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Originally published In Press as doi:10.1074/jbc.M404045200 on July 26, 2004

J. Biol. Chem., Vol. 279, Issue 39, 41085-41094, September 24, 2004
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Protein Kinase C{delta} Mediates Lysophosphatidic Acid-induced NF-{kappa}B Activation and Interleukin-8 Secretion in Human Bronchial Epithelial Cells*

Rhett Cummings{ddagger}, Yutong Zhao{ddagger}, David Jacoby§, E. William Spannhake¶, Motoi Ohba||, Joe G. N. Garcia{ddagger}, Tonya Watkins{ddagger}, Donghong He{ddagger}, Bahman Saatian{ddagger}, and Viswanathan Natarajan{ddagger}**

From the {ddagger}Division of Pulmonary and Critical Care Medicine and the Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, the §Department of Medicine, Oregon Health and Science University, Portland, Oregon 97239, the Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, and the ||Institute of Molecular Oncology, Showa University, Tokyo 142-8555, Japan

Received for publication, April 12, 2004 , and in revised form, July 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lysophosphatidic acid (LPA), a potent bioactive lipid, elicits many of its biological actions via the specific G-protein-coupled receptors LPA1, LPA2, LPA3, and LPA4. Recently, we have shown that LPA-induced transactivation of platelet-derived growth factor receptor-{beta} is regulated by phospholipase D2 in human bronchial epithelial cells (HBEpCs) (Wang, L., Cummings, R. J., Zhao, Y., Kazlauskas, A., Sham, J., Morris, A., Brindley, D. N., Georas, S., and Natarajan, V. (2003) J. Biol. Chem. 278, 39931-39940). Here, we report that protein kinase C{delta} (PKC{delta}) mediates LPA-induced NF-{kappa}B transcription and interleukin-8 (IL-8) secretion in HBEpCs. Treatment of HBEpCs with LPA increased both IL-8 gene and protein expression, which was coupled to Gi and G12/13 proteins. LPA caused a marked activation of NF-{kappa}B in HBEpCs as determined by I{kappa}B phosphorylation and of NF-{kappa}B nuclear translocation and a strong induction of NF-{kappa}B promoter-mediated luciferase activity. Furthermore, LPA-activated PKC{delta} and the LPA-mediated activation of NF-{kappa}B and IL-8 production were attenuated by overexpression of dominant-negative PKC{delta} and rottlerin. Intratracheal administration of LPA in mice resulted in elevated levels of macrophage inflammatory protein-2, a murine homolog of IL-8, and an influx of neutrophils in the bronchoalveolar lavage fluid. These results demonstrate for the first time that LPA is a potent stimulator of IL-8 production in HBEpCs, which involves PKC{delta}/NF-{kappa}B signaling pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The respiratory epithelium, a complex physical and biochemical barrier, has the capacity both to initiate inflammatory responses and to participate in repair. The balance between the protective, physiological inflammatory response and injurious, pathological inflammation involves coordinating the production of chemokines, cytokines, and growth factors (1). The innate immune response is of relatively short duration and involves increased vascular transudation, interstitial edema, and infiltration of inflammatory cells, predominantly of neutrophils. Initiation and maintenance of this leukocyte recruitment requires intercellular communication between infiltrating neutrophils, the endothelium, and respiratory epithelial cells. In this light, the airway epithelium plays a pivotal role in this response through its reaction to and secretion of necessary intercellular regulatory molecules. One such paracrine messenger, interleukin-8 (IL-8),1 the major chemoattractant and activator for neutrophils in the lung, is a key component of the innate immune response (2). Studies supporting the involvement of IL-8 in pulmonary inflammatory disorders have revealed elevated levels of IL-8 in the bronchoalveolar lavage (BAL) fluid of patients with chronic obstructive lung disease, asthma, idiopathic pulmonary fibrosis, pulmonary sarcoidosis, pneumonia, bronchitis obliterans syndrome, and acute lung injury/acute respiratory distress syndrome (3-8). Cultured airway epithelial cells also secrete significant amounts of IL-8 when exposed to tobacco smoke, diesel exhaust particles, ozone, viral infection, and hyperoxia as well as various endogenous mediators such as TNF-{alpha} and IL-1 (9-14).

Derived from membrane glycerophospholipids and sphingolipids, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are released from activated platelets and multiple other cells in response to a wide array of inflammatory stimuli (15). Both LPA and S1P are constitutively found in human serum (~1-20 µM) bound to lipoproteins (16), albumin (17), and gelsolin (18). At target cells, extracellular LPA binds with high affinity and specificity to three of the nine G-protein-coupled lysophospholipid receptors belonging to the Edg family (19), LPA1, LPA2, and LPA3. The LPA receptor isoforms display heterogeneous tissue expression patterns, but we have detected the native expression of all three in primary human bronchial epithelial cells (HBEpCs) (20).

Possessing the growth-related effects of cell proliferation, survival, and differentiation as well as the cytoskeleton-related effects of aggregation, chemotaxis, adhesion, and secretion, LPA and S1P have recently emerged as novel immunomodulating factors (15). Studies in our laboratory have revealed that S1P stimulates significant production and secretion of IL-8 in a human bronchial epithelial cell line (BEAS-2B) (21) and that LPA is capable of transactivating platelet-derived growth factor receptor-{beta} in HBEpCs (20). In guinea pigs, administration of exogenous LPA into the airway enhances the response to acetylcholine (22) and infiltration of neutrophils (23). Other reports have shown that LPA induces airway smooth muscle contractility (24), proliferation (25), and transcription factor activation (26). In ovarian cancer cells, LPA induces both IL-6 and IL-8 expression (27, 28).

Protein kinase C (PKC) belongs to a homologous serine/threonine kinase family that includes three classes of isoenzymes in which the functional specificity is determined by their subcellular localization. The groups consist of the calcium-, phorbol ester-, and phosphatidylserine-activated classical isoforms ({alpha}, {beta}, and {gamma}); the calcium-insensitive and phorbol esterand phosphatidylserine-activated novel isoforms ({delta}, {epsilon}, {eta}, {theta}, and µ); and the phosphatidylserine-activated and calcium- and phorbol ester-insensitive atypical isoforms ({zeta} and {iota}/{lambda}). It has recently been shown that PKC{delta} regulates ICAM-1 expression via NF-{kappa}B activation in human umbilical vein endothelial cells (29) as well as NF-{kappa}B-dependent (and IL-8 promoter-dependent) gene expression in a human bronchial epithelial cell line (30).

These earlier reports support the notion that LPA plays a role in the innate immune response. In this study, we utilized a primary HBEpC culture system to analyze LPA-induced IL-8 secretion. In addition to potent and specific production of IL-8, extracellular LPA stimulated I{kappa}B phosphorylation, NF-{kappa}B-dependent gene expression, and NF-{kappa}B nuclear translocation. The overexpression of dominant-negative PKC{delta} significantly attenuated LPA-mediated I{kappa}B phosphorylation, NF-{kappa}B translocation, and IL-8 secretion. We also supplemented our cell culture experiments with in vivo studies. Intratracheally delivered LPA in C57BL/6J mice resulted in significant elevations of macrophage inflammatory protein-2 (MIP-2) and a neutrophilic influx in the BAL fluid from these animals. Together, these data indicate that PKC{delta} plays a key role in the regulation of LPA-mediated IL-8 production via NF-{kappa}B activation in primary HBEpCs and that extracellular LPA produces an analogous inflammatory response in vivo.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—LPA and S1P were obtained from BIOMOL Research Labs Inc. (Plymouth Meeting, PA). Bovine serum albumin (BSA) was obtained from Sigma. Bisindolylmaleimide and pertussis toxin (PTx) were purchased from EMD Biosciences (San Diego, CA). FuGENE 6 transfection reagent was obtained from Roche Applied Science. Horse-radish peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies was purchased from Molecular Probes, Inc. (Eugene, OR). The enhanced chemiluminescence ECL kit for the detection of proteins by Western blotting was obtained from Amersham Biosciences. All other reagents were of analytical grade.

Cell Culture—Primary HBEpCs were isolated from the lungs of healthy organ donors purchased from Tissue Transformation Technologies (Edison, NJ). Cell isolation was carried out in one of our laboratories (E. W. S.) by a modification of the method described by Schroth et al. (31) and Wu et al. (32). Briefly, airway specimens were rinsed with Hanks' balanced saline solution and then placed in dissociation solution consisting of nutrient mixture F-12 supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and Pronase (1 mg/ml; Roche Applied Science) for 48 h at 4 °C. After incubation, fetal bovine serum was added to a final concentration of 20%, and epithelial cells were detached from the stroma by gentle agitation. The cells were collected by centrifugation at 1000 x g for 10 min, washed, and suspended in serum-free hormone-supplemented basal essential growth medium (BEGM; Bio-Whittaker, Inc., Walkersville, MD). All primary cell cultures were grown in BEGM in 35- or 60-mm dishes at 37 °C in 5% CO2 and 95% air. Experiments with HBEpCs were conducted between passages 0 and 4 at 70-90% confluence.

Measurement of IL-8 Secretion—HBEpCs were pretreated in BEGM with or without PTx (100 ng/ml), rottlerin (5 µM), or bisindolylmaleimide (1 µM) for 1 h prior to stimulation. The pretreatment media were removed, and the cells were treated in BEGM containing 0.1% BSA with or without LPA at the indicated concentrations for the specified lengths of time. Cell supernatants were removed, centrifuged at 5000 x g for 5 min at 4 °C, and frozen at -80 °C for later analysis for IL-8 by enzyme-linked immunosorbent assay (ELISA), which was performed according to the manufacturer's instructions (BIOSOURCE, International, Camarillo, CA).

Immunofluorescence Microscopy—HBEpCs grown on coverslips to ~50% confluence were infected with a vector control or dominant-negative (dn) PKC{delta} (multiplicity of infection (m.o.i.) = 25) for 48 h prior to challenge with LPA (1 µM) for 15 min. In some experiments without infection with adenoviral constructs, ~80% confluent cells were used for LPA challenge. Cells were rinsed twice with phosphate-buffered saline (PBS) at 37 °C and fixed with 3.7% formaldehyde in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) for 15 min at room temperature. The cells were rinsed twice with PBS and incubated for 5 min in PBS containing 0.25% Triton X-100. The cells were stained for 20 min with a 1:200 dilution of either anti-PKC{delta} or anti-NF-{kappa}B subunit (p65RelA) antibody in TBST containing 1% BSA. The cells were thoroughly rinsed with TBST and stained for 30 min with a 1:200 dilution of Alexa Fluor 488 in TBST containing 1% BSA. Cells were examined using a Nikon Eclipse TE 2000-S immunofluorescence microscope and a Hamamatsu digital camera with a x60 oil immersion objective and Meta Vue software (Universal Imaging Corp.).

Cytokine Gene Expression by cDNA Array Analysis—Total cellular RNA was isolated from HBEpCs using an RNeasy kit (QIAGEN Inc., Los Angeles, CA) following vehicle or LPA treatment. Aliquots of total RNA (10 µg) were used to analyze the differential gene expression of common cytokines using Nonrad-GEArray technology (Super Array Inc., Bethesda, MD) according to the manufacturer's instructions. Briefly, biotinylated cDNA probes were prepared using total RNA as a template by reverse transcription with Moloney murine leukemia virus reverse transcriptase. Following denaturation, the cDNA probes were hybridized to a positively charged nylon membrane containing the arrayed DNA. After blocking and incubation with alkaline phosphatase-conjugated streptavidin and chemiluminescent substrate, the membranes were exposed to x-ray film. Gene expression was quantified by scanning densitometry and normalized based on the intensity of hybridization signals to the housekeeping genes {beta}-actin and glyceraldehyde-3-phosphate dehydrogenase. The experiment was performed three times to ensure reproducibility of results.

Preparation of Cell Lysates and Western Blotting—HBEpCs were rinsed two times with ice-cold PBS; scraped in 1 ml of lysis buffer A (20 mM Tris-HCl (pH, 7.4), 150 mM NaCl, 2 mM EGTA, 5 mM glycerophosphate, 1 mM MgCl2, 1% Triton X-100, 1 mM sodium orthovanadate, 10 µg/ml protease inhibitors, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin); incubated at 4 °C for 20 min; and cleared by centrifugation in a microcentrifuge at 10,000 x g for 5 min at 4 °C. After determination of the total protein in the lysates by the BCA method, 6x Laemmli sample buffer was added to cell lysates, and the lysates were boiled for 5 min. Equally loaded proteins were separated on 12% gels by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and subjected to immunoblotting with anti-PKC{alpha} (1:1000 dilution), anti-PKC{delta} (1:1000 dilution), or anti-PKC{epsilon} (1:1000 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-PKC{zeta} (1:1000 dilution), anti-PKC{theta} (1:500 dilution), or anti-PKC{iota}/{lambda} (1:500 dilution) (Transduction Laboratories, San Diego, CA) overnight at 4 °C. The membranes were washed at least three times with TBS containing 0.1% Tween 20 (TBST) and incubated for 2-4 h at room temperature in horseradish peroxidase-conjugated goat anti-rabbit (1:1000 dilution in TBST containing 5% BSA) or goat anti-mouse (1:1000 dilution in TBST containing 5% nonfat milk) secondary antibody. The immunoblots were developed using the ECL kit according to the manufacturer's recommendation.

Preparation of Nuclear Extracts—Nuclear extracts were prepared from HBEpCs according to the manufacturer's instructions (Active Motif North America, Carlsbad, CA). Briefly, cells were collected by scraping in ice-cold PBS with phosphatase inhibitors and pelleted by centrifuging at 1000 x g for 5 min. The pellet was resuspended in 500 µl of 1x hypotonic buffer and incubated on ice for 15 min, followed by addition of 25 µl of detergent and high speed vortexing for 30 s following the manufacturer's recommendation. The suspension was centrifuged at 14,000 x g for 20 min in a microcentrifuge at 4 °C; the nuclear pellet was resuspended in 50 µl of lysis buffer A and incubated on ice for 15 min. This suspension was centrifuged for 10 min at 14,000 x g in a microcentrifuge, and the supernatant (nuclear extract) was aliquoted and stored at -80 °C for further analysis. Protein in the nuclear extract was quantified by BCA protein assay.

Electrophoretic Mobility Shift Assay (EMSA)—The probe for EMSA was a 22-bp double-stranded construct of NF-{kappa}B consensus binding sequence (5'-AGTTGAGGGGACTTTCCCAGGC-3'). End labeling of the probe was performed using T4 kinase and [{gamma}-32P]ATP. EMSAs were performed using nuclear extracts (4 µg) from vector control- or dn-PKC{delta} adenovirus-infected cells with or without LPA treatment and binding buffer containing 10 mM Tris-HCl (pH 7.5), 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 50 µg of the nonspecific blocker poly(dI-dC), and 20,000 dpm of {gamma}-32P-labeled probe at 37 °C for 30 min. Specific competition was performed by adding 200 ng of unlabeled 22-bp double-stranded probe. The protein-DNA complexes were analyzed by electrophoresis on a 4% nondenaturing polyacrylamide gel. The gels were vacuum-dried and subjected to autoradiography.

Transfection and Viral Infection—For transient transfection, HBEpCs grown in 6-well plates (~60% confluent) were transfected with G-protein minigene vectors (Cue Biotech, Chicago, IL). The minigene vectors contain oligonucleotides ligated into pcDNA3.1 that encode a unique peptide that specifically blocks the receptor/G-protein interface. The minigene plasmids (1 µg of cDNA) were mixed with 3 µl of FuGENE 6/well in 1 ml of BEGM, and transfections were performed for 4 h. At the end of 4 h, the transfection medium was aspirated; regular BEGM (2 ml) was added; and cells were incubated for additional 24 h. Infection of HBEpCs (~60% confluent) in 35- or 60-mm dishes was carried out with purified kinase-negative adenoviral vectors of PKC isoforms as described previously (33). Following infection of 50 plaque-forming units/cell in 1 ml of BEGM for 24 h, the virus-containing medium was replaced with complete BEGM, and experiments were performed.

PKC{delta} Kinase Assay—HBEpCs infected with vector or dn-PKC{delta} adenoviruses (m.o.i. = 50) for 24 h were challenged with medium alone or medium containing LPA (1 µM) for 15 min prior to harvesting. Cells were lysed in lysis buffer B (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM {beta}-glycerophosphate, 1 mM Na3VO4, and protease inhibitors from EDTA-free Complete tablets (Roche Applied Science)). Lysates (500 µg of protein) were immunoprecipitated with anti-PKC{delta} antibody (10 µl) overnight at 4 °C, followed by addition of protein A/G (40 µl) and incubation for an additional 2 h at 4 °C. The immunoprecipitates were washed three times with lysis buffer B and two times with kinase buffer (20 mM HEPES (pH 7.4), 25 mM {beta}-glycerophosphate, 1 mM EGTA, 1 mM sodium orthovanadate, and 1 mM dithiothreitol) and resuspended in 200 µl of kinase buffer. The activity of PKC{delta} was measured in 100 µl of kinase buffer containing 25 µg of myelin basic protein as an exogenous substrate to which 5 µCi of [{gamma}-32P]ATP containing unlabeled ATP (10 µM; final specific activity of 10,000 dpm/pmol), 2 µg of diacylglycerol, 12 µg of phosphatidylserine, and 25 µl of immunoprecipitate were added. Incubations were carried out for 15 min at 37 °C and terminated by addition of 100 µl of SDS sample buffer. Aliquots (25-50 µl) of the reaction mixture were spotted onto P-81 filter paper and washed three times with 1% phosphoric acid and once with acetone, and radioactivity was measured by scintillation spectroscopy.

LPA Instillation and Bronchoalveolar Lavage—Wild-type C57BL/6J mice were anesthetized by intraperitoneal injection of 30 µl of 10:1 ketamine (100 mg/kg)/xylazine (10 mg/kg). LPA (10 µM) was administered intranasally after sonication in physiological saline containing tissue culture-grade 0.1% BSA, whereas the control mice received saline plus 0.1% BSA. BAL was performed 3 or 6 h following vehicle or LPA instillation. After the mice were killed with an intraperitoneal overdose of urethane (3 g/kg of body weight), the trachea was cannulated with an 18-gauge intracatheter, and the lungs were lavaged with three 1-ml aliquots of physiological saline (3 ml total). BAL fluids were centrifuged at 200 x g for 10 min, and the pellet was immediately resuspended in physiological saline for total cell counts. Differential cell counts were carried out in the centrifuged cells after Wright-Giemsa staining. The supernatant were frozen at -80 °C for later measurements of MIP-2 and total protein.

Statistical Analysis of Data—S.D. for each data point was calculated from triplicate samples. Unless stated otherwise, data were subjected to one-way analysis of variance, and pairwise multiple comparison was done by Dunnett's method with p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
LPA Activates IL-8 in Primary HBEpCs—We have recently reported that the bioactive sphingolipid S1P stimulates IL-8 production in BEAS-2B bronchial epithelial cells (21). Extracellular LPA has been shown to be an immunological regulator by activating NF-{kappa}B and increasing levels of mRNA transcripts encoding E-selectin, ICAM-1, IL-8, and MCP-1 (monocyte chemoattractant protein-1) in endothelial cells (17). Additionally, LPA has been demonstrated to activate NF-{kappa}B in fibroblasts (10). We examined the ability of both S1P and LPA to modulate IL-8 secretion in HBEpCs. As shown in Fig. 1, treatment of HBEpCs with S1P (1 µM) resulted in significant increases in IL-8 secretion at both 3 and 24 h (2.6- and 1.8-fold over control levels, respectively). Interestingly, LPA treatment (1 µM) produced an even greater induction of IL-8 secretion of 4.8-fold at 3 h and 2.2-fold at 24 h (Fig. 1).



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  		FIG. 1.
Stimulation of IL-8 secretion by LPA and S1P. HBEpCs (~80% confluent in 35-mm dishes) were treated in BEGM with 0.1% BSA (Vehicle) or in BEGM with 0.1% BSA containing LPA (1 µM) or S1P (1 µM) for the indicated times (3 and 24 h). IL-8 secreted into the medium was quantified by ELISA as described under "Experimental Procedures." Data are means ± S.D. of triplicate determinations in three independent experiments. *, significantly different from vehicle controls (p < 0.01); **, significantly different from vehicle controls (p < 0.05).

 
Treatment of HBEpCs with LPA for 3 h resulted in dose-dependent secretion of IL-8 (Fig. 2A) with significant production at 10 nM (1.4-fold over control levels) and a 4.1-fold increase over control levels at 1 µM. These concentrations are within reported physiological concentrations of LPA found in human serum (34). The release of IL-8 into the medium by HBEpCs upon stimulation with LPA (1 µM) significantly increased after 3 h (3.4-fold) and reached a maximum at 12 h (Fig. 2B).



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  		FIG. 2.
Dose response and time course of LPA-induced IL-8 secretion. HBEpCs were grown in 35-mm dishes to ~80% confluence in BEGM. A, cells were treated for 3 h in BEGM with 0.1% BSA (vehicle (Veh)) or in BEGM with 0.1% BSA containing the indicated concentrations of LPA. B, cells were treated in BEGM with 0.1% BSA or in BEGM with 0.1% BSA plus LPA (1 µM) for indicated times (1-24 h). ELISA was performed as described under "Experimental Procedures" to quantify IL-8 secreted into the medium. Data are means ± S.D. of triplicate determinations in three independent experiments. *, significantly different from vehicle controls (p < 0.05).

 
LPA-induced IL-8 Gene Expression—To compare the effect of LPA treatment on production of IL-8 with that of other cytokines, we utilized cytokine-specific cDNA technology. Total RNA from vehicle-treated versus LPA-treated (1 µM, 3 h) HBEpCs was isolated and analyzed for differential cytokine gene expression. As shown in Fig. 3 (A and B), among the 23 different cytokines examined, only the expression of IL-8 was increased (11.5-fold) relative to control cells. HBEpCs produced basal amounts of IL-1{alpha} (spot pair 1) and TNF-{alpha} (spot pair 22), which were not affected by LPA treatment. These striking data indicate that LPA specifically up-regulates IL-8 gene expression in HBEpCs.



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  		FIG. 3.
Expression profile of cytokine genes by cDNA expression array system in HBEpCs. A, total RNA was isolated from HBEpCs (2 x 106 cells) after a 3-h incubation with (right panel) or without (left panel) LPA (1 µM). Total RNA was reverse-transcribed and labeled with biotin, and gene expression was detected using the Nonrad-GEArray cytokine series kit according to the manufacturer's instructions. The numbers on the membranes correspond to IL-1{alpha} (1), IL-1{beta} (2), IL-2 (3), IL-3 (4), IL-4 (5), IL-5 (6), IL-6 (7), IL-7 (8), IL-8 (9), IL-9 (10), IL-10 (11), IL-11 (12), IL-12A (13), IL-12B (14), IL-13 (15), IL-14 (16), IL-15 (17), IL-16 (18), IL-17 (19), IL-18 (20), interferon-{gamma} (21), TNF-{alpha} (22), and TNF-{beta} (Lt-{alpha}) (23). B, IL-8 gene expression was quantified by scanning densitometry, and intensity was normalized to the housekeeping genes {beta}-actin and glyceraldehyde-3-phosphate dehydrogenase. Values are the average of three independent GEArray expression array systems.

 
G-protein Coupling Involved in LPA-induced IL-8 Secretion—We have previously identified the expression of LPA1, LPA2, and LPA3 receptors in HBEpCs using reverse transcription-PCR, Western blotting, and immunocytochemistry (20). To further examine which member(s) of the heterotrimeric G-protein family is responsible for LPA-mediated IL-8 secretion, we investigated the effects of inhibition of LPA receptor/G-protein-coupled signaling. Treatment of HBEpCs with PTx (100 ng/ml) for 3 h, which uncouples G-protein-coupled receptors for Gi, significantly prevented LPA-induced IL-8 secretion (60% inhibition), but had no effect on basal IL-8 secretion (Fig. 4A). In addition to PTx, we also investigated the involvement of Gi, Gq, and G12/13 by transiently transfecting minigenes that encode peptides that specifically block the respective G-protein/receptor interface. As shown in Fig. 4B, expression of G{alpha}i and G12/13 (but not Gq) resulted in attenuation of LPA-mediated IL-8 secretion (G{alpha}i, 30% inhibition; and G12/13, 40% inhibition). These results indicate that endogenously expressed G{alpha}i and G12/13 are responsible for the initial mediating signals that stimulate IL-8 secretion by LPA.



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  		FIG. 4.
PTx and G-protein minigenes attenuate LPA-induced IL-8 secretion. A, HBEpCs (~80% confluent in 35-mm dishes) were treated with PTx (100 ng/ml) for 3 h. Cells were challenged with BEGM and 0.1% BSA (Vehicle) or with BEGM and 0.1% BSA containing LPA (1 µM) for an additional 3 h. IL-8 secreted into the medium was quantified by ELISA as described under "Experimental Procedures." Data are means ± S.D. of three independent determinations. *, significantly different from controls (p < 0.05); **, significantly different from LPA-challenged cells without PTx treatment (p < 0.05). B, HBEpCs (~50% confluent in 35-mm dishes) were transfected with Gi, Gq, G12, and G13 minigene plasmids as described under "Experimental Procedures" for 24 h. Cells were treated in BEGM with 0.1% BSA or in BEGM with 0.1% BSA containing LPA (1 µM) for 3 h. The medium was collected and analyzed for IL-8 by ELISA. Data are means ± S.D. of three independent experiments performed in triplicate. *, significantly different compared with the vehicle control (p < 0.05); **, significantly different compared with LPA challenge in the absence of minigenes Gi and G12/13 (p < 0.05).

 
PKC{delta} Isoform Is Involved in LPA-induced IL-8 Secretion—Recent reports suggest that PKC{delta} plays a key role in the regulation of NF-{kappa}B-dependent gene expression in an airway epithelial cell line (30). To gain further insight into signaling pathways mediated by LPA in enhancing IL-8 secretion in HBEpCs, we investigated the role of PKC isoforms using pharmacological inhibitors and dominant-negative isoforms of PKC. Analysis of the total cell lysates by Western blotting with PKC-specific antibodies revealed the expression of PKC{alpha}, PKC{delta}, PKC{iota}/{lambda}, and PKC{zeta} as the major isotypes present in HBEpCs (Fig. 5A). We then pretreated HBEpCs with the PKC inhibitors bisindolylmaleimide, a general inhibitor of PKC, and rottlerin, a specific inhibitor of PKC{delta}, for 1 h prior to LPA challenge. Bisindolylmaleimide (1 µM) significantly attenuated LPA-induced IL-8 production by 55% without altering the basal values (Fig. 5B). Similarly, rottlerin (5 µM) blocked LPA-mediated generation of IL-8 by 75% (Fig. 5C). In addition to the pharmacological inhibitors, we also tested the effect of the expression of dn-PKC{alpha}, dn-PKC{delta}, dn-PKC{lambda}, and dn-PKC{zeta} adenoviral vectors on LPA-induced IL-8 production. Infection of HBEpCs with the dn-PKC isoforms at varying multiplicities of infection for 12, 24, and 48 h resulted in overexpression of the protein (Fig. 6A). Based on these expression profiles, HBEpCs were infected with the dn-PKC{alpha}, dn-PKC{delta}, dn-PKC{lambda}, and dn-PKC{zeta} adenoviral vectors (m.o.i. = 25) for 24 h before challenging the cells with LPA. As shown in Fig. 6B, overexpression of dn-PKC{delta} significantly blocked LPA-mediated IL-8 secretion. Also, a small but significant attenuation of IL-8 production was seen with dn-PKC{lambda} overexpression (Fig. 6B). Overexpression of dn-PKC{alpha} had no effect on LPA-mediated IL-8 generation; however, overexpression of dn-PKC{zeta} appeared to slightly enhance the effect of LPA on IL-8 secretion.



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  		FIG. 5.
Expression of PKC isoforms and effects of bisindolylmaleimide and rottlerin on LPA-mediated IL-8 secretion. A, cell lysates (20 µg of protein) from HBEpCs were subjected to SDS-PAGE and Western blotting with PKC isoform-specific antibodies. B, HBEpCs (~80% confluent in 35-mm dishes) were treated with bisindolylmaleimide (BIM; 1 µM) for 1 h prior to LPA stimulation in BEGM plus 0.1% BSA for 3 h. C, HBEpCs (~80% confluent in 35-mm dishes) were treated with rottlerin (Rott; 5 µM) for 30 min prior to LPA challenge in BEGM plus 0.1% BSA for 3 h. The medium was collected and analyzed for IL-8 by ELISA as described under "Experimental Procedures." Data are means ± S.D. of three independent experiments performed in triplicate. *, significantly different from vehicle-treated cells (p < 0.05); **, significantly different from LPA-challenged cells without bisindolylmaleimide or rottlerin (p < 0.05).

 



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  		FIG. 6.
Effects of overexpression of dn-PKC isoforms on LPA-induced IL-8 secretion. A, HBEpCs (~50% confluent in 35-mm dishes) were infected with vector or each of the dn-PKC isoforms (DN) in adenoviral constructs at m.o.i. = 25 for varying time periods (12, 24, and 48 h). Cell lysates from vector control- or dn-PKC-infected cells were subjected to SDS-PAGE and Western blotting as described under "Experimental Procedures." Shown is a representative blot of three independent experiments. B, HBEpCs (~50% confluent in 35-mm dishes) were infected with dn-PKC{alpha}, dn-PKC{delta}, dn-PKC{zeta}, and dn-PKC{lambda} adenoviral constructs (m.o.i. = 25) for 48 h. Cells were then treated in BEGM with 0.1% BSA (vehicle (Veh)) or in BEGM with 0.1% BSA plus LPA (1 µM) for 3 h. IL-8 secreted into the medium was quantified by ELISA as described under "Experimental Procedures." Data are means ± S.D. of three independent experiments performed in triplicate. *, significantly different from vehicle controls (p < 0.05); **, significantly different from LPA-treated cells (p < 0.05).

 
Having established a role for PKC{delta} in LPA-induced IL-8 secretion, we investigated the activation of PKC{delta} by LPA. HBEpCs were infected with a vector control or dn-PKC{delta} (m.o.i. = 25) for 48 h, and cell lysates were subjected to immunoprecipitation with anti-PKC{delta} antibody. Immunoprecipitates were analyzed for PKC activity using [{gamma}-32P]ATP and myelin basic protein (MBP) as a substrate. Treatment of cells with LPA (1 µM) for 15 min stimulated PKC{delta} activity by 2.5-fold compared with control cells. Overexpression of dn-PKC{delta} blocked the phosphorylation of MBP mediated by LPA in HBEpCs (Fig. 7A). In these experiments, the phosphorylation of MBP by PKC{delta} was normalized to total ERK in the supernatants after immunoprecipitation of PKC{delta}. In addition, LPA stimulation of HBEpCs also increased phosphorylation of PKC{delta} as evidenced by Western blotting with anti-phospho-PKC{delta} antibody (Fig. 7B). As shown by the immunocytochemical analysis in Fig. 7C, LPA treatment stimulated translocation of PKC{delta} to the plasma membrane compared with control cells. These results suggest that LPA activates the PKC{delta} isoform in HBEpCs, which subsequently regulates LPA-mediated IL-8 production.



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  		FIG. 7.
Effects of dn-PKC{delta} infection on LPA-induced activation of native PKC{delta} HBEpCs (~50% confluence) were infected with either a vector control or dn-PKC{delta} adenoviral constructs (m.o.i. = 25) for 48 h prior to LPA (1 µM) challenge for 15 min. A, cell lysates from vector control- or dn-PKC{delta} adenovirus-infected cells were subjected to immunoprecipitation with anti-PKC{delta} antibody as described under "Experimental Procedures," and immunoprecipitates were tested for activity using MBP as a substrate. Data are expressed as relative counts/min in phosphorylated MBP. B, cell lysates (20 µg of protein) from vector control- or dn-PKC{delta}-infected cells were stimulated with LPA (1 µM) for varying time periods (0-15 min). Total cell lysates were subjected to SDS-PAGE and Western blotting with anti-phospho-PKC{delta} and anti-pan-PKC{delta} antibodies. C, HBEpCs were grown on coverslips to ~70% confluence and infected with a vector control or a dn-PKC{delta} adenoviral construct (m.o.i. = 25) for 48 h prior to challenge with LPA (1 µM) for 15 min. Cells were then subjected to immunostaining with anti-PKC{delta} antibody and examined by fluorescence microscopy as described under "Experimental Procedures." The immunofluorescent image is representative of the monolayer visualized in three independent experiments.

 
PKC{delta} Participates in LPA-mediated NF-{kappa}B Activation—Activation of transcription factors such as NF-{kappa}B and AP-1 (activating protein-1) by pro-inflammatory cytokines and agonists via G-protein-coupled receptors has been reported (28-30). In most mammalian cells, NF-{kappa}B exists as an inactive heterodimer composed of p50 and p65RelA units through its interaction with I{kappa}B proteins, of which there are three major isoforms, I{kappa}B{alpha}, I{kappa}B{beta} and I{kappa}B{epsilon}. Signal-induced phosphorylation of I{kappa}B by I{kappa}B kinase (IKK) results in ubiquitination and subsequent degradation of I{kappa}B by the 26 S proteasome. This releases NF-{kappa}B heterodimers from the inactive complex and allows their translocation to the nucleus for interaction with cognate DNA sequences (35). We examined LPA-mediated activation of NF-{kappa}B utilizing various techniques. As shown in Fig. 8A, LPA treatment (1 µM) for 15 min increased phosphorylation of I{kappa}B in HBEpCs. The LPA-induced phosphorylation of I{kappa}B-{alpha} by LPA was time-dependent, reaching a maximum at 15 min and decreasing thereafter to near basal levels (data not shown). The role of PKC{delta} in LPA-dependent activation of NF-{kappa}B was investigated by infecting HBEpCs with adenoviral constructs containing dn-PKC{delta}. The LPA-induced phosphorylation of I{kappa}B was attenuated by overexpression of dn-PKC{delta} (Fig. 8A). Next, we investigated the ability of LPA to promote migration of NF-{kappa}B to the nucleus. Using an antibody specific to the p65 subunit of human NF-{kappa}B, immunocytochemistry was carried out in control and LPA-treated cells. In control cells, the immunoreactivity of p65 appeared to be in the cytosol. On the other hand, LPA treatment (1 µM) for 15 min induced a rapid and significant accumulation of p65 in the nucleus (Fig. 8B). Furthermore, the LPA-induced translocation of NF-{kappa}B to the nucleus was attenuated by overexpression of dn-PKC{delta} or by rottlerin, suggesting a role for PKC{delta} in NF-{kappa}B nuclear translocation (Fig. 8B). However, dn-PKC{zeta} overexpression did not affect the LPA-mediated translocation of NF-{kappa}B to the nucleus, indicating the specificity of PKC{delta} in regulating the translocation (Fig. 8B). A direct evaluation of NF-{kappa}B activation was carried out by measuring the transcription-activating potential of an NF-{kappa}B-driven luciferase reporter plasmid by LPA. Luciferase activity was measured in HBEpCs transfected with pNF-{kappa}B-luc and pCMV-{beta}-gal treated with LPA (1 µM) for 3 h. Fig. 8C shows that LPA produced a 2-fold enhancement of NF-{kappa}B-driven luciferase activity, which was completely blocked by pretreatment of the cells with rottlerin (5 µM) for 1 h. To further confirm the role of PKC{delta} in LPA-induced NF-{kappa}B activation, HBEpCs were infected with either a vector control or dn-PKC{delta} adenovirus for 24 h; cells were treated with medium alone or containing LPA (1 µM) for 1 h; and nuclear extracts were prepared for EMSAs. LPA treatment increased binding of nuclear proteins to the NF-{kappa}B oligonucleotides and induced supershift of the DNA-binding complex (Fig. 8D). In cells overexpressing dn-PKC{delta} protein, LPA did not induce binding of nuclear proteins to the NF-{kappa}B oligonucleotides and supershift of the DNA-binding complex (Fig. 8D). Together, these results reveal that LPA-induced IL-8 secretion is mediated via PKC{delta}/NF-{kappa}B-dependent signaling processes in HBEpCs.



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  		FIG. 8.
Effects of dn-PKC{delta} and rottlerin on LPA-induced NF-{kappa}B activation. A, HBEpCs (~50% confluent in 35-mm dishes) were infected with vector or a dn-PKC{delta} adenoviral construct (m.o.i. = 25) for 48 h. Cells were stimulated in BEGM with 0.1% BSA (vehicle (Veh)) or in BEGM with 0.1% BSA containing LPA (1 µM) for 15 min. Cell lysates (20 µg of protein) were subjected to SDS-PAGE and Western blotting with anti-phospho-I{kappa}B or anti-pan-ERK1/2 antibody as described under "Experimental Procedures." Shown is a representative Western blot of three independent experiments. The extent of I{kappa}B phosphorylation was quantified by image analysis and normalized to total ERK. Data are means ± S.D. of three independent experiments performed in triplicate. *, significantly different from the vehicle control (p < 0.05); **, significantly different from LPA-challenged cells without dn-PKC{delta} overexpression. B, HBEpCs (~50% confluent) grown on glass coverslips were infected with vector or with dn-PKC{delta} or dn-PKC{zeta} (m.o.i. = 25) for 48 h. Cells were pretreated in BEGM with 0.1% BSA or in BEGM with 0.1% BSA containing rottlerin (5 µM) for 30 min prior to stimulation with LPA (1 µM) for 15 min. The cells were rinsed with PBS, fixed in 3.7% formaldehyde, immunostained with anti-NF-{kappa}B antibody, and examined by fluorescence microscopy as described under "Experimental Procedures." C, HBEpCs were grown in 35-mm dishes to ~50% confluence and transiently transfected with an NF-{kappa}B promoter-driven luciferase reporter plasmid. 18-24 h after transfection, cells were incubated for 3 h in BEGM with 0.1% BSA or in BEGM with 0.1% BSA containing LPA (1 µM). Luciferase activity was measured using a commercial kit, and values were normalized to Renilla luciferase. Data are means ± S.D. of three independent experiments performed in triplicate. *, significantly different from vehicle controls (p < 0.05); **, significantly different from LPA-stimulated cells without rottlerin treatment (p < 0.05). D, EMSA shows the effect of dn-PKC{delta} overexpression on LPA-induced binding of nuclear proteins to the NF-{kappa}B oligonucleotides and supershift of the DNA-binding complex. These data are representative of three separate experiments.

 
Intratracheally Administered LPA Stimulates Airway Neutrophil Infiltration and MIP-2 Secretion in Mice—To examine the in vivo effects of LPA as a pro-inflammatory lipid mediator, we carried out experiments in C57BL/6J mice. Following routine anesthetization, mice were treated intratracheally with 50 µl of saline containing 0.1% BSA with or without LPA (10 µM). The mice were killed, and BAL was performed at the indicated time points. Levels of MIP-2 (the murine homolog of IL-8) and total cell count with differential and total protein concentrations were analyzed in the BAL fluid. As shown in Fig. 9A, intratracheal instillation of LPA stimulated a significant infiltration of neutrophils into the airways (12-fold) 6 h following administration. MIP-2 levels were 40-fold higher 3 h after treatment in the LPA-treated mice (Fig. 9B). Protein concentration, a surrogate of permeability measurement, was similar in vehicle- and LPA-treated mice at 6 h (Fig. 9C). These findings in an animal model parallel our findings in the HBEpC culture system and strongly suggest that extracellular LPA is a mediator of the innate immune response by inducing IL-8 production and subsequent airway neutrophil infiltration.



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  		FIG. 9.
Intratracheal instillation of LPA stimulates secretion of MIP-2 and influx of neutrophils into BAL fluid. Following routine anesthetization, C57BL/6J mice were instilled with 50 µl of either 0.9% saline plus 0.1% BSA or 0.9% saline plus 0.1% BSA containing 10 µM LPA. A, BAL was performed 6 h after LPA instillation, and the cell pellet was obtained immediately as described under "Experimental Procedures." Differential cell counts were performed in centrifuged preparations upon Wright-Giemsa staining. B, BAL was performed 3 h after LPA instillation and centrifuged to pellet the cells. The supernatants were stored at -80 °C and analyzed for MIP-2 by ELISA as described under "Experimental Procedures." C, total protein in BAL fluid obtained as described for A was determined with a Pierce protein assay kit. Data are means ± S.D. of three independent experiments performed in triplicate. *, significantly different from the vehicle-instilled group (p < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Increased expression and release of IL-8, a potent chemoattractant of neutrophils, have been described in bronchial epithelial cells in response to TNF-{alpha}, IL-1, and exposure to tobacco smoke, diesel exhaust particles, ozone, viral infection, and hyperoxia (3-8). The data presented here show for the first time enhanced expression of the IL-8 gene as well as its secretion by LPA in primary cultures of HBEpCs. Our results also suggest that LPA-induced expression and secretion of IL-8 are transcriptionally regulated by NF-{kappa}B. Furthermore, evidence is provided that LPA-mediated activation of PKC{delta} is involved in the activation of I{kappa}B and translocation of NF-{kappa}B to the nucleus.

LPA is a potent bioactive lipid that mediates several cellular responses such as proliferation, differentiation, suppression of apoptosis, tumor metastasis, and cytoskeletal reorganization (15, 21-28). LPA mediates the broad range of cellular responses through the G-protein-coupled receptors LPA1/Edg-2, LPA2/Edg-4, and LPA3/Edg-7 (19). Recently, a fourth LPA receptor, LPA4/p2y9-GPR23, which shares ~20-24% homology with LPA1, LPA2, and LPA3, was identified from an analysis of the expressed sequence tag data base and cloned from human genomic DNA (36). Although LPA4 is also a G-protein-coupled receptor, only very limited information is available regarding its biological functions. LPA binds with high affinity to LPA1, LPA2, and LPA3, which are coupled to heterotrimeric G-proteins Gi, Gq/G11, and G12/13. Earlier studies showed that LPA-induced transactivation of platelet-derived growth factor receptor-{beta} and ERK activation are sensitive to PTx, suggesting a role for Gi-dependent signaling in HBEpCs (20). In human aortic smooth muscle cells, ERK activation by LPA is attenuated by PTx (37), whereas in fibroblasts, LPA-mediated cell growth is PTx-sensitive (18). Our present results demonstrate that LPA-induced IL-8 secretion is PTx-sensitive, suggesting coupling of LPA receptors to Gi. Additionally, transfecting cells with minigenes for Gq and G12/13 also demonstrated possible involvement of G12/13 (but not Gq) in IL-8 release mediated by LPA in HBEpCs. LPA1, LPA2, and LPA3 are present in the lung and HBEpCs (20). It is unclear which LPA receptor is coupled to Gi and/or G12/13, resulting in downstream activation of NF-{kappa}B and IL-8 secretion in HBEpCs. Studies using small interfering RNA to differentially block individual LPA receptors will be necessary to dissect out exact coupling between LPA receptors and heterotrimeric G-protein signaling.

This study demonstrates that LPA-elicited PKC{delta} signaling is coupled to IL-8 expression and secretion in primary cultures of HBEpCs. The role of PKC in S1P-induced IL-8 production has been reported previously (21); however, little information is available on the PKC isoform(s) and downstream target(s) of PKC regulating IL-8 generation. The PKC family of serine/threonine kinases includes three major types of isoenzymes. The classical isoforms ({alpha}, {beta}1, {beta}2, and {gamma}) are activated by diacylglycerol, calcium, and phosphatidylserine, whereas the novel isoforms ({delta}, {epsilon}, {eta}, {theta}, and µ) are calcium-insensitive, but are activated by diacylglycerol and phosphatidylserine. The atypical isoforms ({zeta} and {iota}/{lambda}) are insensitive to calcium and diacylglycerol, but are dependent on phosphatidylserine. In agreement with published results (38), we detected the presence of PKC{alpha}, PKC{delta}, PKC{zeta}, PKC{theta}, and PKC{iota}/{lambda} (but not PKC{beta}, PKC{gamma}, PKC{epsilon}, and PKC{eta}) in HBEpCs (Fig. 5). By infecting cells with adenoviral constructs of dn-PKC{alpha}, dn-PKC{delta}, dn-PKC{zeta}, and dn-PKC{iota}/{lambda}, we observed that the LPA-mediated IL-8 secretion was dependent on PKC{delta} and, to a much lesser degree, on PKC{iota}/{lambda} isoforms (Fig. 6). Furthermore, using MBP as a substrate for PKC{delta}, it was shown that LPA activated PKC{delta} in HBEpCs. Experiments with rottlerin also suggested a role for PKC{delta} in LPA-mediated IL-8 production. Besides the role of PKC{delta} signaling, our data also show the significance of NF-{kappa}B activation by LPA in IL-8 expression and secretion in HBEpCs. This work concurs with other studies suggesting regulation of epithelial cell cytokine secretion by NF-{kappa}B (30). In cells of the A549 respiratory line, rhinovirus-, TNF-{alpha}-, and lipopolysaccharide-mediated IL-8 generation is dependent on NF-{kappa}B activation and translocation to the nucleus (13, 39). In unstimulated cells, NF-{kappa}B exists as p50/p65 heterodimers and is sequestered in the cytoplasm by the I{kappa}B family of kinases, of which I{kappa}B{alpha} is the best characterized. Upon stimulation, IKKs phosphorylate I{kappa}B with subsequent ubiquitination and degradation, leading to the release of NF-{kappa}B and its translocation to the nucleus. The experiments presented here show that LPA stimulates phosphorylation of I{kappa}B, enhances NF-{kappa}B-luciferase reporter gene expression, and translocation of NF-{kappa}B to the nucleus. These data demonstrate very clearly a role for NF-{kappa}B transcriptional regulation of IL-8 secretion by LPA in HBEpCs.

A major finding of this study is the PKC{delta}-dependent activation of NF-{kappa}B and IL-8 secretion induced by LPA in HBEpCs. Earlier reports have implicated several atypical ({zeta} and {iota}/{lambda}) and novel ({delta}, {epsilon}, and {theta}) PKC isoforms in the stimulation of NF-{kappa}B signaling in mammalian cells. In NIH 3T3 cells, PKC{zeta} directly activates IKK{beta} in vitro and regulates the transcriptional activity of NF-{kappa}B (40). A role for PKC{zeta}/NF-{kappa}B signaling in TNF-{alpha}-mediated ICAM expression was shown in endothelial cells (41), whereas targeted disruption of PKC{zeta} attenuates TNF-{alpha}- and IL-1-dependent phosphorylation of p65 and NF-{kappa}B transcriptional activity in mice (42). In contrast to the above studies, we observed that overexpression of dn-PKC{zeta} in HBEpCs had no effect on LPA-induced NF-{kappa}B translocation to the nucleus and slightly increased IL-8 production (Fig. 6B). In this study, selective inhibition of PKC{delta} effectively blocked LPA-dependent phosphorylation of I{kappa}B, NF-{kappa}B translocation to the nucleus, and luciferase reporter gene activity. Consistent with our results in primary cultures of HBEpCs, a recent report suggests a key role for PKC{delta} in the regulation of TNF-{alpha}- and phorbol ester-mediated transcription of granulocyte/macrophage colony-stimulating factor, RANTES (regulated on activation normal T cell expressed and secreted), ICAM-1, and IL-8 in the 16HBE14o human airway epithelial cell line (30). The mechanism(s) by which PKC{delta} activates NF-{kappa}B is unclear. In 16HBE14o cells, overexpression of a dominant-negative mutant of IKK{beta} (IKK{beta}-AA) attenuates PKC{delta} catalytic domain-mediated NF-{kappa}B transactivation, suggesting regulation via IKK signaling (30). However, in thrombin-mediated ICAM-1 gene expression, transactivation of NF-{kappa}B is dependent on the PKC{delta}/p38 MAPK pathway (29). A similar regulation by p38 MAPK in thrombin-induced NF-{kappa}B stimulation and VCAM-1 (vascular cell adhesion molecule-1) expression was observed in human umbilical vein endothelial cells (43). Alternatively, PKC{delta} may directly phosphorylate NF-{kappa}B, resulting in enhanced DNA binding and transcriptional activity. Recent studies suggest that endogenous PKC{delta} translocates to the nucleus after etoposide treatment in C5 cells, suggesting regulation of nuclear event(s) by nuclear import of PKC{delta} (44). Although our results indicate that LPA-induced I{kappa}B phosphorylation and NF-{kappa}B activation are dependent on PKC{delta}, it is unclear whether PKC{delta} activates I{kappa}B directly or via the IKK-mediated pathway. Furthermore, expression of a dominant-negative mutant of IKK{beta} (IKK{beta}-AA) does not completely attenuate bryostatin-1 or PKC{delta} catalytic domain-induced NF-{kappa}B activation in 16HBE14o bronchial epithelial cells, suggesting that PKC{delta} also activates NF-{kappa}B via IKK{beta}-independent pathways (30). Therefore, further studies are necessary to delineate the signaling pathways of LPA-induced NF-{kappa}B activation by PKC{delta} in HBEpCs.

IL-8 is a potent chemoattractant for neutrophils as well as eosinophils, basophils, and T lymphocytes in airways. In airway diseases, severity of inflammation has been correlated with levels of IL-8 in the BAL fluids of various respiratory diseases. We have recently observed that LPA levels in BAL fluids from segmental allergen-challenged asthmatics were significantly higher compared with non-segmental allergen-challenged asthmatics and, furthermore, that the elevation of LPA levels correlated with higher eosinophils in the BAL fluid.2 In this study, increased influx of neutrophils in the alveolar space within the first 6 h of instillation of LPA into the mouse trachea was observed. Interestingly, the neutrophil infiltration was preceded by an elevation of MIP-2, the murine homolog of human IL-8, in BAL fluid within 3 h of LPA instillation. At later time periods (>12 h), the levels of MIP-2 and neutrophils returned to near normal values. These results suggest that accumulation of LPA in the airway induces accumulation of neutrophils and other inflammatory cells at local sites of injury and inflammation. LPA (1-10 µg/ml) instilled into guinea pig airways substantially increased eosinophils and neutrophils in BAL fluid after 4 h of inhalation, which was blocked by Y-27632, an inhibitor of Rho-associated protein kinase (23). The in vivo studies in murine and guinea pig models of airway inflammation indicate that generation of LPA at or near the site of injury can contribute to the activation of inflammatory cells as well the initial process of inflammation. Further studies in our laboratory are in progress to address the source, pathway(s), and amounts of LPA generated in the airway during allergic responses or bronchial asthma.

In summary, our results provide a definitive link between LPA and secretion of the pro-inflammatory cytokine IL-8 in HBEpCs and are reproducible in a murine model of lung injury. We have demonstrated for the first time that LPA-induced IL-8 secretion in HBEpCs is dependent on PKC{delta}-mediated activation of NF-{kappa}B. The importance of LPA as a pro-inflammatory lipid mediator is evidenced by instillation of LPA into the trachea and the demonstration of increased influx of neutrophils and higher MIP-2 levels in the BAL fluid. These results suggest that LPA may be a potent lipid mediator that induces secretion of IL-8 from the bronchial epithelial cells, followed by chemotaxis of neutrophils and other inflammatory cells into the airway. Better understanding of the mechanisms of LPA production by different inflammatory cells in the airway may be relevant to treatment of asthma and other airway inflammatory diseases.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant HL71121 (to V. N.) and Grants HL54659 and HL61013 (to D. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: Dept. of Medicine, Div. of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, MFL 675, Center Tower, 5200 Eastern Ave., Baltimore, MD 21224. Tel.: 410-550-7748; Fax: 410-550-8571; E-mail: vnataraj{at}jhmi.edu.

1 The abbreviations used are: IL-8, interleukin-8; BAL, bronchoalveolar lavage; TNF-{alpha}, tumor necrosis factor-{alpha}; LPA, lysophosphatidic acid; HBEpCs, human bronchial epithelial cells; S1P, sphingosine 1-phosphate; Edg, endothelial differentiation gene; PKC, protein kinase C; ICAM-1, intercellular adhesion molecule-1; NF-{kappa}B, nuclear factor-{kappa}B; MIP-2, macrophage inflammatory protein-2; BSA, bovine serum albumin; PTx, pertussis toxin; BEGM, basal essential growth medium; ELISA, enzyme-linked immunosorbent assay; dn, dominant-negative; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; EMSA, electrophoretic mobility shift assay; MBP, myelin basic protein; ERK, extracellular signal-regulated kinase; IKK, I{kappa}B kinase; MAPK, mitogen-activated protein kinase. Back

2 E. Berdyshev, S. Georas, A. J. Morris, R. Cummings, W. Hubbard, M. Liu, and V. Natarajan, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Shigeo Ohno (Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama City, Japan) for providing the dn-PKC{lambda} adenoviral construct. We thank Dr. Sekhar Reddy (Johns Hopkins University Bloomberg School of Public Health) for helpful discussions regarding the NF-{kappa}B EMSA protocol. The secretarial assistance of Leslie Gregg is appreciated.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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