IL-1beta enhances beta 2-adrenergic receptor expression in human airway epithelial cells by activating PKC

Wei Bin, Mark O. Aksoy, Yi Yang, and Steven G. Kelsen

Pulmonary Division, Department of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania 19140


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Protein kinase C (PKC)-activated signal transduction pathways regulate cell growth and differentiation in many cell types. We have observed that interleukin (IL)-1beta upregulates beta 2-adrenergic receptor (beta 2-AR) density and beta 2-AR mRNA in human airway epithelial cells (e.g., BEAS-2B). We therefore tested the hypothesis that PKC-activated pathways mediate IL-1beta -induced beta -AR upregulation. The role of PKC was assessed from the effects of 1) the PKC activator phorbol 12-myristate 13-acetate (PMA) on beta -AR density, 2) selective PKC inhibitors (calphostin C and Ro-31-8220) on beta -AR density, and 3) IL-1beta treatment on the cellular distribution of PKC isozymes. Recombinant human IL-1beta (0.2 nM for 18 h) increased beta -AR density to 213% of control values (P < 0.001). PMA (1 µM for 18 h) increased beta -AR density to 225% of control values (P < 0.005), whereas Ro-31-8220 and calphostin C inhibited the IL-1beta -induced upregulation of beta -AR in dose-dependent fashion. PKC isozymes detected by Western blotting included alpha , beta II, epsilon , µ, zeta , and lambda /iota . IL-1beta increased PKC-µ immunoreactivity in the membrane fraction and had no effect on the distribution of the other PKC isozymes identified. These data indicate that IL-1beta -induced beta -AR upregulation is mimicked by PKC activators and blocked by PKC inhibitors and appears to involve selective activation of the PKC-µ isozyme. We conclude that signal transduction pathways activated by PKC-µ upregulate beta 2-AR expression in human airway epithelial cells.

cytokines; gene expression; airway inflammation; signal transduction; interleukin-1beta ; protein kinase C


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INTERLEUKIN (IL)-1beta , a pleiotypic cytokine released into the airway in asthma (1, 2, 7), produces a variety of effects on respiratory tract cells (6, 8, 12, 23). In particular, IL-1beta profoundly affects the expression and function of the beta 2-adrenergic receptor (beta 2-AR) system expressed by airway epithelial and smooth muscle cells (6, 8, 23). For example, IL-1beta enhances the expression of beta 2-AR protein and mRNA in airway epithelial cells (8). At the same time, IL-1beta decreases the responsiveness of these cells to a beta 2-AR agonist.

IL-1beta binds to a discrete receptor on the cell surface and activates a spectrum of second messenger pathways composed of G proteins, a variety of tyrosine and serine/threonine kinases, and transcription factors (4). The signal transduction pathways activated by IL-1beta are cell-type dependent (4, 5). In some cell types, the effects of IL-1beta are mediated by activation and translocation of protein kinase C (PKC) (3, 18). PKC is a threonine/serine kinase that, when activated, plays a key regulatory role in a variety of cellular functions such as stimulation or inhibition of growth, changes in morphology, and modulation of gene expression (3, 18, 21). In particular, PKC activation mediates a variety of effects important for the differentiated function of airway epithelial cells (11-13, 16, 24).

The purpose of this study was to test the hypothesis that PKC-activated signal transduction pathways mediate IL-1beta -induced upregulation of beta 2-AR gene expression. We also sought to characterize the spectrum of PKC isozymes expressed by human airway epithelial cells. The role of PKC was assessed from the effects of 1) the PKC activator phorbol 12-myristate 13-acetate (PMA) on beta -AR density; 2) PKC inhibitors, e.g., calphostin C and Ro-31-8220, on IL-1beta -induced beta -AR upregulation; and 3) IL-1beta on the cellular distribution of PKC isozymes. The PKC isozymes expressed by airway epithelial cells and their pattern of cellular distribution were assessed by immunoblotting of cytosolic and membrane fractions.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Cell culture. Experiments were performed on cultured BEAS-2B cells, a transformed normal human airway epithelial cell line (gift of Dr. Curtis Harris, National Cancer Institute, Bethesda, MD) (20). Cells (passages 18-32) were grown in RPMI 1640 medium (Sigma, St. Louis, MO) plus 10% FCS in 5% CO2 at 37°C until 90-100% confluent (5-6 days).

At confluence, 0.2 nM IL-1beta or 1 µM PMA was added to the culture medium for 18 h. The concentration of IL-1beta chosen was that which produced a maximum increase in beta -AR density based on previous findings by Kelsen et al. (8). Vehicle-treated cells served as controls (0.1% BSA in PBS for IL-1beta and DMSO for PMA-treated cells).

Several subsequent experiments were performed to examine 1) the time course of changes in beta -AR density and PKC activation with 0.2 nM IL-1beta or 1 µM PMA and 2) the effect of varying the duration of PMA exposure in pulse-chase experiments. In the former group of experiments, cells were exposed to 0.2 nM IL-1beta or 1 µM PMA for 2, 6, or 18 h before harvest. In the latter group of experiments, cells were exposed to 1 µM PMA for 2 or 18 h and then either returned to medium or, in the case of the 18-h exposure, harvested. In these experiments, in contrast to the time-course experiments, cells were harvested at a standard time, i.e., 18 h after first exposure to PMA.

The PKC inhibitors calphostin C and Ro-31-8220 were added to confluent cells 30 min before IL-1beta or PMA treatment (6). Because in some cell types IL-1beta mediates its effects by production of prostanoids, we assessed the contribution of cyclooxygenase (COX) by using the COX inhibitor indomethacin. Indomethacin (10 µM) was added to the medium 30 min before IL-1beta or PMA treatment.

Cells allocated for measurements of beta -AR binding were harvested with trypsin-EDTA at 37°C and washed twice with PBS. Cell number and viability were determined with a hemocytometer and trypan blue (0.4%) exclusion. Cells allocated for measurement of PKC immunoreactivity were harvested with a rubber policeman.

beta -AR density. beta -AR density was determined in cell suspensions by radioligand binding with the use of the beta -AR subtype nonselective antagonist [125I]iodopindolol ([125I]PIN; specific activity 2,200 Ci/mmol; NEN Life Sciences, Boston, MA) as previously described (8-10, 19). A single concentration of [125I]PIN that saturated specific binding sites (i.e., ~300 pM) was used. Cell aliquots (~100,000 epithelial cells) were delivered to polypropylene tubes containing 10 mM Tris · HCl, 2 mM MgCl2 buffer, pH 7.4, and [125I]PIN. Separate tubes containing these reactants plus 40 µM alprenolol, a beta -AR subtype nonselective antagonist, were used to determine nonspecific binding. Tubes were then vortexed and mechanically shaken for 120 min at 28°C. Incubations were quenched by the addition of ice-cold 10 mM Tris · HCl-2 mM MgCl2 buffer followed by filtration on Whatman GF/B glass fiber filters with a cell harvester filtration unit (Brandel Biomedical Research and Development Laboratories, Gaithersburg, MD). Activity was measured in triplicate tubes with a gamma counter (LKB Wallac model 1282, 78% efficiency). Specific binding was taken as the difference between total and nonspecific binding.

Western blot for PKC immunoreactivity. Cells (8-10 × 106) were scraped from the culture dish with a rubber policeman and placed in 800-1,000 µl of ice-cold cell lysis buffer [20 mM Tris · HCL, pH 7.5, 5 mM EGTA, 5 µg/ml of 4-(2-aminoethyl) benzenesulfonyl fluoride, 5 µg/ml of leupeptin, 1 µg/ml of aprotinin, and 1 µg/ml of pepstatin A]. Cell lysates were then homogenized by ultrasonic probe (3 × 15-s pulses). The cell lysate was centrifuged at 1,200 g for 20 s to eliminate unbroken cells. A portion of the supernatant was removed as a source of whole cell lysate, and the remainder was centrifuged at 100,000 g for 1 h at 4°C. The resulting clear supernatant was collected and taken as the cytosolic fraction. Pellets solubilized in cell lysis buffer were then taken as the membrane fraction. Protein concentration was assayed with the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA).

Protein (20-40 µg) from whole cell, cytosolic, and membrane fractions was electrophoresed on a 7.5% SDS-PAGE gel in Tris-glycine buffer at pH 8.3. Separated proteins were then transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA) at 1-A constant current (Hoefer model PS250; Bio-Rad) for 1 h in transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol). Rat brain and Jurkat cell lysates (5 µg) were used as PKC isozyme standards for all isoform-specific antibodies. Rat brain lysates were used with antibodies against PKC-µ, -beta , -beta II, -delta , -epsilon , -zeta , -lambda , and -iota . Jurkat cell lysates were used with antibodies against PKC-µ and -theta . Rat brain or Jurkat cell lysates and molecular size markers (Bio-Rad) encompassing the range of interest (72-115 kDa) were electrophoresed and transferred in parallel with the BEAS-2B cell samples. Membranes were washed with Tris-buffered saline (TBS), pH 7.5, and blocked with 5% nonfat dry milk in TBS, pH 7.5, containing 0.05% Tween 20 (TBS-T) overnight at 4°C.

The membranes were subsequently incubated with PKC isoform-specific antibodies. Mouse anti-human antibodies for PKC-alpha , -beta , -delta , -epsilon , -zeta , -lambda , -iota , -µ, and -theta were purchased from Transduction Laboratories (Lexington, KY), and rabbit anti-human antibody for PKC-beta II was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PKC-eta was not tested for because previous studies (16, 24) indicated that it was not detected in human airway epithelial cells.

Primary antibodies were diluted 1:100 in TBS-T and incubated with blots for 1 h at room temperature. The membranes were washed six times with TBS-T and incubated with 1:20,000 diluted secondary antibody for 1 h at room temperature. Goat anti-mouse IgG conjugated to horseradish peroxidase was used as a secondary antibody for all but PKC-beta II (Pierce, Rockford, IL). For PKC-beta II, a goat anti-rabbit IgG conjugated to horseradish peroxidase was used as the secondary antibody (Pierce). Membranes were washed six times in TBS-T and visualized by SuperSignal chemiluminescent substrate-stable peroxide solution (Pierce) on conventional X-ray film. Several exposure times were used to ensure that the images obtained were in the linear range of detection. Immununoreactivity was quantitated by densitometry. Equality of protein loading was assessed by staining the gel with Coomassie brilliant blue R (Pierce).

Reagents. Calphostin C was purchased from Calbiochem (La Jolla, CA). Ro-31-8220 was a gift from Dr. Christopher Hill (Roche Products, Welwyn Garden City, Herts, UK). PMA, trypsin-EDTA, FCS, and RPMI 1640 medium were purchased from Sigma. [125I]PIN was purchased from NEN/Life Sciences.

Data analysis. Group values are presented as means ± SE. beta -AR density is expressed as receptor sites per cell or as a percentage of control value when vehicle-treated cells were taken as control. Significance of differences in normalized responses of epithelial cells was assessed by the Mann-Whitney rank sum test. Significance between group means was accepted at the P < 0.05 level.


    RESULTS
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IL-1beta (0.2 nM) and PMA (1 µM) produced significant increases in beta -AR density after 18 h of exposure (P < 0.005 for both; Fig. 1) and a trend for beta -AR density to increase 6 but not 2 h postexposure (data not shown). In addition, in pulse-chase experiments, PMA exposure for 2 and 6 h produced increases in beta -AR density at 18 h similar to those observed with a full 18 h of PMA exposure. These latter experiments suggest that increasing the duration of PKC activation beyond 2 h does not enhance the magnitude of beta -AR gene expression.


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Fig. 1.   Effect of interleukin (IL)-1beta (for 18 h) and the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA; for 18 h) on beta -adrenergic receptor (beta -AR) density (n = 8 experiments/group). Values are percentages of the vehicle-treated control responses. Both agonists significantly increased beta -AR density, P < 0.005.

The PKC inhibitors calphostin C and Ro-31-8220 both inhibited IL-1beta -induced beta -AR upregulation in a dose-dependent fashion (P < 0.001 for both; Figs. 2 and 3). Ro-31822 had no effect at concentrations of 0.1 and 1 µM but profoundly decreased IL-1beta expression at a concentration of 10 µM (Fig. 2). In contrast, calphostin C inhibited beta -AR upregulation at concentrations of 1 and 3 µM (Fig. 4). In contrast to the effect of PKC inhibitors, indomethacin at a concentration of 10 µM had no effect on beta -AR density (P > 0.5; data not shown).


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Fig. 2.   Effect of increasing concentrations of Ro-31-8220 on IL-1beta -induced beta -AR upregulation (n = 7, 3, 3, and 6 experiments for IL-1beta alone and 0.1, 1.0, and 10 µM Ro-31-8220 treatments, respectively). Ro-31-8220 inhibited IL-1beta -induced beta -AR upregulation in dose-dependent fashion, P < 0.001.



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Fig. 3.   Effect of increasing concentrations of calphostin C on IL-1beta -induced beta -AR upregulation (n = 8, 6, and 5 experiments for IL-1beta alone and 1.0 and 3.0 µM calphostin C treatments, respectively). Calphostin C inhibited IL-1beta -induced beta -AR upregulation in a dose-dependent fashion, P < 0.001.



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Fig. 4.   Effect of IL-1beta on PKC isozyme immunoreactivity in cytosolic (CYT) and membrane (MEM) fractions from BEAS-2B cells (1 experiment representative of 5). Results of several selected isozymes are shown to indicate the various isozyme-specific results. Rat brain or Jurkat cell lysates were used as standards. As shown in vehicle-treated cells (control), the PKC isoforms alpha , iota , zeta , and µ were variously distributed in BEAS-2B cells in the cytosolic fraction (e.g., alpha ) or in both membrane and cytosolic fractions (e.g., µ, iota , and zeta ). IL-1beta increased PKC-µ immunoreactivity in the membrane compartment. IL-1beta had no effect on the immunoreactivity or cellular distribution of any of the other PKC isozymes shown.

PKC isozymes expressed in airway epithelial cells. BEAS-2B cells expressed a spectrum of PKC isozymes (Table 1). Isozymes detected included species from the conventional (alpha , beta II), novel (epsilon , µ), and atypical (zeta , lambda /iota ) categories. The PKC isozymes beta I, delta , and theta  were not detected. We did not test for PKC-eta .

                              
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Table 1.   PKC isoform expression and distribution
in BEAS-2B cells

PKC isozymes were detected in the cytosolic or both the cytosolic and membrane fractions (Table 1). PKC-alpha , -zeta , and -lambda /iota were present largely in the cytosol. In contrast, PKC-µ, -beta II, and -epsilon were found largely in the membrane fraction.

IL-1beta treatment significantly increased the immunoreactivity of PKC-µ in membrane fractions without affecting the activity in the cytosolic fraction. In IL-1beta -treated cells, PKC-µ immunoreactivity was 643 ± 318% (SE) of the control value (P < 0.02) in the membrane and 156 ± 41% (SE) of control value in the cytosolic fraction (P > 0.3). No other consistent changes in the intensity or distribution of any of the other PKC isozymes were observed with IL-1beta treatment.

Changes in PKC-µ in the membrane and cytosol with the 2- and 18-h IL-1beta treatments were similar (data not shown).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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In a previous study, Kelsen et al. (8) showed that IL-1beta increased beta 2-AR gene expression in human airway epithelial (BEAS-2B) cells. IL-1beta increased beta 2-AR mRNA and beta 2-AR protein monotonically over time and in biphasic fashion with increasing dose. Increases in beta 2-AR protein were maximum at ~36 h and were blocked by the protein synthesis inhibitor cycloheximide, indicating a requirement for new protein synthesis. In this study, we assessed the role of PKC in mediating the IL-1beta -induced increase in beta 2-AR gene expression. The role of PKC was assessed from the response to 1) phorbol esters and 2) the PKC inhibitors calphostin C and Ro-31-8220 and from 3) the effect of IL-1beta on the cellular distribution of PKC isozymes expressed by these cells.

The response to PMA has been widely used as a probe for PKC-mediated effects (3, 18, 21). In this study, PMA increased beta -AR expression and mimicked the effects of IL-1beta . Furthermore, the two PKC inhibitors inhibited the beta -AR response to IL-1beta in a dose-dependent fashion and achieved virtually complete inhibition. Finally, IL-1beta treatment of airway epithelial cells selectively increased PKC-µ immunoreactivity in the membrane fraction. Taken together, these several lines of evidence strongly support the notion that the effects of IL-1beta are mediated by PKC activation, probably via the PKC-µ isotype. Of note, we did not test for the PKC-eta subtype, and the antibody for PKC-gamma was not selective, so possible effects of IL-1beta on these two isotypes cannot be ruled out.

Of note, upregulation of beta -AR expression was first evident at 6 h and increased progressively over 18 h. Accordingly, increases in beta -AR density were considerably slower than activation of PKC (<2 h), probably reflecting the slower time course required for synthesis of new receptor protein and its insertion into the cell membrane. In addition, in pulse-chase experiments, upregulation of beta -AR expression was maximal with 2 h of PMA exposure, suggesting that increasing the duration of PKC activation beyond 2 h did not enhance the magnitude of beta -AR gene expression.

In contrast to the effects of PKC inhibitors, the COX inhibitor indomethacin had no effect on the response to IL-1beta , suggesting that arachidonic acid-derived mediators play no role in the response to IL-1beta in airway epithelial cells.

Our study of PKC isozyme expression in a transformed airway epithelial cell line immortalized by SV40 adenovirus transfection closely resembles results obtained in primary cultures of both human and bovine airway epithelial cells (13, 15, 16, 24). For example, a study by Liedkte et al. (15) reported that primary cultures of human airway epithelial cells express PKC isozymes from the conventional, novel, and atypical groups (i.e., alpha , beta II, delta , epsilon , and zeta ) as is the case in BEAS-2B cells. Wyatt et al. (24) detected PKC-alpha , -beta II, -delta , and -epsilon in primary cultures of bovine airway epithelial cells. Unlike the studies of Liedkte et al. (15) and Wyatt et al. (24), we did not detect PKC-delta , perhaps because our studies were performed in a transformed cell line. Unlike the present study, neither Liedkte et al. (15) nor Wyatt et al. (24) tested for theta , µ, or lambda /iota isotypes, which are expressed in BEAS-2B cells.

In agreement with our study, Liedkte et al. (15) observed a differential cellular distribution of the isozymes. PKC-alpha was confined to the cytosol, whereas PKC-epsilon , -zeta , and -lambda /iota were evenly distributed between the cytosolic and particulate fractions and PKC-µ and -beta II were found primarily in the membrane fraction.

PKC is a threonine/serine kinase that, when activated, plays a key regulatory role in a variety of cellular functions such as stimulation or inhibition of growth, changes in morphology, and modulation of gene expression (3, 18, 21). In particular, PKC activation has been observed to mediate a variety of effects important for the differentiated function of airway epithelial cells. For example, PKC-activated pathways are involved in mucin secretion (11), activation of the chloride channel (16), and the sodium-potassium-chloride cotransporter at the basolateral membrane (13, 16).

In addition, the PKC signal transduction pathway is involved in the airway epithelial cell response to injury and repair and modulates the airway epithelial response to proinflammatory cytokines. For example, PKC mediates the airway cell migration response to fibronectin (24) and the shedding of high-affinity tumor necrosis factor (TNF)-alpha receptors in response to IL-1beta and phorbol esters (12). TNF-alpha receptor shedding mediated by PKC activation presumably serves to downregulate the airway epithelial cell response to TNF-alpha .

Our observation that IL-1beta may selectively activate a subset of the repertoire of PKC isozymes is in keeping with previous observations in a variety of other cell types and in airway epithelial cells in particular (6, 13-15, 18, 21). For example, in human airway epithelial cells, PKC-delta and -zeta are selectively activated in response to the alpha 1-AR agonist methoxamine, which activates Na-K-2Cl basolateral to apical cotransport (13, 15). Moreover, antisense mRNA against PKC-delta , but not PKC-zeta , inhibits the response to methoxamine, suggesting that methoxamine acts solely through this isozyme (13).

Our data indicate that the PKC signal transduction pathway is involved in the regulation of expression of the beta 2-AR gene, a gene vitally important in airway homeostasis and airway epithelial cell function, including mucin production and salt and water exchange (17). In the clinical setting, increased concentrations of IL-1beta are present in the airway secretions of patients with asthma and are thought to promote the inflammatory process in this disease. In fact, constitutive activation of alveolar macrophage PKC is suggested in patients with asthma (22). These latter observations and the results of the present study suggest that the PKC signal transduction pathway plays an extensive role in regulating the phenotypic behavior of a variety of respiratory cell types.


    FOOTNOTES

Address for reprint requests and other correspondence: S. G. Kelsen, Rm. 761 Parkinson Pavilion, Temple Univ. Hospital, 3401 N. Broad St., Philadelphia, PA 19140 (E-mail: kelsen{at}vm.temple.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 24 February 2000; accepted in final form 14 September 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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