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B Activation and IL-8/CXCL8 Expression in Lung Epithelial Cells1
* Graduate Institute of Medical Sciences,
Graduate Institute of Biomedical Technology,
Department of Microbiology and Immunology,
School of Respiratory Therapy, College of Medicine, and
¶ Graduate Institute of Nursing, College of Nursing, Taipei Medical University, Taipei, Taiwan
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Abstract |
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B activation and IL-8/CXCL8 expression by thrombin in human lung epithelial cells (EC). Thrombin caused a concentration-dependent increase in IL-8/CXCL8 release in a human lung EC line (A549) and primary normal human bronchial EC. In A549 cells, thrombin, SFLLRN-NH2 (a protease-activated receptor 1 (PAR1) agonist peptide), and GYPGQV-NH2 (a PAR4 agonist peptide), but not TFRGAP-NH2 (a PAR3 agonist peptide), induced an increase in IL-8/CXCL8-luciferase (Luc) activity. The thrombin-induced IL-8/CXCL8 release was attenuated by D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (a thrombin inhibitor), U73122 (a phosphoinositide-phospholipase C inhibitor), Ro-32-0432 (a protein kinsase C 
(PKC) inhibitor), an NF-B inhibitor peptide, and Bay 117082 (an IB phosphorylation inhibitor). Thrombin-induced increase in IL-8/CXCL8-Luc activity was inhibited by the dominant-negative mutant of c-Src and the cells transfected with the B site mutation of the IL-8/CXCL8 construct. Thrombin caused time-dependent increases in phosphorylation of c-Src at tyrosine 416 and c-Src activity. Thrombin-elicited c-Src activity was inhibited by Ro-32-0432. Stimulation of cells with thrombin activated IB kinase 
(IKK), IB phosphorylation, IB degradation, p50 and p65 translocation from the cytosol to the nucleus, NF-B-specific DNA-protein complex formation, and B-Luc activity. Pretreatment of A549 cells with Ro-32-4032 and the dominant-negative mutant of c-Src DN inhibited thrombin-induced IKK activity, B-Luc activity, and NF-B-specific DNA-protein complex formation. Further studies revealed that thrombin induced PKC, c-Src, and IKK complex formation. These results show for the first time that thrombin, acting through PAR1 and PAR4, activates the phosphoinositide-phospholipase C/PKC/c-Src/IKK signaling pathway to induce NF-B activation, which in turn induces IL-8/CXCL8 expression and release in human lung EC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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70 cm2 in surface area, is now recognized as not only the boundary of ambient air along the bronchial tree, but also as an active biomodulator via its production of a variety of proinflammatory mediators (1, 2, 3). Type II alveolar epithelial cells (EC)3are known to indirectly contribute to immune responses during bacterial infection through its product surfactant, protein A, which enhances the phagocytosis of bacteria by macrophages (4). In addition, lung EC also directly contribute to immune responses by secreting bioactive substances. Lung EC have been shown to secrete IL-6, PGE2, and IL-8/CXCL8 when exposed to stimulation by inflammatory mediators (5, 6, 7, 8). Among the bioactive substances produced by lung EC, IL-8/CXCL8 has been found to be one of the most important mediators of airway inflammation (9, 10).
IL-8/CXCL8 is a member of the CXC chemokine family that plays a pivotal role in controlling neutrophil and monocyte chemotaxis toward sites of infection and induced airway inflammation (9, 10, 11). IL-8/CXCL8 is abundant in sputum from patients with chronic bronchitis, bronchiectasis, and cystic fibrosis, and in bronchoalveolar lavage (BAL) fluid from patients with diffuse panbronchiolitis (10, 12) and is related to the neutrophil chemoattractant activity exhibited in these diseases. IL-8/CXCL8 expression and secretion have been shown to be dependent on transcriptional activation of the IL-8/CXCL8 gene in a number of cell types (13, 14). Many studies have revealed that a sequence spanning –1 to –133 nucleotides within the 5'-flanking region of the IL-8/CXCL8 gene contains binding sites for a number of important transcription factors including NF-B, NF-IL-6, and AP-1 (15). NF-B, which comprises a family of Rel-related proteins that are normally retained in the cytoplasm bound to inhibitor IBs, is the most important transcription factor to regulate IL-8/CXCL8 expression (16). Following cellular activation, IB kinases (IKKs) are activated and phosphorylate IB at serine (Ser)32 and Ser36 and IB at Ser19 and Ser23 (17, 18, 19) to produce ubiquitination of IB at lysine residues and degradation by the 26S proteasome (20). This process releases active NF-B, which is then translocated from the cytosol to the nucleus, to bind the IL-8/CXCL8 B-binding site at position –80 to –71 and induce IL-8/CXCL8 transcription (16). A previous report showed that thrombin-induced NF-B activation is mediated through protease-activated receptor 1 (PAR1)-dependent multiple signaling molecules, including protein kinase C-
(PKC-), p38 MAPK, and IKK signaling pathways (21, 22, 23), but little information is available about the role of c-Src in regulating epithelial NF-B activation and IL-8/CXCL8 expression following thrombin stimulation.
Thrombin, a multifunctional serine protease generated at sites of vascular injury and well known for its pivotal role in the coagulation cascade, not only contributes to tissue repair, but also promotes a wide range of cellular responses including modulation of lung inflammation (24). Vascular injury, which is an early event in most inflammatory lung diseases, is characterized by disruption of endothelial cells and damage to the intima of blood vessels (25). During this state, thrombin may leave the circulation and become activated when it becomes part of the BAL fluid. For example, thrombin levels and activities are increased in BAL fluid from patients with acute respiratory distress syndrome and pneumonia, and in asthma patients promptly after inhalation of an allergen (26, 27). Thrombin also plays an important role in acute lung inflammation such as inducing the accumulation of neutrophils into the airway and elevation of TNF- levels in BAL fluid from thrombin-inhaled mice (28). Moreover, thrombin stimulates contraction of human bronchial rings (29). In addition, several reports have shown that thrombin can induce PGE2, IL-6, and IL-8/CXCL8 in lung EC (6, 8). Thus, the presence of thrombin in airway fluids appears to be a common feature of a variety of inflammatory lung diseases and plays critical roles in lung inflammation. However, little is known about how thrombin regulates IL-8/CXCL8 expression and release in lung EC. Many of the biological activities mediated by thrombin are attributed to cleavage of the G protein-coupled thrombin G protein-coupled receptors (GPCR) named PARs. To the present, four different PARs (PAR1–4) have been cloned (30, 31, 32, 33), and each receptor has been shown to modulate a variety of pathophysiological processes such as cytokine and chemokine release, platelet aggregation, and cellular proliferation (34). Thrombin activates PAR1, PAR3, and PAR4, whereas PAR2 does not seem to be activated by thrombin but by trypsin, tryptase, and cofactor Xa (35). Thrombin or other peptidases activate PARs via cleavage of the extracellular N-terminal domain, which then enables the new N terminus to interact distally with the same molecule to activate G protein-coupled signal pathways and subsequently induce cellular responses (35). A previous report showed that thrombin can induce IL-8/CXCL8 release in human lung EC (6, 8). However, the signal transduction events, especially the PI-PLC/PKC/c-Src/IKK pathway, which lead to NF-B activation and IL-8/CXCL8 expression by thrombin are unclear. In the present study, our results demonstrate for the first time that thrombin acts on PAR1 and PAR4 to induce PKC, c-Src, and IKK complex formation, and subsequently activates the IKK and NF-B signaling pathways and ultimately induces IL-8/CXCL8 expression and secretion in lung EC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Thrombin (from bovine plasma) and enolase were purchased from Sigma-Aldrich. TNF- was obtained from PeproTech. SFLLRN-NH2 (a PAR1 agonist peptide), TFRGAP-NH2 (a PAR3 agonist peptide), and GYPGQV-NH2 (a PAR4 agonist peptide) were purchased from Bachem. D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone(PPACK), U73122, and Ro-32-0432 were obtained from Calbiochem. Bay 117082 was obtained from Alexis. The NF-B inhibitor peptide (in a cell-permeable form) was purchased from BIOMOL. A dominant-negative mutant of c-Src (c-Src DN) was purchased from Upstate Biotechnology. pGL2-ELAM-luciferase (Luc) (which is under the control of one NF-B binding site) and pBK-CMV-LacZ were provided by Dr. W.-W. Lin (National Taiwan University, Taipei, Taiwan). IL-8/CXCL8 wild-type (–133)-Luc (IL-8/CXCL8 wt-Luc) and the B site mutant of IL-8/CXCL8-Luc (B-mt-IL-8/CXCL8-Luc) were provided by Dr. N. Mukaida (Kanazawa University, Kanazawa, Japan). DMEM/Ham’s F-12, FCS, penicillin/streptomycin, and Lipofectamine Plus reagent were purchased from Invitrogen Life Technologies. Lung EC A549 were obtained from American Type Culture Collection. Primary normal human bronchial EC (NHBEC) and BEC growth medium were obtained from Clonetics. An Ab specific for PKC was purchased from Transduction Laboratories. An Ab specific for c-Src phosphorylated at typrosine (Tyr)416 was purchased from Cell Signaling and Neuroscience. IL-8/CXCL8, keratinocyte-derived chemokine (KC), and MIP-2 ELISA kits were obtained from R&D Systems. Protein A/G beads, IB protein (amino acids 1–317), rabbit IgG, Abs specific for IB, IB phosphorylated at Ser32, IKK, IKK, c-Src, and anti-mouse and anti-rabbit IgG-conjugated HRP were purchased from Santa Cruz Biotechnology. [
-32P]ATP (6000 Ci/mmol) was purchased from Amersham Biosciences. All materials for SDS-PAGE were purchased from Bio-Rad.
Cell culture
A549 cells were cultured in DMEM/Ham’s F-12 nutrient mixture containing 10% FCS, 100 U/ml penicillin G, and 100 µ/ml streptomycin in a humidified 37°C incubator. After reaching confluence, cells were seeded onto 6-cm dishes for immunoblotting, kinase assays, or EMSA; 12-well plates for cell transfection, B-Luc, and IL-8/CXCL8-Luc assays; or 24-well plates for IL-8/CXCL8 assays. The NHBEC were expanded and cultured according to the protocol provided by Clonetics. NHBEC at passages 3–7 were seeded on T-25 culture flasks (2 x 104 cells/cm2) and grown in NHBEC growth medium supplemented with 35 µg/ml bovine pituitary extract, 5 µg/ml insulin, 0.07 µg/ml hydrocortisone, 10 µg/ml transferrin, 6.73 ng/ml triiodothyronine, 0.5 µg/ml epinephrine, 5 ng/ml human epidermal growth factor, 0.1 µg/ml retinoic acid, and 50 ng/ml gentamicin in a humidified 37°C incubator. The medium was changed every 48 h until cells were 80% confluent. Cells were then passaged and seeded onto 24-well plates for the IL-8/CXCL8 assays.
IL-8/CXCL8 measurement
A549 cells or NHBEC (1 x 105 cells per well) were seeded onto 24-well plates in DMEM/Ham’s F-12 with 10% FCS or NHBEC growth medium supplemented with growth factors, respectively, overnight. The next day, the growth medium was removed and replaced with 0.5 ml of basal medium devoid of FCS or growth factors before drug treatment. IL-8/CXCL8 released into the culture medium after thrombin (10 U/ml) or TNF- (10 ng/ml) treatment or after being pretreated with specific inhibitors as indicated followed by thrombin stimulation was assayed using the IL-8/CXCL8 ELISA kit following the manufacturer’s instructions. After finishing, cells were detached, and cell numbers were determined by a trypan blue assay. Lung EC exposed to the various inhibitors were analyzed for cell cytotoxic reactions using the MTT assay as described previously (36).
Assessment of the KC or MIP-2 concentration in BAL fluid
Male specific pathogen-free BALB/c mice, aged 6–8 wk and weighing 20–25 g, were obtained from the Laboratory Animal Center, National Taiwan University (Taipei, Taiwan) and maintained in a specific pathogen-free environment in the animal house of Taipei Medical University. The use of animals was approved by the Institutional Animal Care and Use Committee of Taipei Medical University. Mice were anesthetized with sodium pentobarbital (55 mg/kg body weight) given i.p. Saline or thrombin (1000 U/kg body weight in 15 µl) was administrated by a single intratracheal injection as described previously (28, 37). At 24 h after administration, the mice were killed by i.p. overdoses of sodium pentobarbital to take the BAL fluid. The trachea was exposed and cannulated for BAL with 2 x 0.5-ml volumes of PBS, and 0.7–0.8 ml of BAL fluid was retrieved per mouse. The BAL fluid was centrifuged at 3000 x g for 5 min, and the supernatant was then collected for KC or MIP-2 measurement. KC or MIP-2 levels in BAL fluid were measured by using mouse specific KC or MIP-2 ELISA kit, respectively, according to manufacturer’s instructions.
Transfection and B- and IL-8/CXCL8-Luc assays
A549 cells (2 x 105 cells per well) were seeded onto 12-well plates, and cells were transfected the following day using Lipofectamine Plus with 0.5 µg of pGL2-ELAM-Luc, 0.2 µg of IL-8/CXCL8 wt-Luc, or 0.2 µg of B-mt-IL-8/CXCL8-Luc and 0.5 µg of pBK-CMV-LacZ. After 24 h, the medium was aspirated and replaced with fresh DMEM/Ham’s F12 containing 10% FBS and then stimulated with thrombin (10 U/ml), SFLLRN-NH2 (300 µM), TFRGAP-NH2 (300 µM), or GYPGQV-NH2 (300 µM) for another 24 h before being harvested. To assess the effects of the indicated inhibitors, drugs were added to cells 20 min before thrombin addition. To assay the effect of c-Src DN, cells were cotransfected with c-Src DN, pGL2-ELAM-Luc, IL-8/CXCL8-wt-Luc, and pBK-CMV-Lac Z. Luc activity was determined with a Luc assay system (Promega), and was normalized on the basis of Lac Z expression. The level of induction of Luc activity was compared as a ratio to cells with and without stimulation.
Western blot analysis
To determine the expression of IKK phosphorylation at Ser180 (IKK) or Ser181 (IKK), IB phosphorylation at Ser32, IKK/, IB, c-Src phosphorylation at Tyr416, and c-Src in A549 cells, proteins were extracted, and Western blot analysis was performed as described previously (38). Briefly, A549 cells were cultured in 6-cm dishes. After reaching confluence, cells were treated with vehicle and thrombin, or pretreated with specific inhibitors as indicated followed by thrombin. After incubation, cells were washed twice in ice-cold PBS and solubilized in extraction buffer containing 10 mM Tris (pH 7.0), 140 mM NaCl, 2 mM PMSF, 5 mM DTT, 0.5% Nonidet P-40, 0.05 mM pepstatin A, and 0.2 mM leupeptin. Samples of equal amounts of protein (80 µg) were subjected to SDS-PAGE, then transferred onto a polyvinylidene difluoride membrane which was then incubated in TBST buffer (150 mM NaCl, 20 mM Tris-HCl, and 0.02% Tween 20; pH 7.4) containing 5% BSA. Proteins were visualized by specific primary Abs and then incubated with HRP-conjugated secondary Abs. The immunoreactivity was detected using ECL following the manufacturer’s instructions. Quantitative data were obtained using a computing densitometer with scientific imaging systems (Kodak).
Analysis of PKC translocation
For the detection of PKC translocation, cytosolic and membrane fractions were separated as described previously (39). Briefly, A549 cells were treated with thrombin for the indicated time intervals. After incubation, cells were placed on ice, rinsed with PBS, resuspended in homogenization buffer (20 mM Tris-HCl, 0.5 mM EGTA, 2 mM EDTA, 2 mM DTT, 0.5 mM PMSF, and 10 µg/ml leupeptin (pH 7.5)) and sonicated. The lysate was separated into cytosolic and membrane fractions by centrifugation at 40,000 x g for 45 min. Protein levels of PKC in the cytosolic and membrane fractions were determined by Western blot analysis performed as described above.
Immunoprecipitation and protein kinase assays
A549 cells were grown in 6-cm dishes. After reaching confluence, cells were treated with 10 U/ml thrombin for the indicated time intervals or pretreated with specific inhibitors as indicated followed by thrombin. After incubation, cells were washed twice with ice-cold PBS, lysed in 1 ml of lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 125 mM NaCl, 1% Triton X-100, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 25 mM -glycerophosphate, 50 mM NaF, and 100 µM sodium orthovanadate, and centrifuged. The supernatant was then immunoprecipitated with polyclonal Abs against IKK, IKK, or c-Src in the presence of A/G-agarose beads overnight. The beads were washed three times with lysis buffer and two times with kinase buffer containing 20 mM HEPES (pH 7.4), 20 mM MgCl2, and 2 mM DTT. The kinase reactions were performed by incubating immunoprecipitated beads with 20 µl of kinase buffer supplemented with 20 µM ATP and 3 µCi of [-32P]ATP at 30°C for 30 min. To assess the IKK and c-Src activities, 0.5 µg of GST-IB protein (aa 1–317) and 1 µg of acetic acid-denatured enolase (40, 41) were respectively added as the substrates. The reaction mixtures were analyzed by 12% SDS-PAGE followed by autoradiography.
Preparation of nuclear extracts and the EMSA
A549 cells were cultured in 6-cm dishes. After reaching confluence, cells were treated with vehicle or 10 U/ml thrombin for various time intervals, and then cells were scraped and collected. In some experiments, cells were transfected with the c-Src DN for 24 h before thrombin treatment. The cytosolic and nuclear protein fractions were then separated as described previously (42). Briefly, cells were washed with ice-cold PBS and pelleted. Cell pellets were resuspended in hypotonic buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 0.5 mM DTT, 10 mM aprotinin, 10 mM leupeptin, and 20 mM PMSF) for 15 min on ice, and vortexed for 10 s. Nuclei were pelleted by centrifugation at 15,000 x g for 1 min. Supernatants containing cytosolic proteins were collected. A pellet containing nuclei was resuspended in hypertonic buffer (20 mM HEPES (pH 7.6), 25% glycerol, 1.5 mM MgCl2, 4 mM EDTA, 0.05 mM DTT, 10 mM aprotinin, 10 mM leupeptin, and 20 mM PMSF) for 30 min on ice. Supernatants containing nuclear proteins were collected by centrifugation at 15,000 x g for 2 min and then stored at –70°C. The protein levels of p50 and p65 in the cytosolic and nuclear fractions were determined by Western blot analysis performed as described.
A double-stranded oligonucleotide probe containing NF-B sequences (5'-AGTTGAGGGGACTTTCCCAGGC-3'; Promega) was purchased and end-labeled with [-32P]ATP using T4 polynucleotide kinase. The nuclear extract (2.5–5 µg) was incubated with 1 ng of a 32P-labeled NF-B probe (50,000–75,000 cpm) in 10 µl of binding buffer containing 1 µg of poly(dI:dc), 15 mM HEPES (pH 7.6), 80 mM NaCl, 1 mM EDTA, 1 mM DTT, and 10% glycerol at 30 °C for 25 min. DNA/nuclear protein complexes were separated from the DNA probe by electrophoresis on 5% polyacrylamide gels. Gels were vacuum dried and subjected to autoradiography with an intensifying screen at –80°C. For competition experiments, 1 ng of the labeled oligonucleotide was mixed with 50 ng of unlabeled competitor oligonucleotides before protein addition. For the supershift experiments, either 4 µg of Abs specific for p65 or p50 or 4 µg of rabbit polyclonal IgG was mixed with the nuclear extract proteins.
Statistical analysis
Results are presented as the mean ± SE from at least three independent experiments. One-way ANOVA followed by, when appropriate, Bonferroni’s multiple range test was used to determine the statistical significance of the difference between means. Values of p <0.05 were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Incubation of A549 cells or NHBEC for 24 h with thrombin (0.3–10 U/ml) produced a concentration-dependent increase in IL-8/CXCL8 release. In A549 cells or NHBEC, 24 h of thrombin (10 U/ml) treatment induced IL-8/CXCL8 release from 720 ± 85 to 2275 ± 199 pg/ml or from 313 ± 25 to 627 ± 27 pg/ml, respectively (n = 3) (Fig. 1, A and B). Similarly, 10 ng/ml TNF-, a potent lung epithelium IL-8/CXCL8 stimulator (43), increased IL-8/CXCL8 release by 325 ± 15% (n = 3) (Fig. 1A). There is a lack of IL-8/CXCL8 gene in mice, and it has been shown that the mouse KC and MIP-2 are functional homologs of human IL-8/CXCL8 (44, 45). Therefore, we further determine whether thrombin elicited KC and MIP-2 release in BAL fluid in mice, thrombin (1000 U/kg body weight) was introduced into the trachea lumen by intratracheal administration. As shown in Fig. 1C, thrombin induced an increase in KC and MIP-2 release in BAL fluid from 172 ± 28 to 395 ± 27 pg/ml and 363 ± 76 to 885 ± 82 pg/ml, respectively (Fig. 1C). Thrombin-induced IL-8/CXCL8 release obtained with A549 cells being similar to that of the primary NHBEC response supported the use of A549 cells for more detailed studies. To further confirm whether thrombin can induce IL-8/CXCL8 expression, A549 cells were transiently transfected with a human IL-8/CXCL8 wild-type (–133)-Luc (IL-8/CXCL8 wt-Luc) plasmid as an indicator of IL-8/CXCL8 expression. As shown in Fig. 1D, A549 cells treated with 10 U/ml thrombin for 24 h showed an increase in IL-8/CXCL8-Luc activity of 320 ± 34% (n = 3). Moreover, both SFLLRN-NH2 (a PAR1 agonist peptide, 300 µM) and GYPGQV-NH2 (a PAR4 agonist peptide, 300 µM) also induced increases in IL-8/CXCL8-Luc activity of 235 ± 21% and 227 ± 26%, respectively (n = 3). In contrast, TFRGAP-NH2 (a PAR3 agonist peptide, 300 µM) did not induce IL-8/CXCL8-Luc activity (Fig. 1D). These results suggest that PAR1 and PAR4 may be involved in thrombin-induced epithelium IL-8/CXCL8 expression and release.
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Previous studies demonstrated that thrombin couples with PARs which induce the activation of phospholipase C- (PLC-), triggering PI turnover which results in increasing intracellular calcium mobilization and activation of PKC (35). To understand the connection between IL-8/CXCL8 release of thrombin and its PI turnover signaling pathway, the thrombin inhibitor, PPACK (46), and the PI-PLC inhibitor, U73122 (47), were tested. As shown in Fig. 2A, treatment of cells with 100 nM PPACK and 10 µM U73122 inhibited thrombin-induced IL-8/CXCL8 release by 63 ± 4% and 83 ± 3%, respectively (n = 3). A previous report showed that PKC activation was implicated in the control of IL-8/CXCL8 release by IL-1 (48). To further explore whether PKC might play a crucial role in thrombin-induced IL-8/CXCL8 release, the selective PKC inhibitor, Ro-32-0432 (49), was used. Fig. 2A shows that thrombin-induced IL-8/CXCL8 release was inhibited by Ro-32-0432 (3 and 10 µM) in a concentration-dependent manner. When cells were treated with 10 µM Ro-32-0432, thrombin-induced IL-8/CXCL8 release was inhibited by 63 ± 4% (n = 3) (Fig. 2A). However, 10 µM of Ro-32-0432 alone did not affect the basal IL-8/CXCL8 level. In addition, treatment of cells with PPACK (100 nM), U73122 (10 µM), and Ro-32-0432 (10 µM) did not affect cell viability, which was assessed by the MTT assay (data not shown). We next attempted to determine whether thrombin activates PKC by examining the translocation of PKC from the cytosolic to the membrane fraction using Western blot analysis. Stimulation of cells with 10 U/ml thrombin for 0–30 min resulted in translocation of PKC from the cytosolic fraction to the membrane fraction beginning at 5 min, and the effect was sustained to 30 min (Fig. 2B).
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To examine whether c-Src, a target protein for PKC (50), might play a crucial role in thrombin-induced IL-8/CXCL8 expression, the c-Src DN plasmid was used. As shown in Fig. 3A, transfection of A549 cells with c-Src DN inhibited the thrombin-induced increase in IL-8/CXCL8-Luc activity, with 1 µg of c-Src DN reducing the basal IL-8/CXCL8-Luc activity and thrombin response by 58 ± 12% and 90 ± 10% (n = 3), respectively. Regulation of c-Src activation occurs as a result of multiple phosphorylation sites on specific residues, including Tyr416 and Tyr527 (51). The major phosphorylation site of c-Src at the Tyr416 residue results in activation from c-Src autophosphorylation (51). Thus, phosphorylation of c-Src at Tyr416 is a critical step in c-Src activation. We further examined c-Src phosphorylation at Tyr416 by thrombin stimulation in A549 cells using the anti-phospho-c-Src Ab at Tyr416. As shown in Fig. 3B, treatment of A549 cells with 10 U/ml thrombin resulted in a time-dependent phosphorylation of c-Src at Tyr416. The c-Src phosphorylation at Tyr416 began at 3 min and was sustained until 30 min after thrombin stimulation (Fig. 3B, upper panel). The protein level of c-Src was not affected by thrombin treatment (Fig. 3B, lower panel). Next, we directly examined c-Src kinase activity in response to thrombin. In vitro c-Src kinase activity was measured using enolase as a c-Src exogenous substrate (40, 41). Fig. 3C shows that treatment of A549 cells with 10 U/ml thrombin induced an increase in c-Src activity in a time-dependent manner. The c-Src activity began at 5 min, peaked at 10–20 min, and then declined to 30 min after thrombin stimulation (Fig. 3C, upper panel). The protein level of c-Src was not affected by thrombin treatment (Fig. 3C, lower panel). In addition, we found that thrombin-induced c-Src activity was inhibited in cells pretreated with Ro-32-0432 (10 µM) (Fig. 3D). Based on these results, we suggest that PKC-dependent c-Src activation is required for thrombin-induced IL-8/CXCL8-Luc activity.
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B is involved in thrombin-induced IL-8/CXCL8 release
As mentioned previously, NF-B activation is important for IL-8/CXCL8 expression (52). To examine whether NF-B activation is involved in thrombin-induced IL-8/CXCL8 release, a cell-permeable NF-B inhibitor peptide was used. Fig. 4A shows that A549 cells pretreated with 10 µg/ml the NF-B inhibitor peptide inhibited thrombin-induced IL-8/CXCL8 release by 79 ± 4% (n = 3). Furthermore, A549 cells pretreated with 10 µM Bay 117082, an IB phosphorylation inhibitor (53), abolished thrombin-induced IL-8/CXCL8 release by 94 ± 5% (n = 3) (Fig. 4A). In addition, treatment of cells with the NF-B inhibitor peptide (10 µg/ml) and Bay 117082 (10 µM) did not affect cell viability (data not shown). NF-B activation was evaluated by the translocation of NF-B from the cytosol to the nucleus and a gel shift DNA-binding assay. Treatment of A549 cells with 10 U/ml thrombin resulted in marked translocation of p50 and p65 from the cytosol to the nucleus in a time-dependent manner, with a maximal effect after 30–60 min of treatment (Fig. 4B). Similarly, stimulation of cells with 10 U/ml thrombin resulted in an increase in NF-B-specific DNA protein complex formation, which began at 20 min and peaked at 60 min (Fig. 4C). To identify the specific subunits involved in the formation of the NF-B complex, supershift assays were performed using Abs specific for anti-p65 or anti-p50. Incubation of nuclear extracts with anti-p65 or anti-p50 attenuated NF-B-specific DNA-protein complex formation (Fig. 4C, lanes 8 and 9). However, incubation of nuclear extracts with control rabbit IgG had no effect on the formation of NF-B-specific DNA-protein complex (Fig. 4C, lane 7). These results indicated that the components of p50 and p65 exist in the NF-B complex. This gel shift assay detected a specific band produced in the presence of thrombin, which was outcompeted by an unlabeled 50x competitive probe, compared with thrombin treatment for 60 min (Fig. 4C, lanes 6 and 10). To directly determine NF-B activation after thrombin treatment, A549 cells were transiently transfected with pGL2-ELAM-B-Luc as an indicator of NF-B activation. Stimulation of A549 cells with thrombin (0.3–10 U/ml) induced an increase in B-Luc activity in a concentration-dependent manner, with an increase in B-Luc activity of 311 ± 16% at 10 U/ml thrombin treatment (data not shown). To further confirm that NF-B is necessary for thrombin-induced IL-8/CXCL8 expression, the IL-8/CXCL8 wt-Luc plasmid or B site mutant of the IL-8/CXCL8-Luc (B-mt-IL-8/CXCL8-Luc) plasmid was transfected into A549 cells. As shown in Fig. 4D, 10 U/ml thrombin induced a 3.3-fold increase in IL-8/CXCL8-Luc activity in cells transfected with the IL-8/CXCL8 wt-Luc construct. Thrombin-induced IL-8/CXCL8-Luc activity was reduced by 71 ± 10% (n = 3) in cells transfected with the B-mt-IL-8/CXCL8-Luc construct. These results indicate that NF-B activation is necessary for thrombin-induced IL-8/CXCL8 expression and release in A549 cells.
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activity, IB phosphorylation, and IB degradation
To further determine the upstream molecules involved in thrombin-induced NF-B activation, stimulation of cells with 10 U/ml thrombin induced an increase in IKK phosphorylation and activity in a time-dependent manner, reaching maximums after 10–30 and 20–30 min of treatment, respectively (Fig. 5, A and B). Similar to stimulation of IL-8/CXCL8-Luc activity, 300 µM SFLLRN-NH2 and 300 µM GYPGQV-NH2 induced an increase in IKK phosphorylation, but 300 µM TFRGAP-NH2 had no effect (Fig. 5C). In parallel with IKK phosphorylation and activity, 10 U/ml thrombin-induced IB phosphorylation increased beginning at 10 min and peaked at 30 min, and this was sustained to 60 min (Fig. 5D). IB degradation was apparent after 10 min of treatment with 10 U/ml thrombin, and the IB protein was resynthesized after 60 min of treatment (Fig. 5E).
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and c-Src mediate thrombin-induced IKK and NF-B activities
To further investigate whether thrombin-induced IKK and NF-B activities occur through PKC and c-Src, A549 cells were pretreated for 30 min with Ro-32-0432 (10 µM) or transiently transfected with 1 µg of c-Src DN, which inhibited the thrombin-induced increase in IKK activity as shown in Fig. 6, A and B. Neither of these inhibitors affected the basal IKK expression. Furthermore, the thrombin-induced increase in B-Luc activity was also inhibited by treatment with Ro-32-0432 (3 and 10 µM) in a concentration-dependent manner. When cells were treated with 10 µM Ro-32-0432, the thrombin-induced increase in B-Luc activity was inhibited by 61 ± 4% (n = 3) (Fig. 6C). Similarly, the thrombin-induced increase in B-Luc activity and NF-B-specific DNA-protein complex formation also were inhibited by c-Src DN in a concentration-dependent manner. c-Src DN (1 µg) almost completely inhibited the thrombin-induced increase in B-Luc activity and NF-B-specific DNA-protein complex formation (Fig. 6, D and E). In addition, thrombin-induced c-Src phosphorylation at Tyr416 was markedly inhibited by transfection of cells with 0.5 or 1 µg of c-Src DN (Fig. 6F). Furthermore, pretreatment of A549 cells with PPACK (100 nM) and U73122 (10 µM) inhibited thrombin-induced B-Luc activity by 41 ± 3% and 77 ± 6%, respectively (n = 3) (Fig. 6C). Taken together, these data suggest that PKC-dependent c-Src activation is required for thrombin-induced IKK and NF-B activities in lung EC A549.
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is associated with IKK by c-Src upon thrombin stimulation
Next, we attempted to investigate whether thrombin can induce the interaction between PKC and IKK. As shown in Fig. 7, treatment of A549 cells with 10 U/ml thrombin led to the association of PKC and IKK, as detected by immunoblotting using the Ab to PKC after immunoprecipitation of IKK. The association of PKC and IKK occurred at 5 min and was sustained to 30 min (Fig. 7A). A previous report showed that c-Src can interact with IKK and IKK to mediate TNF--induced cyclooxygenase-2 (COX-2) expression (41). Therefore, we further examined the association between c-Src and IKK in thrombin stimulation. Fig. 7B shows that 10 U/ml thrombin treatment increased the complex formation of IKK and c-Src at 5 min, which was sustained for 20 min. The interaction between c-Src and IKK was further confirmed by converse experiments in which the c-Src and IKK complex was immunoprecipitated with a c-Src Ab and immunoblotted with an IKK Ab (Fig. 7C). We also found that treatment of A549 cells with thrombin increased the formation of PKC and c-Src at 5 min, which declined from 10 to 30 min (Fig. 7D). These results suggest that thrombin induces IKK activation by interacting with PKC and c-Src in lung EC.
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/c-Src/IKK/NF-B signal pathway. It is known that increased thrombin production occurs in the lungs of most patients with pulmonary inflammatory diseases and that thrombin plays a critical role in both physiological and pathological processes. For example, in the lungs and airway, generated thrombin modulates the tissue repair response by altering the vascular permeability, stimulating the secretion of proteases, and promoting fibroblast and smooth muscle cell adhesion, spread, and proliferation (54). In addition, thrombin plays a pathological role in lung inflammation by stimulating the secretion of proinflammatory mediators such as IL-6, IL-8/CXCL8, and PGE2 in lung EC and fibroblasts (5, 6, 8). In this present study, we showed that thrombin induced increases in IL-8/CXCL8 release and IL-8/CXCL8-Luc activity in lung EC. Two murine functional homologues of IL-8/CXCL8 (KC and MIP-2) have been implicated as the dominant mediators of lung inflammation (55). In this study, we also found that KC and MIP-2 were released into BAL fluid by thrombin-treated mice in vivo.
In humans, the IL-8/CXCL8 gene promoter contains multiple potential regulatory transcription factor binding sites, including NF-B, AP-1, and NF-IL-6 (15). Recent studies have suggested that NF-B plays a critical role in the regulation of pulmonary EC IL-8/CXCL8 gene expression. In human colonic EC, neurotensin-induced IL-8/CXCL8 expression requires 
-dependent NF-B activation (56). In A549 cells, deletion of the NF-B promoter sequence was shown to reduce respiratory syncytial virus- and rhinovirus-mediated IL-8/CXCL8 transcriptional activities (57, 58). The results of this study showed that NF-B activation is essential for IL-8/CXCL8 expression and secretion stimulated by thrombin. This is based on the thrombin-induced increases in IKK activation, IB phosphorylation, IB degradation, p50 and p65 translocation from the cytosol to the nucleus, NF-B-specific DNA-protein complex formation, and B-Luc activity. Furthermore, the NF-B inhibitor peptide and Bay 117082, both inhibitors of the NF-B signaling pathway, reduced thrombin-induced IL-8/CXCL8 expression and release. Moreover, mutational analysis demonstrated that thrombin-induced IL-8/CXCL8-Luc was attenuated by transfection with the B-mt-IL-8/CXCL8-Luc construct. Therefore, these results suggest that NF-B activation is required for IL-8/CXCL8 expression by thrombin in human airway EC. Thrombin is known to activate three PARs, including PAR1, PAR3, and PAR4. Upon thrombin stimulation, activated PARs couple with the G protein and activate downstream signaling molecules to mediate IL-8/CXCL8 expression in many cell types. Several reports have demonstrated that thrombin activates PAR1 to induce IL-8/CXCL8 expression (46, 59). In this study, we found that SFLLRN-NH2 (a PAR1 agonist peptide) and GYPGQV-NH2 (a PAR4 agonist peptide) mimic the thrombin-induced increase in IL-8/CXCL8-Luc activity and IKK phosphorylation. A previous study demonstrated that PAR3 is expressed in A549 cells (8). However, the PAR3 agonist peptide, TFRGAP-NH2, did not induce significant IL-8/CXCL8-Luc activity or IKK phosphorylation. These results suggest that PAR1 and PAR4 are involved in thrombin-induced IL-8/CXCL8 expression and release from A549 cells. Consistent with our findings, Asokananthan et al. (8) showed that in A549, BEAS-2B, and primary HBEC, PAR1 and PAR4, but not PAR3, agonist peptides induce increases in IL-6 and IL-8/CXCL8 release. Moreover, Kataoka et al. (60) also reported that in mouse vascular EC, both PAR1 and PAR4 appear to account for thrombin induction of PI turnover, increases in intracellular calcium, ERK phosphorylation, and c-fos expression Thus, we suggest that thrombin may mediate the induction of IKK phosphorylation, NF-B activation, and IL-8/CXCL8 expression in A549 cells through PAR1 and PAR4.
Thrombin-induced cellular effects in different kinds of cells, including human lung EC, are primarily mediated by means of GPCR known as PAR. Proteolytic cleavage of PARs by thrombin leads to activation of PI-PLC, causing the rapid hydrolysis of PIP2 and generation of two important second messengers: IP3 and DAG. Two bifurcate signaling cascades involved in thrombin-triggered PI-breakdown, i.e., IP3-mediated intracellular calcium and DAG-mediated PKC activation, have been established and are known to regulate a variety of cellular functions (35). In this study, we showed that the PI-PLC downstream signaling pathway is involved in thrombin-induced NF-B activation and IL-8/CXCL8 expression in human lung EC. A549 cells pretreated with PPACK, a thrombin inhibitor, and U73122, a PI-PLC inhibitor, inhibited thrombin-induced B-Luc activity and IL-8/CXCL8 release, suggesting that thrombin responses are PI-PLC dependent.
To date, at least 12 PKC isoforms have been characterized at the molecular level, and these were found to mediate several cellular responses (59, 61, 62). Ludwicka-Bradley et al. (59) reported that thrombin-induced IL-8/CXCL8 production in lung fibroblasts is regulated by a PKC-dependent pathway. In 16HBE14o-human EC, bryostatin, a PKC activator, induces IL-8/CXCL8 expression, which is dependent on NF-B activation (62). However, downstream signaling pathways, especially that of calcium-dependent PKC, and in particular, the identity of the IKK signal pathway for NF-B activation and IL-8/CXCL8 expression in thrombin-induced lung EC are still unknown. A previous report showed that PKC directly binds IKK to mediate PMA-generated signals to the IKK complex (63). Recently, PKC was shown to be required for BCR-mediated NF-B activation through the recruitment of the IKK complex into lipid rafts, the activation of IKK, and the degradation of IB (64). In this study, we addressed the role of PKC involvement in thrombin-induced IKK activity, NF-B activation, and IL-8/CXCL8 expression. We found that Ro-32-0432 (a specific PKC inhibitor) inhibited thrombin-induced IL-8/CXCL8 release by A549 cells. Furthermore, thrombin can rapidly induce translocation of PKC from the cytosol to the membrane. We also demonstrated that thrombin-induced IKK activity and B-Luc activity were inhibited by Ro-32-0432. Taken together, our results indicate that thrombin-induced IL-8/CXCL8 expression is mediated through PKC-dependent activation of the IKK and NF-B signaling pathway in A549 cells.
Src, a tyrosine kinase, plays a critical role in the induction of chemokine transcription (65, 66). In human aortic EC, oxidized phospholipids induce IL-8/CXCL8 expression through a c-Src-dependent pathway (65). In chicken fibroblasts, thrombin-induced 9E3/CEF4 chemokine expression requires c-Src activation (66). Because c-Src has been reported to be a downstream effector of GPCR (67, 68), we examined the potential role of c-Src in the signaling pathway by thrombin-induced IL-8/CXCL8 expression. In this study, we found that treatment of A549 cells with thrombin induced increases in c-Src phosphorylation at Tyr416 and in c-Src kinase activity. We also found that pretreatment of cells with Ro-32-0432 inhibited thrombin-induced c-Src activity. In addition, A549 cells transfected with c-Src DN inhibited thrombin-induced IL-8/CXCL8-Luc activity. Recent reports have shown that c-Src is involved in IKK phosphorylation and NF-B activation in HeLa cells, NCI-H292 EC, and lymphoblastoma Reh cells (41, 67, 68). Our findings showed that c-Src DN inhibited thrombin-induced IKK activity, NF-B-specific DNA-protein complex formation, and B-Luc activity. Taken together, these results suggest that the c-Src signal pathway is very important for thrombin-induced IKK activity, NF-B activation, and IL-8/CXCL8 expression in A549 cells.
The IKKs can be stimulated by various proinflammatory stimuli, including IL-1, peptidoglycan, bradykinin, and thrombin (23, 42, 69, 70). These extracellular signals activate the IKK complex, which is comprised of catalytic subunits (IKK and IKK) and a linker subunit (IKK/NEMO). This kinase complex, in turn, phosphorylates IB at Ser32 and Ser36 and signals for ubiquitin-related degradation (18). The released NF-B is then translocated into the nucleus where it promotes NF-B-dependent transcription (71). There also is strong evidence that IKK and IKK are themselves phosphorylated and activated by one or more upstream activating kinases. One such upstream kinase, NF-B-inducing kinase, has been identified (72). In addition, Huang et al. (41, 73) showed that the direct interaction between c-Src and the IKK complex leads to the phosphorylation of IKK at Tyr188 and Tyr189, which, in turn, induces phosphorylation of IB and activation of NF-B. The findings of our experiments showed that thrombin rapidly induced PKC and c-Src after 5 min of treatment, and subsequently PKC interacted with IKK following thrombin treatment. Simultaneously, we also found that thrombin induced the association of c-Src and IKK during the interaction of PKC and IKK. Based on these findings, we suggest that the complex formation of PKC, c-Src, and IKK is involved in thrombin-induced IKK activation.
In conclusion, our study for the first time provides fundamental information on the regulatory molecular mechanisms of thrombin-induced IL-8/CXCL8 expression through the PI-PLC/PKC-dependent c-Src signaling pathway in increasing IKK activity, NF-B activation, and IL-8/CXCL8 expression and release in lung EC. Fig. 8 is a schematic representation of the signaling pathway involved in the enhanced expression and increased production of IL-8/CXCL8 in response to thrombin in A549 cells. Our results provide a compelling mechanism linking thrombin and the proinflammatory chemokine IL-8/CXCL8, and provide additional support for the notion that thrombin has important pathophysiological roles in inflammatory diseases of the lung.
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1 This work was supported by grants from the National Science Council of Taiwan (NSC91-2320-B-038-048, NSC92-2314-B-038-059, and NSC94-2320-B-038-047). 
2 Address correspondence and reprint requests to Dr. Bing-Chang Chen, School of Respiratory Therapy, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. E-mail address: bcchen{at}tmu.edu.tw
3 Abbreviations used in this paper used in this paper: EC, epithelial cell; BAL, bronchoalveolar lavage, COX-2, cyclooxygenase-2; GPCR, G protein-coupled receptor; IKK, IB kinase; PAR1, protease-activated receptor 1; PI, phosphoinositide; PLC, phospholipase C; PKC-, protein kinase C-, PPACK, D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone; c-Src DN, domainant-negative mutant of c-Src; NHBEC, normal human bronchial EC; KC, keratinocyte-derived chemokine; Ser, serine; Tyr, tyrosine; Luc, luciferase; wt, wild type.
Received for publication May 12, 2005. Accepted for publication June 14, 2006.
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