. The Km constants observed for both the sugar nucleotide donor (UDP-GlcNAc) and two synthetic trisaccharide acceptors were indistinguishable. The Vmax values toward three synthetic acceptors were also determined first for the BHK GlcNAc-T V, and they varied by over 5-fold. When these values were measured for the variant and transformed cell enzymes, however, similar 5-fold differences were still observed, although the absolute values for these acceptors were all higher or lower for the RS-BHK and LP3.3 enzymes, respectively. In addition, we have synthesized a deoxygenated analog of the specific GlcNAc-T V acceptor, beta GlcNAc(1,2) alpha Man(1,6) beta ManOR, where the reactive 6'-OH group has been removed, and the resulting trisaccharide was found to be a competitive inhibitor of the enzyme. The Ki for this inhibitor was near 70 microM for the GlcNAc-T V from all three sources. These kinetic comparisons demonstrate that the enzymes from the three cell types have kinetically indistinguishable active sites. These results suggest that the differences in the apparent Vmax values among the cell types are most likely due to alterations in the number of active molecules rather than in the modulation of either their catalytic activities or specificities.     

Aberrant O-linked oligosaccharide biosynthesis in lymphocytes and platelets from patients with the Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immunodeficiency affecting B lymphocytes, T lymphocytes, and platelets. Previous studies on lymphocytes from WAS patients have revealed that leu-kosialin (CD43), a cell-surface glycoprotein bearing approximately 90 O-linked oligosaccharide chains, shows an aberrant electrophoretic mobility. To determine whether this finding reflects a different pattern of O-linked glycosylation in WAS cells, we have compared healthy individuals and WAS patients with respect to glycosyltransferase activities in T lymphocytes, platelets, and Epstein-Barr virus (EBV)-immortalized B cell lines. Stimulation of peripheral T cells from normal individuals in vitro with anti-CD3 antibodies and interleukin-2 was associated with a 3-fold increase in UDP-GlcNAc:Gal beta 3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T) from 0.8 to 2.2 nmol/mg/h. In contrast, peripheral T lymphocytes from WAS patients showed an inversion of this phenotype with high core 2 GlcNAc-T activity in unstimulated cells (2.3 nmol/mg/h) and a 2-3-fold decrease in activity following stimulation. Core 2 GlcNAc-T activity was also three times higher in platelets from WAS patients than in normal platelets. Glycosyltransferase activities were measured in immortalized B cell lines established from WAS and normal subjects by infection with EBV. Core 2 GlcNAc-T was less than 0.4 nmol/mg/h in WAS EBV-B cell lines compared to 2.4 nmol/mg/h in EBV-B cell lines from healthy individuals, In contrast, CMP-SA:SA alpha 2-3Gal beta 1-3GalNAc-R (where SA represents sialyl (sialic acid to GalNAc) alpha 6-sialyltransferase II activity was 2.0 nmol/mg/h in the WAS EBV-B cell and less than .01 nmol/mg/h in EBV-B cell lines derived from normal subjects. Eleven other glycosyltransferase activities were measured and found to be similar in EBV-B cell lines from WAS and normal individuals. Polylactosamine sequences were much reduced in the O-linked oligosaccharides of CD43 from WAS EBV-B cells consistent with decreased core 2 GlcNAc-T activity and expression of core 1 oligosaccharides in the cells. In conclusion, B cells, T cells, and platelets in WAS patients show abnormal expression of two developmentally regulated glycosyltransferases, consistent with the idea that the WAS immunodeficiency is due to a failure of normal lymphocyte maturation.     

Cell-density-dependent changes in cell-surface glycopeptides and in adhesion of cultured intestinal epithelial cells

We studied mannose-containing glycopeptides and glycoproteins of subconfluent and confluent intestinal epithelial cells in culture. Cells were labelled with d-[2-³H]mannose for 24h and treated with Pronase or trypsin to release cell-surface components. The cell-surface and cell-residue fractions were then exhaustively digested with Pronase and the resulting glycopeptides were fractionated on Bio-Gel P-6, before and after treatment with endo-β-N-acetylglucosaminidase H to distinguish between high-mannose and complex oligosaccharides. The cell-surface glycopeptides were enriched in complex oligosaccharides as compared with residue glycopeptides, which contained predominantly high-mannose oligosaccharides. Cell-surface glycopeptides of confluent cells contained a much higher proportion of complex oligosaccharides than did glycopeptides from subconfluent cells. The ability of the cells to bind [³H]concanavalin A decreased linearly with increasing cell density up to 5 days in culture and then remained constant. When growth of the cells was completely inhibited by either retinoic acid or cortisol, no significant difference was observed in the ratio of complex to high-mannose oligosaccharides in the cell-surface glycopeptides of subconfluent cells. Only minor differences were found in total mannose-labelled glycoproteins between subconfluent and confluent cells by two-dimensional gel analysis. The adhesion of the cells to the substratum was measured at different stages of growth and cell density. Subconfluent cells displayed a relatively weak adhesion, which markedly increased with increased cell density up to 6 days in culture. It is suggested that alterations in the structure of the carbohydrates of the cell-surface glycoproteins are dependent on cell density rather than on cell growth. These changes in the glycopeptides are correlated with the changes in adhesion of the cells to the substratum.     

Physiological State of Human Embryonic Lung Cells Affects Their Response to Human Cytomegalovirus

Cultures of human embryonic lung (HEL) cells in different physiological states were studied for their susceptibility to infection with human cytomegalovirus (CMV) with respect to production of infectious virus, synthesis of viral antigens, and virus-induced stimulation of cellular DNA synthesis. In general, subconfluent, actively growing cells yielded higher amounts of infectious virus than did confluent contact-inhibited cells. The higher yield of infectious virus was correlated with a greater percentage of cells producing viral antigens within the first 48 h after infection. In confluent cultures, 25 to 50% of the cells produced viral antigens within the first 48 h postinfection. This proportion did not change over a 10-fold range of multiplicity of infection, indicating that many of the cells in confluent cultures did not support productive infection. However, virtually all the cells in subconfluent cultures were susceptible. Also, in contrast to herpes simplex virus and pseudorabies virus, infectious CMV is not produced by cells treated with 5-fluorouracil and thymidine. Virus-induced stimulation of cellular DNA synthesis in cells infected at high multiplicities of infection could be detected only in confluent cultures, in which cellular DNA synthesis had been previously suppressed, but could not be detected in similarly treated cultures of subconfluent cells. The lack of detectable stimulation of cellular DNA synthesis in the latter was related to the fact that practically all the cells in the culture synthesized viral antigens within the first 48 h after infection, productive infection and detectable synthesis of cellular DNA being mutually exclusive.     

Cell density modulates apoptosis in human articular chondrocytes

This study addresses the effects of cell density and serum on CD95 (APO-1/Fas) and CD95L (Fas Ligand) expression and on the induction of CD95-dependent apoptosis in human articular chondrocytes from normal knees. Subsets of articular chondrocytes in first passage monolayer culture expressed CD95 and CD95L on the cell surface. The expression of both molecules was influenced by cell density: 22.3% of chondrocytes plated at subconfluent density expressed CD95L while expression in confluent cultures was reduced to 8.2%. CD95 expression was 32.1% under subconfluent and 12.2% under confluent conditions. Induction of specific apoptosis by agonistic antibody to CD95 was 15 times higher in confluent cultures than in subconfluent cultures despite higher levels of CD95 and CD95L expression in subconfluent cells, suggesting that protective antiapoptotic mechanisms were activated in low-density cultures. In subconfluent cultures, serum withdrawal had no effect on the sensitivity of the cells toward CD95 antibody-induced apoptosis. However, in confluent cultures, serum withdrawal led to a significant reduction of CD95-dependent apoptosis. Together, these findings demonstrate that cell density is an important modulator of CD95/CD95L expression and susceptibility to CD95-mediated apoptosis in cultured human chondrocytes. J. Cell. Physiol. 180:439–447, 1999. © 1999 Wiley-Liss, Inc.     

The lateral mobility of the (Na+,K+)-dependent ATPase in Madin-Darby canine kidney cells

Fluorescence microphotolysis (recovery after photobleaching) was used to determine the lateral mobility of the (Na+,K+)ATPase and a fluorescent lipid analogue in the plasma membrane of Madin-Darby canine kidney (MDCK) cells at different stages of development. Fluorescein-conjugated Fab' fragments prepared from rabbit anti-dog (Na+,K+)ATPase antibodies (IgG) and 5-(N-hexadecanoyl)aminofluorescein (HEDAF) were used to label the plasma membrane of confluent and subconfluent cultures of MDCK cells. Fractional fluorescence recovery was 50% and 80-90% for the protein and lipid probes, respectively, and was independent of developmental stage. The estimated diffusion constants of the mobile fraction were approximately 5 X 10(-10) cm2/s for the (Na+,K+)ATPase and approximately 2 X 10(-9) cm2/s for HEDAF. Only HEDAF diffusion showed dependency on developmental stage in that D for confluent cells was approximately twice that for subconfluent cells. These results indicate that (Na+,K+)ATPase is 50% immobilized in all developmental stages, whereas lipids in confluent MDCK cells are more mobile than in subconfluent cells. They suggest, furthermore, that the degree of immobilization of the (Na+,K+)ATPase is insufficient to explain its polar distribution, and they support restricted mobility of the ATPase through the tight junctions as the likely mechanism for preventing the diffusion of this protein into the apical domain of the plasma membrane in confluent cell cultures.     

The biosynthesis of highly branched N-glycans: studies on the sequential pathway and functional role of N-acetylglucosaminyltransferases I, II, III, IV, V and VI

At least 6 N-acetylglucosaminyltransferases (GlcNAc-T I, II, III, IV, V and VI) are involved in initiating the synthesis of the various branches found in complex asparagine-linked oligosaccharides (N-glycans), as indicated below: GlcNAc beta 1-6 GlcNAc-T V GlcNAc beta 1-4 GlcNAc-T VI GlcNAc beta 1-2Man alpha 1-6 GlcNAc-T II GlcNAc beta 1-4Man beta 1-4-R GlcNAc T III GlcNAc beta 1-4Man alpha 1-3 GlcNAc-T IV GlcNAc beta 1-2 GlcNAc-T I where R is GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAcAsn-X. HPLC was used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct (I. Brockhausen, J.P. Carver and H. Schachter (1988) Biochem. Cell Biol. 66, 1134-1151). The following sequential rules have been established: GlcNAc-T I must act before GlcNAc-T II, III and IV; GlcNAc-T II, IV and V cannot act after GlcNAc-T III, i.e., on bisected substrates; GlcNAc-T VI can act on both bisected and non-bisected substrates; both Glc-NAc-T I and II must act before GlcNAc-T V and VI; GlcNAc-T V cannot act after GlcNAc-T VI. GlcNAc-T V is the only enzyme among the 6 transferases cited above which can be essayed in the absence of Mn2+. In studies on the possible functional role of N-glycan branching, we have measured GlcNAc-T III in pre-neoplastic rat liver nodules (S. Narasimhan, H. Schachter and S. Rajalakshmi (1988) J. Biol. Chem. 263, 1273-1281). The nodules were initiated by administration of a single dose of carcinogen 1,2-dimethyl-hydrazine.2 HCl 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. The nodules had significant GlcNAc-T III activity (1.2-2.2 nmol/h/mg), whereas the surrounding liver, regenerating liver 24 h after partial hepatectomy and control liver from normal rats had negligible activity (0.02-0.03 nmol/h/mg). These results suggest that GlcNAc-T III is induced at the pre-neoplastic stage in liver carcinogenesis and are consistent with the reported presence of bisecting GlcNAc residues in N-glycans from rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (A. Kobata and K. Yamashita (1984) Pure Appl. Chem. 56, 821-832).     

Changes in galactosyltransferase activity in chick pectoral muscle during embryonic development.

The two major vertebrate galactosyltransferases have been investigated in developing chick muscle in ovo and in vitro, and in cultured chick fibroblasts. The two enzymes were UDP-galactose-N-acetylglucosamine galactosyltransferase (galactosyltransferase I) and UDP-galactose-N-acetylgalactosamine galactosyltransferase (galactosyltransferase II). Both activities fell during muscle development in ovo. Galactosyltransferase I activity was constant from day 7 to day 16, after which it declined 5-fold, whereas galactosyltransferase II activity fell markedly from day 9 to 13 and 16 to 20, displaying an overall 8-fold decrease. In primary muscle cultures, galactosyltransferase I activity fell slightly during 7 days in culture, whereas galactosyltransferase II increased 2-fold during the same period. No significant change in activity of either galactosyltransferase was observed during intercellular recognition and fusion. Analysis of muscle cultures treated with cytosine arabinoside and of fibroblast cultures revealed that the majority of galactosyltransferase I activity in primary muscle cultures is associated with fibroblasts, whereas the majority of galactosyltransferase II activity is muscle-associated. The addition of 5-bromodeoxyuridine to primary muscle cultures resulted in a 3-fold rise in activities of both transferases.     

N—乙酰氨基葡萄糖转移酶Ⅲ,Ⅳ及Ⅴ在7721人肝癌细胞周期中的变化

In order to investigate the changes of N-acetylglucosaminyl transferase (GlcNAc-T) III, IV and V in cell cycle, the synchronization of 7721 human hepatocellular carcinoma cells was performed using serum hunger method. The percentages of cells in different phases during cell cycle were measured by flow cytometry (FCM) and the cell cycle was checked by determining the activity of cellular p34cdc2 kinase. It was found that the activities of GlcNAc-T III increased in G0/G1 cell peak phase and had correlation with the cell percentage of G0/G1 phase (r = 0.760, P < 0.05), while GlcNAc-T V showed the highest activity when G2/M cells were most abundant and had an apparent correlation with the cell percentage of G2/M phase (r = 0.868, P < 0.001). The changes of GlcNAc-T IV activity seemed not related to the cell cycle. The changes in opposite directions of relative activities (percentage of total GlcNAc-T III, IV, V) of GlcNAc-T III and GlcNAc-T V were observed during cell cycle (r = -0.951, P < 0.001), suggesting that these two enzymes might be regulated differently and functioned oppositely in the cells: GlcNAc-T V may be related to the proliferation of 7721 cells, while GlcNAc-T III may be related to the non-mitotic silence phase of the cells, or, it may be a factor against proliferation. Immunohistochemical results showed that the protein content of GlcNAc-T V was not significantly changed during cell cycle, and had no correlation with the activity of GlcNAc-T V, suggesting that the changes of GlcNAc-T V activity in cell cycle might not be resulted from the alteration of enzyme protein synthesis. The correlation between the activities of GlcNAc-T V and p34cdc2 kinase (r = 0.752, P < 0.05) was observed in cell cycle, implicating that GlcNAc-T V might possibly be regulated by p34cdc2 kinase.     

Beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase III activity in human B and T lymphocyte lines and in tonsillar B and T lymphocytes

beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase III (GlcNAc-T III) catalyzes the incorporation of a "bisecting" N-acetylglucosamine (GlcNAc) residue in beta 1-4 linkage to the beta-linked mannose of the core of asparagine linked-protein bound oligosaccharides (N-glycans). The activity of GlcNAc-T III was determined in Triton X-100 extracts of four human Epstein-Barr virus (EBV)-infected B-cell lines, in four T-cell lines originally established from lymphocytes of patients with acute lymphatic leukemia, and in human tonsillar B and T lymphocytes. The four EBV-transformed B-cell lines showed appreciable GlcNAc-T III activities (ranging from 3.4 to 19.0 nmol.h-1.mg protein-1), while the tonsillar resting B lymphocytes had much less activity (0.68 nmol.h-1.mg protein-1). The four T-cell lines and the tonsillar T lymphocytes had negligible GlcNAc-T III activities (ranging from 0.02 to 0.25 nmol.h-1.mg protein-1). Enzyme product was identified by high resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. This is the first demonstration of GlcNAc-T III activity in human lymphocytes. The presence of GlcNAc-T III in B-cell lines correlates with the reported occurrence of bisecting GlcNAc residues in the oligosaccharides of human immunoglobulins G, A1, M, and D, tonsillar class II antigens, and membrane glycoproteins from B lymphocytes. The negligible GlcNAc-T III activity of the four human T-cell lines and of tonsillar T lymphocytes agrees with the reported absence of bisected structures in N-glycans from human T lymphocyte membrane glycoproteins.     

Heterogeneity in the replicating population of cultured human epidermal keratinocytes

We studied the replication of keratinocytes in stratified squamous epithelia. Other studies have revealed functional and morphological heterogeneity in the replicating population of such cells. To examine possible kinetic heterogeneity, we determined the cell-cycle lengths of replicating cells in cultures of human epidermal keratinocytes. A double-label assay was developed, which measures the time between two successive cycles of DNA synthesis. The first cycle of DNA synthesis was marked by pulse labeling cultures for a brief period with 14C-thymidine (dThd), and the second cycle was detected by labeling at a later time with bromodeoxyuridine (BrdUrd). The time taken for the 14C-labeled DNA to become doubly labeled with BrdUrd was shown to correspond to the length of the cell cycle. In subconfluent cultures in which the cell number increased at an exponential rate, the average cell-cycle time was 21.5 h. In confluent cultures in which desquamation was balanced by cell renewal, the average cell cycle was 31.5 h. However, in confluent cultures, three populations of replicating cells were evident, these having cycle times of 22, 33, and 40 h. In subconfluent cultures, there was no clear evidence for cell-cycle heterogeneity of the replicating cells, although the most rapidly cycling cells in these cultures had a cycle time (16 h) considerably less than the most rapidly cycling cells in the confluent cultures (21 h). It is possible that the rapidly cycling cells seen in the subconfluent cultures were stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of sodium-linked nucleoside transport activity in monolayer cultures of IEC-6 intestinal epithelial cells

Mature, confluent monolayer cultures of IEC-6 rat intestinal epithelial cells in conventional growth media express both Na(+)-linked, concentrative nucleoside transport (NT) activity and equilibrative, inhibitor-sensitive NT activity, but do not show morphologic differentiation. Na(+)-dependent fluxes of Ado and formycin B were minor in early subconfluent IEC-6 monolayers, but increased severalfold to become the major component of influx of these agents in confluent monolayers grown in medium containing Nu-Serum, a commercial medium supplement with a low serum content. In monolayers cultured in medium with fetal bovine serum, cell proliferation rates were similar to those in medium supplemented with Nu-Serum, but expression of Na(+)-linked NT activity was 6-8-fold lower than in monolayers grown in the latter medium. Inclusion of hydrocortisone in growth medium with Nu-Serum caused a 2-fold increase in the expression of Na(+)-linked NT activity. Experiments in which components of medium supplementation were withheld showed that insulin and epidermal growth factor were important in expression of the Na(+)-linked NT activity. Because the Na(+)-linked NT system has a brush border location in fresh intestinal epithelium, it is concluded that the regulated expression of this activity in the IEC-6 monolayers is a differentiative change.     

Differential expression of cytochrome P-450 in proliferating and quiescent cultures of murine lung epithelial cells.

Expression of the cytochrome P-450 monooxygenase activity 7-ethoxyresorufin O-deethylase (7-ERD) was surveyed in proliferating and quiescent cultures of murine cell line C-10, a non-tumorigenic line of presumed alveolar type II origin. 7-ERD activities were undetectable in subconfluent/proliferating cultures but became detectable once the cultures had become confluent and their growth had arrested due to contact inhibition. Serum deprivation of subconfluent cultures resulted in a rapid inhibition of cell proliferation and the subsequent expression of 7-ERD. These results suggest that 7-ERD expression is regulated as a function of the proliferative status of C-10 cells.