Stimulation of protein kinase C activity by tumor necrosis factor-alpha in bovine bronchial epithelial cells

Todd A. Wyatt, Harumasa Ito, Thomas J. Veys, and John R. Spurzem

Research Service, Department of Veterans Affairs Medical Center, Omaha 68105; and Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Bronchial epithelial cell migration, attachment, and proliferation are important processes in response to airway injury. We have shown that tumor necrosis factor (TNF)-alpha stimulates the migration of bovine bronchial epithelial cells (BBEC) in vitro. We hypothesized that protein kinase C (PKC) may be one of the intracellular signaling mediators of TNF-alpha in BBEC. In this study, we have identified multiple PKC isoforms in BBEC and measured total cellular PKC activity. Polyclonal antibodies to the PKC-alpha , -beta 2, -delta , and -epsilon isoforms recognized protein bands around 80-90 kDa. BBEC primary cultures treated with either 500 U/ml TNF-alpha for 2-4 h or 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate for 15 min resulted in three- to fivefold increases in PKC activity in the particulate fractions of crude cell lysates. This activity was inhibited by 1 µM calphostin C or 10 µM H-7. Similarly, TNF-alpha -stimulated BBEC migration was reduced at least twofold in the presence of H-7 or calphostin C. These studies suggest that the activation of PKC is necessary for TNF-alpha -stimulated BBEC migration.

protein kinase C isoenzymes; epithelial cell migration; enzyme activation

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

BRONCHIAL EPITHELIAL CELL migration, attachment, and proliferation are important processes in the physiological response to wound healing as well as in the pathogenic response to such lung diseases as chronic bronchitis and asthma. Various growth factors and cytokines have been identified that modulate epithelial cell growth rates, extracellular matrix production, and cellular morphology. Dissecting the molecular physiology of those enzymes that regulate signal transduction is essential to understanding the relationship between pharmacological stimuli and the morphological responses observed in these cells.

Protein kinase (PK) C is stimulated by numerous calcium-elevating agents and tumor promoters and is a major activator of the serine-threonine phosphorylation pathway (19). PKC is represented by a family of isoenzymes that have been identified to coexist in many cell types (3, 8). Some of these isoforms have been demonstrated to be calcium and/or phospholipid independent (11). Therefore, each isoenzyme, although similar in structure and sequence, may play a unique role in a compartmentalized state within the cell.

Immune reactions are effected by soluble cytokine mediators such as tumor necrosis factor (TNF; see Ref. 1). These cytokines are produced primarily, but not exclusively, by monocytes and act upon their target cells to induce further proinflammatory mediators. Two classes of TNF-alpha receptors (high and low affinity) are found to exist ubiquitously throughout most tissues, although the relationship between these receptors and the molecular mechanism of TNF-alpha signal transduction is unknown.

It has been suggested that PKC is one of the intracellular signaling mediators of TNF-alpha effects (22). However, it has also been reported that cell treatment with TNF-alpha results in the activation of adenylate cyclase with an accompanying increase in adenosine 3',5'-cyclic monophosphate production (27). We have previously shown that TNF-alpha stimulates the migration of bovine bronchial epithelial cells (BBEC) in cultured monolayers (12), but the signal transduction mechanisms for cytokine-mediated bronchial epithelial cell migration are not understood, and the role of PKC in these cells has not been investigated extensively. It is our hypothesis that PKC activation plays a role in the cytokine modulation of bronchial epithelial cell migration.

We investigated the relationship between in vitro bronchial epithelial cell migration and PKC activity using kinase inhibitors, immunocytochemical methods, and direct assay of kinase activity. We have identified, for the first time, several PKC isoforms in BBEC and correlated BBEC PKC activity with the activation of cell migration in response to the bioactive cytokine TNF-alpha .

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Reagents. Laboratory of Human Carcinogenesis (LHC) basal medium was purchased from Biofluids (Rockville, MD). RPMI 1640, Dulbecco's modified Eagle's medium (DMEM), minimum essential media (MEM), streptomycin-penicillin, and fungizone were purchased from GIBCO (Chagrin Falls, OH). Extraction of frozen bovine pituitaries from Pel Freez (Rogers, AR) was performed as previously described and yielded an extract containing 10 mg/ml protein (15). Recombinant human TNF-alpha was purchased from Genzyme (Cambridge, MA). Purified PKC, PKC isoform marker peptides, and polyclonal rabbit antisera to the PKC isoenzymes were obtained from Calbiochem (San Diego, CA). Peroxidase-conjugated goat anti-rabbit immunoglobulin G was purchased from Cappel (Durham, NC). All other reagents not specified were purchased from Sigma Chemical (St. Louis, MO).

Cell preparation. As previously described (25), the cells were prepared from bovine lung obtained fresh from a local abattoir. Bronchi were necropsied from the lung, cleaned of adjoining lung tissue, and incubated overnight at 4°C in 0.1% bacterial protease (type IV) in MEM. After the overnight incubation, the bronchi were rinsed in DMEM with 10% fetal calf serum repeatedly to collect the cells lining the lumen. These cells were then filtered through a 250-µm nylon mesh and were washed again in DMEM. This technique typically produces a high-viability cell preparation of >95% epithelial cells (24). The cells were then washed in DMEM, counted with a hemacytometer, and plated in 1% collagen-coated 100-mm polystyrene culture dishes at a density of 1 × 104 cells/cm2 in a 1:1 medium mixture of LHC-9 and RPMI (15). Cell incubations were performed at 37°C in humidified 95% air-5% CO2. Confluent monolayers of cells were obtained every 3 days. At this time, each 100-mm dish contained ~2 mg of total cellular protein. Primary cultures of BBEC were used for these studies because it has been suggested that tissue culture artifacts may induce the downregulation of enzyme activity in the late-passaged cell (7).

DEAE chromatography of BBEC fractions. Because the relative amounts of some PKC isoforms may exist in concentrations beyond detectability by Western blot of crude whole cell fractions, we attempted large-scale fractionation and concentration of PKC by ion-exchange chromatography. Chromatography of PKC isoforms was performed as follows. Confluent monolayers of BBEC (>1 × 108 cells) were extracted in 10 ml of buffer containing 10 mM KH2PO4, 1 mM EDTA, and 25 mM 2-mercaptoethanol (KPEM). The cells were sonicated and centrifuged at 10,000 g for 30 min at 4°C. The pellet fraction was sonicated further in 12 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and 0.1% Triton X-100. Both supernatants were applied to a DEAE-Sephacel column (0.9 × 10 cm) equilibrated in KPEM buffer. The column was washed with 100 ml of KPEM and was developed with a 30-ml NaCl linear gradient (0-500 mM), and 1-ml fractions were collected. Individual fractions collected were analyzed for protein content [by the method of Bradford (5)], NaCl concentration, and immunoreactivity for PKC isoforms.

Western blotting. PKC distribution was determined by Western blot of BBEC subcellular fractions. After stimulation of the cells, the medium was removed, and the cells were sonicated (20 s, 1 pulse) at 4°C in 0.5 ml of lysis buffer consisting of 35 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.4), 0.2 mM phenylmethylsulfonyl fluoride, 10 mM MgCl2, 10 mM 2-mercaptoethanol, and 5 µg/ml leupeptin. The lysis buffer also contained 3 mM CaCl2 or 9 mM EGTA in the presence or absence of 1% Triton X-100, depending upon which fraction was being homogenized. Sonicates were then centrifuged for 30 min at 10,000 g at 4°C. Cell fractions were solubilized in sodium dodecyl sulfate (SDS)-2-mercaptoethanol reducing buffer, and equal amounts of protein per sample (20-200 µg) were resolved on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Transferred gels were stained with Coomassie R-250 to determine completeness of transfer. Blots were blocked overnight with a blocking buffer of 3% bovine serum albumin in 50 mM Tris (pH 7.5), 150 mM NaCl, and 0.1% NaAz at 4°C with constant rocking. Membranes were incubated with rabbit anti-PKC-alpha , -beta 1, -beta 2, -gamma , -delta , -epsilon , -theta , -eta , or -zeta (1:2,000 dilution in blocking buffer) for 1 h at room temperature. As a control, membranes were incubated in either rabbit preimmune serum or in the absence of primary antibody. Membranes were washed three times in blocking buffer with 0.02% Nonidet P-40 for 20 min during each wash. Blots were incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody diluted 1:10,000 in blocking buffer for 30 min at room temperature. Membranes were washed again as described, and immunoreactivity was visualized by chemiluminescence as detected on autoradiographic film. Any immunoreactive bands were identified as occurring in the cytosolic or particulate (membrane) fraction, and their relative molecular masses (kDa) were calculated for each isoform blotted.

PKC activity assay. PKC activity was determined in both DEAE fractions as well as in crude whole cell fractions of bronchial epithelial cells. The assay employed was a modification of procedures previously described (9, 10) using 50 µg/ml PKC substrate peptide, 12 mM Ca(C2H3O2)2, 8 µM phosphatidyl-L-serine, 24 µg/ml 12-O-tetradecanoyl-phorbol-13-acetate (PMA), 30 mM dithiothreitol, 150 µM ATP, 45 mM Mg(C2H3O2)2, and 10 µCi/ml [gamma -32P]ATP in a Tris · HCl buffer (pH 7.5). Samples (20 µl) were added to 40 µl of the above reaction mixture and were incubated for 15 min at 30°C. Incubations were halted by spotting 50 µl of each sample onto P-81 phosphocellulose papers (Whatman). Papers were then washed five times for 5 min each in phosphoric acid (75 mM) and one time in ethanol and then were dried and counted in nonaqueous scintillant as previously described (21). Negative controls consisted of similar assay samples with 12 mM EGTA or without substrate peptide. A positive control of 0.35 mg/ml purified rat brain PKC (Calbiochem) was included as a sample. Kinase activity was expressed in relation to total cellular proteins assayed and was calculated in picomoles per minute per milligram. All samples were assayed in triplicate, and no less than three separate experiments were performed per unique parameter. Data were analyzed for statistical significance using Student's t-test.

Bronchial epithelial cell migration. Bronchial epithelial cell migration assay was performed with the Boyden chamber technique (4, 6, 20, 23) using a 48-well multiwell chamber (Neuroprobe, Bethesda, MD). Polycarbonate membranes with 8-µm pores (Neuroprobe) were used. Membranes were coated with 0.1% gelatin (Bio-Rad, Richmond, CA) as previously described (6, 20, 23). Varying concentrations of fibronectin were used in the bottom wells as attractants. Bronchial epithelial cells were placed into each of the top wells above the filter. The chambers were then incubated at 37°C with 5% CO2 for 6 h. After incubation, cells on the top of the filter were removed by scraping. The filter was then stained with a modified Wright stain (Leukostat; Fisher Scientific, Fairlawn, NJ). Epithelial cell migration activity was quantified as the number of migrated cells on the lower surface of the filter in 10 high-power fields (HPF) using a light microscope at ×400 magnification. The results represent means ± SE for triplicate wells.

As an additional control, the above assays incorporated the treatment of epithelial cells with various concentrations of TNF-alpha or PMA in the presence of PKC inhibitors (H-7 and calphostin C) for various time points. Controls included unstimulated cells and incubation of cells with PKC inhibitors without TNF-alpha or PMA stimulation. Triplicates were performed for each assay. Data are expressed as mean ± SE cells migrated per HPF from triplicates assayed. Statistical significance was determined by Student's t-test and was considered significant at P < 0.01.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Identification of PKC isoforms by Western blot. We have utilized Western blotting to identify the various PKC isoforms expressed in DEAE-Sephacel fractions of primary BBEC. Monolayers of BBEC were lysed and Triton extracted, and supernatants were loaded onto a DEAE-Sephacel column. The column was then developed in a linear NaCl gradient of 0-0.5 M. The subsequent chromatograph revealed three distinct peaks of PKC activity eluting at ~0.1, 0.3, and 0.4 M NaCl. Utilizing polyclonal antibodies to the specific unique peptide regions of nine different PKC isoenzymes, we have identified the presence of alpha -, beta 2-, and delta -isoforms as major immunoreactive bands ranging in molecular mass from 78 to 82 kDa in the BBEC (Fig. 1). Additionally, we have identified a 90-kDa immunoreactive band for the epsilon -isoform (Fig. 1). The presence of multiple bands within the same cell type was not surprising and has been previously reported in other cell types (3, 14, 17).


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Fig. 1.   Western blot for protein kinase C (PKC) isoenzymes present in bovine bronchial epithelial cells (BBEC). Polyclonal antibodies raised against specific-peptide amino-terminal regions of various PKC isoenzymes were used to detect those isoforms present in DEAE-Sephacel chromatography fractions of fresh primary BBEC. The presence of major bands recognized by antibodies to alpha  (82 kDa)-, beta 2 (80 kDa)-, delta  (78 kDa)-, and epsilon  (90 kDa)-isoforms were identified by this technique. Positive control (Peptide) consists of purified peptide antigen corresponding to the specific isoform probed. Negative control (No 1° Ab) consists of the same BBEC protein fractions probed without the primary antibody to the specific PKC isoform.

PKC activation assays. In an attempt to identify PKC activity in crude cell fractions, we have assayed PKC activity in cytosolic and particulate fractions of BBEC. Primary cultures of BBEC were treated with various concentrations of TNF-alpha (0-500 U/ml) for various time points (2-48 h) in multiple 100-mm tissue culture dishes. The cells were then separated into a cytosolic fraction and a detergent-extracted fraction from the particulate. Both fractions were assayed for PKC kinase activity. PKC activity changes in TNF-alpha -stimulated epithelial cells were compared with unstimulated control cells. We determined the concentration curve for this TNF-alpha -mediated response to maximally stimulate PKC activity at a concentration of 500 U/ml (Fig. 2). At this concentration of TNF-alpha , no significant cell death was observed because BBEC viability was determined to be >96% by trypan blue exclusion assay.


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Fig. 2.   PKC activity concentration curve in whole cell lysates of BBEC stimulated with tumor necrosis factor (TNF)-alpha for 2 h. Cells were fractionated by sonication and were separated into a 10,000-g supernatant cytosolic fraction and a Triton-extracted pellet particulate fraction. Maximal stimulation of PKC translocation to the particulate fraction occurs in cells treated for 2 h with 500 U/ml TNF-alpha .

Additionally, we observed that a time-dependent translocation (2-4 h) of peak PKC activity occurs in primary cultures of BBEC treated with 500 U/ml TNF-alpha from the cytosol to the particulate fraction (Fig. 3). TNF-alpha -stimulated PKC activity reached its maximal levels from 2 to 4 h and subsided to near-unstimulated levels by 24 h. We observed that PKC inhibitors such as calphostin C and H-7 abrogate this TNF-alpha -mediated PKC activity (Fig. 4). As expected, calphostin C (1 µM) completely inhibited the activity of PKC in both the cytosolic and particulate fractions, whereas H-7 (10 µM) reduced PKC activity well below unstimulated resting cells. At these concentrations of calphostin C and H-7, no significant cell death was observed by the cellular uptake of trypan blue dye (viability >92%). Compared with early primary cells (>1 wk old), older cultures of BBEC (<2 wk old) as well as passaged cells demonstrate a distinct loss of particulate fraction PKC activity in response to TNF-alpha stimulation (data not shown).


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Fig. 3.   PKC activity time course in whole cell lysates of primary BBEC stimulated with TNF-alpha . Cells were fractionated by sonication and were separated into a 10,000-g supernatant cytosolic fraction and a Triton-extracted pellet particulate fraction. Stimulation of cells with 500 U/ml TNF-alpha results in localization of the greatest kinase activity to the particulate fraction by 60 min.


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Fig. 4.   Inhibition of TNF-alpha -stimulated PKC activity in whole cell lysates of BBEC by PKC inhibitors. Cytosolic and particulate BBEC fractions were assayed for PKC activity in the presence and absence of the PKC inhibitors H-7 and calphostin C (CP). Kinase activity was significantly reduced in both unstimulated cells (R) and cells treated with 500 U/ml TNF-alpha for 2 h in the presence of 1 µM calphostin C or 10 µM H-7.

As a positive control, cells were treated with PMA (100 ng/ml for 15 min) to activate PKC. We observed a significant increase in PKC activity in the particulate fraction of BBEC treated with PMA compared with the cytosolic fraction, suggesting that a translocation of PKC from the cytosol to a component of the particulate fraction was occurring in those BBEC under conditions of activated PKC (data not shown).

TNF-alpha -stimulated BBEC migration. To examine the role of PKC in TNF-alpha -stimulated bronchial epithelial cell migration to fibronectin, bronchial epithelial cells were treated with TNF-alpha for various times and were evaluated for migration in Boyden blind-well chamber assays. Bronchial epithelial cells treated with TNF-alpha for 4 h before assay showed significantly increased migration to fibronectin compared with control cells (Fig. 5). Exposure of the cells for up to 24 h with TNF-alpha resulted in the stimulation of increased BBEC migration to fibronectin. However, long-term stimulation of the cells with TNF-alpha (24-50 h) led to a significant decrease in cell migration compared with 4 h of TNF-alpha treatment. Similar to the inhibition of PKC activity, BBEC pretreated with the PKC inhibitors H-7 (10 µM) or calphostin C (1 µM) for 30 min before TNF-alpha stimulation resulted in a 50% reduction in cell migration (Fig. 6). Incubation of BBEC with these inhibitors alone resulted in no significant change in migration from control unstimulated cells.


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Fig. 5.   Effect of TNF-alpha on BBEC migration to fibronectin. Bronchial epithelial cells were treated with TNF-alpha (500 U/ml) for 2-48 h, and cells were incubated for migration to fibronectin (30 µg/ml) for 6 h at 37°C. Bronchial epithelial cells treated with TNF-alpha for 4 and 24 h showed an increase in migration. Values are means ± SE of 3 experiments. * P < 0.005 compared with control. HPF, high-power field.


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Fig. 6.   Effect of PKC inhibitors on TNF-alpha -stimulated BBEC migration to fibronectin. Bronchial epithelial cells were pretreated with 10 µM H-7 or 1 µM calphostin C for 30 min before treatment with TNF-alpha (500 U/ml) for 2 h. Cells were then incubated for migration to fibronectin (30 µg /ml) for 6 h at 37°C in the presence or absence of PKC inhibitors. Both H-7 and calphostin C inhibited migration in BBEC treated with TNF-alpha . Values are means ± SE of 3 experiments. * P < 0.05 compared with control.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The presence of multiple PKC isoforms in BBEC may represent a multifaceted role for both calcium-dependent and calcium-independent PKC signaling in highly compartmentalized BBEC. Heterogeneous subpopulations of BBEC may exist in our cultures, each containing unique isoenzymes. However, the presence of various numbers of ciliated and nonciliated BBEC in these primary cultures has not contributed to significant differences in the magnitude or response of our BBEC cultures to TNF-alpha -mediated PKC activation. Continued studies designed to answer this question consist of immunofluorescence microscopy of both primary cultures of BBEC as well as immunohistochemistry of Formalin-fixed bronchial thin sections utilizing antibodies to the PKC isoforms.

Our data suggest the presence of at least two distinct peaks of calcium-dependent kinase activity in the elution profile of BBEC DEAE fractions. These peaks may represent the distinct elution of the calcium-dependent isoform-alpha and -beta 2. Another smaller peak of PKC activity corresponding to PKC-delta demonstrated calcium-independent activity. The identification of PKC-epsilon primarily comes from Western blots of crude cell lysates. This isoform appears to be localized to the particulate fraction, possibly the cytoskeleton, and therefore may be lost in the pellet fraction before the DEAE column was loaded. It has been previously reported that certain PKC isoforms are localized on the microfilaments and intermediate filaments (18).

The presence of conventional PKC isoforms is also demonstrated by the calcium-dependent kinase activity observed in BBEC extracts. The presence of PKC-alpha and PKC-beta 2 isoforms could be responsible for the translocatable PKC activity observed in the particulate fractions of BBEC stimulated with TNF-alpha . Additionally, such isoforms as PKC-delta and PKC-epsilon could be responsive to phorbol ester stimulation. Maximal or peak PKC activity is detected in the particulate fractions of cells treated with the phorbol ester PMA or with TNF-alpha . This suggests that a translocatable form of PKC is present in the BBEC, which may represent alpha - and/or beta 2-enzyme activity in the BBEC. However, this study does not establish that translocation of one or more PKC isoforms is entirely responsible for TNF-alpha -stimulated migration. We feel that the coincident activation of PKC with the stimulation of migration and the inhibition of that migration by PKC inhibitors link the requirement of PKC activation with TNF-alpha -stimulated migration. Whether or not the targeting of specific isoforms to specific regions of the cells occurs must be addressed in a future study.

The time of maximal BBEC-stimulated migration (~4-6 h) correlates well with the time of peak PKC particulate fraction activity. However, as PKC activity begins to subside after 6 h of continual stimulation with TNF-alpha , migration of the cells to fibronectin continues at an increased rate up to 24 h of TNF-alpha treatment. Inhibition of PKC activity by calphostin C or H-7 resulted in the inhibition of TNF-alpha -stimulated migration. Together, these data suggest that PKC activation is required for migration to be initiated in response to TNF-alpha . PKC activation is apparently required as an upstream initiator of BBEC migration but is not necessary for continued long-term migration in response to TNF-alpha . Although TNF-alpha has been shown to activate other kinases in other cell types, we found no inhibition of TNF-alpha -stimulated cell migration in the presence of the protein tyrosine kinase inhibitor genistein (25-100 µM) nor did we detect any TNF-alpha -stimulated activation of PKA or PKG in our control studies (data not shown).

When BBEC are stimulated with TNF-alpha over long periods of time (24-48 h), the rate of cell migration to fibronectin begins to decrease. TNF-alpha receptor shedding may play a role in this longer time course TNF-alpha insensitivity. It has been reported that normal human airway epithelial cells shed soluble type I TNF-alpha receptors upon activation of PKC by PMA (16). Levine et al. (16) demonstrated that maximal TNF-alpha receptor shedding occurred at 24 h of stimulation by PMA and that receptor shedding could be halted by PKC inhibitors. This delayed loss of TNF-alpha receptors was observed despite the fact that PKC was most likely fully activated within minutes of PMA treatment. Our findings in BBEC support those of Levine et al. (16). Activation of PKC by TNF-alpha may result in an eventual decrease in PKC activity over a period of several hours due to the downregulation of the TNF-alpha receptor. Thus the very signal produced by TNF-alpha stimulation (PKC activation) may lead to the downregulation of TNF-alpha responsiveness by stimulating TNF-alpha receptor shedding. As the maximal amount of TNF-alpha receptors are shed beginning at 24 h, TNF-alpha is no longer able to stimulate BBEC migration, and the decrease in cell migration to fibronectin is observed at these extended time points.

Alternatively, the loss of TNF-alpha -stimulated cell migration over very long periods of time could be explained through the loss of expressed PKC activity in BBEC. It has been previously reported that BBEC extrude filopodia in response to PKC-activating agents such as phorbol esters and calcium ionophores (2). It was noted that a steady loss in the filopodia-inductive response occurred over the first several days in culture. Although no kinases were assayed, the loss of phorbol ester-stimulated filopodia could be the result of a downregulation of PKC expression or a loss of PKC activity. The loss of an enzyme activity over the course of several passages of certain cultured cell lines has been previously reported in numerous studies because cell adaptation is often associated with changes in specific proteins (7). In this report, our studies were limited to primary cultures of BBEC used at ~3 days ex vivo. However, we have observed diminished PKC activity in response to TNF-alpha as well as to PMA in passaged cells and in cells that are >1 wk old in culture (unpublished observation). This possible artifact of tissue culture leading to decreased PKC activity over time may be involved in the decreased stimulation of cell migration over several days of TNF-alpha treatment.

The widespread application of kinase inhibitors instead of direct kinase activity assays has often led to a lack of kinase specificity and cytotoxicity in the cellular model (13, 26). Because of these limitations, we have chosen to measure the PKC activation state of BBEC in the presence of both TNF-alpha and PKC inhibitors rather than only assaying BBEC migration in the presence of PKC inhibitors. As isoform-specific substrates and inhibitors become available, it will be important to dissect the role of these PKC isoenzymes in regulating TNF-alpha -stimulated migration, receptor shedding, and substrate phosphorylation. Until activity assays specific for individual PKC isoforms can be performed, it is difficult to make any quantitative comparison of isoform activity within the intact cell in response to an agent such as TNF-alpha . Although the separation of the individual isoforms is possible by immunoprecipitation, our preliminary studies have shown that the binding of specific antibody to PKC isoforms inhibits PKC activity (data not shown).

This report represents the initial observation of multiple PKC isoforms in airway epithelial cells and the composite PKC activity in response to TNF-alpha stimulation. Our data also show that inhibition of PKC activity results in the inhibition of BBEC migration to fibronectin in response to TNF-alpha . This observation, combined with previous studies of other PKC effects on the airway epithelial cell (i.e., receptor shedding), suggests a multifunctional role for multiple PKC isoenzymes found in the BBEC. Elucidating those roles for PKC in the bronchial epithelial cell may contribute to a better understanding of how cytokines affect the airway cells in both the physiological and pathological states. Understanding the mechanisms that regulate epithelial cell migration are tantamount to elucidating the processes of airway injury and repair. These mechanisms are thought to reflect complex interactions between the inflammatory cells, proinflamatory cytokines, and epithelial cells. Determining which intracellular kinases are involved in the responses of bronchial epithelial cells to cytokines and under what conditions those kinases are activated may lead to the development of pharmacological agents that can be employed to modulate such responses at the level of phosphate signaling.

    ACKNOWLEDGEMENTS

This work was supported by a Department of Veterans Affairs Merit Review grant (to J. R. Spurzem) and by the American Lung Association (T. A. Wyatt).

    FOOTNOTES

Address for reprint requests: T. A. Wyatt, Dept. of Internal Medicine, Pulmonary and Critical Care Medicine Section, University of Nebraska Medical Center, 600 S. 42nd St., Omaha, NE 68198-5300.

Received 9 January 1997; accepted in final form 7 August 1997.

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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 273(5):L1007-L1012
1040-0605/97 $5.00 Copyright © 1997 the American Physiological Society




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