Protein Kinase C Mediates Cigarette Smoke/Aldehyde- and Lipopolysaccharide-induced Lung Inflammation and Histone Modifications*

Hongwei Yao, Jae-woong Hwang, Jorge Moscat, Maria T. Diaz-Meco, Michael Leitges, Nandini Kishore, Xiong Li, and Irfan Rahman From the Department of Environmental Medicine, Lung Biology and Disease Program, University of Rochester Medical Center, Rochester, New York 14642, the Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45237, and the Department of Biological Sciences, Pfizer Research Laboratories, St. Louis, Missouri 63167

Cigarette smoke (CS) 2 is the major etiological factor in the pathogenesis of chronic obstructive pulmonary diseases (COPD), which is characterized by chronic lung inflammation and accelerated decline in lung function. Previous studies showed that infiltration of immune inflammatory cells, such as macrophages, neutrophils, and lymphocytes, and release of nuclear factor-B (NF-B)-dependent pro-inflammatory mediators are increased in lungs of patients with COPD and in rodent lungs exposed to CS (1)(2)(3)(4)(5)(6). It is well known that the transcription of pro-inflammatory genes is up-regulated by activation of NF-B signaling pathway and modifications of histones, such as acetylation on lysine residues of histones, leading to increased accessibility for transcription factor NF-B binding to coactivator complex (7,8). Recently we have shown that the recruitment of the RelA/p65 subunit of NF-B and the increased acetylation of histone proteins H3 and H4 on the promoters of pro-inflammatory genes in rodent lungs in response to CS exposure (1,9,10). This is corroborated by the findings that increased acetylation of histones occurs in lungs of smokers and patients with COPD (7,11,12). However, the molecular mechanism that underlies CS-mediated chromatin modifications (e.g. increased acetylation of histone proteins and subsequent NF-B-dependent gene transcription) is not known.
It has been suggested that protein kinase C (PKC) , an atypical PKC, participates in inflammatory response to diverse stimuli in vitro and in vivo (13)(14)(15)(16)(17). We have recently shown that PKC regulates NF-B transcriptional activity by phosphorylating RelA/p65 at serine 311 and activating IB kinase leading to translocation of RelA/p65 into the nucleus (18,19). Separate evidence suggests that phosphorylation of RelA/p65 on serine 311 enhances its binding with CREB-binding protein (CBP), a transcriptional coactivator possessing intrinsic histone acetyltransferase (HAT) activity, leading to acetylation of RelA/ p65 (20,21). Hence, it is likely that PKC regulates the transcriptional activity of NF-B through recruitment and associa-tion of CBP with RelA/p65 as well as CBP-mediated acetylation of histone proteins on promoters of pro-inflammatory genes.
Recently, a role of PKC in allergic airway inflammation is shown by the finding that disruption of PKC attenuated ovalbumin-induced airway inflammation in mice (15). Importantly, the expression of PKC is elevated in lungs of patients with COPD implicating the critical role of PKC in chronic airway diseases (22). However, the role of endogenous PKC in regulation of lung inflammatory response to CS exposure or any inflammatory agents is not known. We hypothesized that down-regulation of PKC protects lungs against inflammatory response and chromatin modifications by CS, its reactive aldehydes as well as other pro-inflammatory agents, such as lipopolysaccharide (LPS), because endotoxin present in tobacco smoke is an important etiological factor for the pathogenesis of COPD/emphysema and exacerbates asthma from smoking (23)(24)(25). To test this hypothesis, PKC-deficient (PKC Ϫ/Ϫ ) and wild-type (WT) mice were exposed to CS and LPS, and the role of PKC in lung inflammatory response, NF-B signaling, and histone modifications was determined in lungs. Furthermore, human monocyte-macrophage cells (MonoMac6 cells) and primary mouse alveolar macrophages were used to determine the role and molecular mechanism of PKC in CS-, aldehyde-, and LPS-mediated inflammatory response.

EXPERIMENTAL PROCEDURES
Mice and CS Exposure-The generation of PKC Ϫ/Ϫ mice were described previously with their background WT mice being the 129/SVJ strain (18). The mice were housed in the Vivarium Facility of the University of Rochester with a 12-h light/dark cycle (light on at 6:00 a.m.). All animal procedures described in this study were approved by the University Committee on Animal Research at the University of Rochester. Tenweek-old male PKC Ϫ/Ϫ and WT mice were used for CS exposure as described previously (3,4). In brief, research grade cigarettes (2R4F; Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington) were used to generate smoke, and mice were exposed to CS according to the Federal Trade Commission protocol (1 puff/min of 2 s duration and 35-ml volume) using a Baumgartner-Jaeger CSM2072i automatic CS generating machine (CH Technologies, Westwood, NJ). Mainstream CS was diluted with filtered air and directed into the exposure chamber. The smoke exposure (total particulate matter in per cubic meter of air) was monitored in real time with a MicroDust Pro-aerosol monitor (Casella CEL, Bedford, UK) and verified daily by gravimetric sampling (2,3,9,26,27). The smoke concentration was set at a nominal value of ϳ300 mg/m 3 total particulate matter (corresponding to human consumption of 1-1.5 packs per day (28)), by adjusting the flow rate of the diluted medical air, and the level of carbon monoxide in the chamber was 350 ppm (3,9,26,27,29). The level of carboxyhemoglobin in blood was 17%. Mice received two 1-h exposures (1 h apart) daily for 3 consecutive days and were sacrificed at 2 and 24 h post-last exposure. Control mice were exposed to filtered air in an identical chamber according to the same protocol described for CS exposure.
LPS Aerosolization-Age-matched adult male WT and PKC-deficient mice (8 -10 weeks age) were exposed to an aero-sol of saline alone or saline containing Escherichia coli LPS (1 mg/ml; Sigma) for 8 min as described previously (4,26). Two and 24 h after LPS aerosolization, mice were anesthetized and sacrificed.
Bronchoalveolar Lavage (BAL)-BAL in mouse was performed as described previously (3,4,30). The total cell numbers in BAL fluid were determined by counting on a hemocytometer. Differential cell counts were performed on cytospin-prepared slides (Thermo Shandon, Pittsburgh, PA) stained with Diff-Quik (Dade Behring, Newark, DE). The remaining BAL cells were kept at Ϫ80°C for later analysis.
Lung Tissue Preparation, Hematoxylin and Eosin, and Macrophage Immunohistochemical Staining-Mouse lungs (which had not been lavaged) were inflated with 1% low melting agarose at a pressure of 25 cm of H 2 O and then fixed with 4% neutral buffered paraformaldehyde. The fixed lung tissues were dehydrated and embedded in paraffin and sectioned using a rotary microtome (MICROM International GmbH, Walldorf, Germany) into 4-m sections. Hematoxylin and eosin staining was performed on lung midsagittal sections to investigate the influx of inflammatory cells in lungs (30). For immunohistochemical staining of macrophages, the deparaffinized and rehydrated lung sections were exposed to 3% H 2 O 2 in methanol so as to quench endogenous peroxidase activity. Rat anti-mouse Mac-3 monoclonal antibody (Pharmingen) at a titer of 1:50 was used to detect macrophages. The number of Mac-3-positive cells in the lung sections (five random microscopic fields per lung section in three different sections) was counted manually under a microscope with ϫ200 magnification and averaged (30).
Myeloperoxidase Assay-Myeloperoxidase activity was assayed in lung homogenates as described previously (4). The lung tissues were homogenized in 10 volumes of 100 mM phosphate buffer (pH 7.4) with protease inhibitors. Myeloperoxidase activity in enzymatic extract was determined using tetramethylbenzidine (Sigma) as substrate. The activity was expressed in the samples as units/mg protein.
Preparation of Aqueous Cigarette Smoke Extract (CSE)-CSE (10%) was prepared by bubbling smoke from one research grade cigarette 3R4F (University of Kentucky) into 10 ml of culture media at a rate of one cigarette/min as described previously (4,30). The pH of the CSE was adjusted to 7.4, and was sterilefiltered through a 0.45-m filter (25 mm Acrodisc; Pall Corp., Ann Arbor, MI). CSE preparation was standardized by measuring the absorbance (0.85 Ϯ 0.05) at a wavelength of 320 nm. CSE (10%), containing up to 394 M acrolein (31) equivalent to 39.4 M in 1% of CSE, was freshly prepared and diluted with culture media (RPMI 1640 medium) to indicated concentrations used for all experiments within 15 min of preparation. Control medium was prepared by bubbling air through 10 ml of serum-free RPMI 1640 medium, adjusting pH to 7.4, and sterile-filtering as described above.
Cell Culture, Transfection, and Treatments-MonoMac6 cells were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) at 37°C in humidified atmosphere under 5% CO 2 (2,30). Cells were seeded at a density of 2.0 ϫ 10 6 cells in 100-mm culture plates in a total volume of 5 ml of RPMI 1640 medium containing 0.5% fetal bovine serum for 12 h. Cells were pretreated with a myristoylated PKC pseudosubstrate peptide inhibitor (PS-PKC, 0.3, 1, and 3 M) (Invitrogen) for 2 h prior to incubation with or without CSE (0.25, 0.5, and 1%), LPS (1 and 10 ng/ml) (Sigma),or acrolein (10 M, Sigma) for 12 h. In certain experiments, MonoMac6 cells were transfected with WT CBP-HAT plasmid or mutated CBP without HAT activity. The plasmids for WT and mutated CBP (Leu 1690 /Cys 1691 mutated into Lys 1690 /Leu 1691 ) were the kind gifts from Dr. Sankar Ghosh (Columbia University Medical Center) (20,32). Transient transfection was performed in MonoMac6 cells with 8 g of plasmids in the presence of Lipofectamine 2000 transfection reagent (Invitrogen) for 24 h before the aforementioned PKC inhibitor pretreatment and subsequent CSE, acrolein, and LPS exposures in a 6-well plate. Transfection efficiency in the case of both plasmid transfections was more than 80%. After treatment, cells were washed with ice-cold sterile Ca 2ϩ /Mg 2ϩ -free phosphate-buffered saline, and whole cell and nuclear proteins were extracted as described below.
Alveolar macrophages from C57BL/6J mice were isolated as described previously (26). Briefly, the lungs were lavaged with sterile Ca 2ϩ /Mg 2ϩ -free phosphate-buffered saline. BAL cells were seeded in a 6-well plate with RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% CO 2 . After 2 h of incubation, wells were gently pipetted to remove nonadherent cells, and adherent cells were cultured in medium containing 0.5% fetal bovine serum for 4 h. After starvation, the cells were treated with CSE (0.25% equivalent to 10 M of reactive aldehydes; see Refs. [31][32][33][34][35], LPS (10 ng/ml), acrolein (5 and 10 M, Sigma), and acetaldehyde (10 and 30 M, Sigma) for 12 h followed by incubation with or without PS-PKC (1 M) for 2 h at 37°C with 5% CO 2 . Both acrolein and acetaldehyde were used in this study because they are the main aldehyde components present in CS, and one cigarette yields ϳ500 g of acrolein and ϳ1000 g of acetaldehyde depending on cigarette type and determination methods (31,(33)(34)(35). Culture media from these adherent alveolar macrophages were collected and stored at Ϫ80°C for KC assay. The cells were washed with cold sterile Ca 2ϩ /Mg 2ϩ -free phosphate-buffered saline and lysed for the determination of NF-B activation.

Preparation of Whole Cell Lysate and Isolation of Nuclear Proteins from Lung Tissues, BAL Cells, and Cultured
Macrophages-The preparation of whole cell lysate and isolation of nuclear proteins from lung tissues and cells were described previously (3,4,30). Briefly, 100 mg of lung tissue was mechanically homogenized with 0.5 ml of RIPA buffer, and the tissue homogenates were kept on ice for 45 min to allow total cell lysis. Likewise, MonoMac6 and BAL cells were resuspended with RIPA buffer, kept on ice for 30 min, and then were vortexed for 15 s. Following centrifugation at 13,000 ϫ g for 5 min, the supernatant was collected as a whole cell lysate. As for isolation of nuclear proteins, 100 mg of lung tissue was mechanically homogenized in 0.5 ml of ice-cold buffer A. The homogenate was centrifuged at 2000 ϫ g for 30 s at 4°C to remove cellular debris. The supernatant was further centrifuged for 30 s at 13,000 ϫ g at 4°C. Similarly, cultured macrophages, including MonoMac6 cells and primary mouse alveolar macrophages, were lysed with 0.4 ml of buffer A for 15 min, and lysates were centrifuged for 30 s at 13,000 ϫ g at 4°C. The pellet was resuspended in 50 -100 l of buffer C and placed on the rotating rocker in the cold room for 30 min. Following centrifugation at 13,000 ϫ g for 5 min, the supernatant was collected as the nuclear extract, and stored at Ϫ80°C until it was used.
Acid Extraction of Histone Proteins from Lung Tissues-Acid extraction of histone protein was performed as described previously (1,10). In brief, pellets from the aforementioned nuclear fraction were resuspended in 150 l of deionized water containing 0.2 N HCl and 0.36 N H 2 SO 4 . The histone proteins were precipitated from the supernatant, agitated overnight at 4°C using a rotating rocker, and then centrifuged at 13,000 ϫ g for 10 min. The supernatants were decanted into a fresh tube. Icecold acetone precipitation samples were incubated overnight at Ϫ80°C and centrifuged, and the air-dried pellets were resuspended in 50 l of deionized water.
Protein Assay-Protein level in samples was measured with a BCA kit (Pierce). Linear regression was used to determine the actual protein concentration of the samples.
CBP Immunoprecipitation and Its Interaction with PKC or RelA/p65-Whole cell lysate were used for CBP immunoprecipitation with an antibody against CBP (1:40 dilution; Santa Cruz Biotechnology), which was added to 100 g of sample proteins in a final volume of 400 l, and incubated for 1 h. Protein-A/G-agarose beads (20 l) (Santa Cruz Biotechnology) were added to each sample and kept overnight at 4°C on a rotating rocker. The beads were washed three times and then resuspended in 40 l of RIPA buffer. For immunoblot, the immunoprecipitated CBP-agarose bead suspension was resolved by SDS-PAGE. Negative alone (beads only) was used as a negative control. To assess the interaction of CBP protein with PKC or RelA/p65, the membranes of immunoprecipitated CBP were blotted against PKC or RelA/p65 (Santa Cruz Biotechnology), respectively.
Chromatin Immunoprecipitation (ChIP) Assay-ChIP was performed as described previously (1,10) to investigate the role of PKC in recruiting transcription factor RelA/p65 and others coactivators on the promoters of pro-inflammatory genes in response to CS and LPS exposures. Lung tissue was homogenized in phosphate-buffered saline containing 1 mg/ml bovine serum albumin and protease inhibitor (Roche Applied Science) and cross-linked with 1% formaldehyde for 10 min, and then 0.5 ml of 2.5 M glycine was added. After a brief centrifugation, cell pellets were resuspended with SDS-lysis buffer (50 mM Tris-HCl, 1% SDS, 5 mM EDTA, 5 mM sodium butyrate, protease inhibitors). Sonication of the nuclear pellet containing chromatin was performed four times for 30 s and one time for 15 s at a maximum speed using Misonix-3000 Sonicator (Misonix Inc, Farmingdale, NY). Supernatants were collected and diluted (1:10 dilution) with buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM sodium butyrate, and protease inhibitor) followed by preclearing the extract with 60 l of protein A-agarose/salmon sperm DNA (catalog no. 16-157, Upstate Biotechnology, Inc.) for 3 h at 4°C (1,10,36). Immunoprecipitation was mixed with 1 g of specific antibodies at 4°C overnight. After immunoprecipitation, 40 l of protein A agarose/salmon sperm DNA was added and incubated for 2 h, followed by brief centrifugation. Precipitates were washed sequentially with Paro buffer I, Paro buffer II, and Paro buffer III for 5 min at 4°C. Precipitates were again washed twice with Tris buffer for 5 min each. The antigen-antibody complexes were extracted two times with 50 l of elution buffer (0.2 g/l proteinase K, 1% SDS, and 0.1 M NaHCO 3 ). The eluted samples were incubated at 65°C overnight to reverse formaldehyde cross-linking. The recovered DNA was purified with a QIAquick PCR purification kit (Qiagen, Valencia, CA) (10,36). Samples of input DNA were also prepared in the same way as described above. PCR amplification was performed under the following conditions: 94°C for 180 s; 30 -38 cycles at 94°C for 45 s, 60°C for 60 s, and 72°C for 60 s; and final elongation at 72°C for 10 min. PCR for the input reaction was performed using 100 ng of genomic DNA. The following primers were used in PCR: mip-2, 5Ј-CAA CAG TGT ACT TAC GCA GAC G-3Ј (sense) and 5Ј-CTA GCT GCC TGC CTC ATT CTA C-3Ј (antisense); il-6, 5Ј-GAC ATG CTC AAG TGC TGA GTC AC-3Ј (sense) and 5Ј-AGA TTG CAC AAT GTG ACG TCG-3Ј (antisense). PCR products were analyzed on a 1.5-2.0% agarose gel.
Statistical Analysis-Statistical analysis of significance was calculated using one-way analysis of variance followed by Tukey's post hoc test for multigroup comparisons using STATVIEW. The results are shown as the mean Ϯ S.E. The p Ͻ 0.05 is considered as statistically significant.

PKC Is Activated and Translocated into Nucleus in Lungs
of WT Mice Exposed to CS and LPS-We determined whether PKC was activated in mouse lung exposed to CS and LPS. Immunoblot was performed to determine the phos-phorylation of PKC on Thr 410 in lung tissues and BAL cells by exposure to CS or LPS. We found that the levels of phosphorylated PKC (Thr 410 ), which is shown to be required for activation of PKC (37), were significantly increased in lungs and BAL cells of WT mice exposed to CS and LPS (Fig. 1, A   FIGURE 1. PKC was activated and translocated into nucleus in lungs of WT mice exposed to CS and LPS. WT and PKC-deficient mice were exposed to CS and LPS and were sacrificed at 2 and 24 h of post-last exposures. Protein levels of PKC and its phosphorylation on the Thr 410 residue were analyzed by immunoblot in lung tissue (A) and BAL cell pellets (B) of whole cell lysates and in lung nuclear protein (C) of WT and PKC-deficient mice exposed to CS and LPS. PKC was activated and translocated into the nucleus in lungs and BAL cells of WT mice exposed to CS and LPS. There was no expression of PKC and its phosphorylated form in lungs and BAL cells of PKC-deficient mice. Blots are representative of three separate experiments. ***, p Ͻ 0.001, significant compared with respective air-or saline-exposed group. and B). As expected, there was no expression of phosphorylated and total PKC in lungs and BAL cells of PKC-deficient mice (Fig. 1, A and B). Nuclear translocation and activation of PKC are shown to regulate cell function in response to diverse stimuli (38 -41). Therefore, it is possible that CS and LPS exposures can induce the translocation and phosphorylation of PKC in the nucleus leading to increased transcription of proinflammatory mediators in lungs. We determined the levels of phosphorylated (Thr 410 ) and total PKC in lung nuclear protein by immunoblotting. Phosphorylated (Thr 410 ) and total levels of PKC were significantly increased in lung nuclear proteins of WT mice exposed to CS and LPS (Fig. 1C). PKC and its phosphorylated form were not detected in lung nuclear protein of PKC Ϫ/Ϫ mice (Fig. 1C). Hence, PKC was activated and translocated into nucleus of mouse lung in response to both CS and LPS exposures.

Inflammatory Response in Lungs Is Decreased in PKC Ϫ/Ϫ
Mice in Response to CS Exposure and LPS Aerosolization-In light of activation of PKC by CS and LPS, and the pro-inflammatory effect of PKC (14 -17), we hypothesized that CS-and LPS-induced lung inflammation is mediated by PKC activation. To test this hypothesis, PKC Ϫ/Ϫ and WT mice were exposed to CS, and influx of inflammatory cells into BAL fluid and lung interstitium was assessed by the stainings of Diff-Quik, hematoxylin and eosin, and immunohistochemistry (for macrophages). CS exposure resulted in a significant influx of neutrophils into BAL fluids of WT mice, which was significantly decreased in PKC Ϫ/Ϫ mice at both the 2-and 24-h time points ( Fig. 2A, panels i and ii). However, the deficiency of PKC had no effect on the number of macrophages in BAL fluid of mice exposed to CS and LPS (data not shown). This may be due to the insensitivity of the influx of macrophages in BAL FIGURE 2. Lung inflammation was decreased in PKC ؊/؊ mice in response to CS exposure and LPS aerosolization. Neutrophil influx in BAL fluid of mice was determined on cytospin-prepared slides stained with Diff-Quik (A). Immunohistochemical detection of macrophages was performed using rat anti-mouse Mac-3 antibody on mouse lung sections (B). Myeloperoxidase activity in lung tissue was determined spectrophotometrically (C). Number of neutrophils in BAL fluid (A) was decreased in PKC-deficient mice in response to CS exposure (panels i and ii) and LPS aerosolization (panels iii and iv) compared with respective WT mice at 2 h (panels i and iii) and 24 h (panels ii and iv) of post-last exposure. Deficiency of PKC decreased CS (panel i)-and LPS (panel ii)-induced macrophage influx into the lungs (B) at both the 2-and 24-h time points. PKC-deficient mice showed decreased myeloperoxidase activity in lung (C) at both 2-h (panels i and iii) and 24-h (panels ii and iv) time points as compared with WT mice exposed to CS (panels i and ii) and LPS (panels iii and iv). Data are shown as mean Ϯ S.E. (n ϭ 4 to 5 per group). **, p Ͻ 0.01; and ***, p Ͻ 0.001, significant compared with respective air-or saline-exposed groups; ϩ, p Ͻ 0.05; ϩϩ, p Ͻ 0.01; and ϩϩϩ, p Ͻ 0.001, significant compared with respective CS-or LPS-exposed WT mice. Immunohistochemical pictures represent three separate experiments. Arrows indicate macrophages. Original magnification, ϫ200. fluid to reflect lung inflammation under the present exposure protocol (3,4). The staining of hematoxylin and eosin and immunohistochemistry showed that CS-mediated infiltration of inflammatory cells (e.g. macrophages and neutrophils) into the interstitium of lungs was also attenuated in PKC Ϫ/Ϫ mice exposed to CS as compared with WT mice (Fig. 2B, panel i, and  supplemental Fig. S1A). Likewise, disruption of PKC decreased the influx of inflammatory cells into lungs in response to LPS aerosolization compared with that of WT mice (Fig. 2, A, panels iii and iv, and B, panel ii, and supplemental Fig.  S1B). Furthermore, deficiency of PKC attenuated the activity of myeloperoxidase, a marker for neutrophilic inflammation (42), in mouse lungs in response to CS and LPS exposures (Fig.  2C). The levels of pro-inflammatory mediators, such as granulocyte-macrophage colony-stimulating factor, interferon-␥, IL-13, IL-17, IL-18, IL-1␤, IL-6, KC, MCP-1, MIP-2, and tumor necrosis factor-␣, were also significantly decreased in lungs of PKC-deficient mice compared with WT mice exposed to CS and LPS (supplemental Fig. S2, A-D). These results suggest that CS and LPS exposure-induced lung inflammation is dependent on PKC activation.
Inhibition of PKC-attenuated CSE-, Aldehyde-, and LPS-induced Pro-inflammatory Mediator Release and PKC Activation in Monocytes/Macrophages-In the light of the above findings that disruption of PKC attenuated CS-and LPS-mediated lung inflammatory response, and both CS and LPS exposures activated PKC in BAL cells, which mainly consist of alveolar macrophages, we hypothesized that PKC in macrophages plays an important role in CS-and LPS-mediated lung inflammatory response. To test this hypothesis, MonoMac6 cells were pretreated with PS-PKC, a specific PKC inhibitor, for 2 h prior to incubation with CSE (0.25, 0.5, and 1%) or LPS (1 and 10 ng/ml) for 12 h. Pretreatment with PS-PKC (0.3-3 M) significantly decreased CSE-and LPS-induced release of IL-8 from MonoMac6 cells (Fig. 3A). Furthermore, CSE-and LPS-mediated phosphorylation of PKC (Thr 410 ) was attenuated in the nucleus of MonoMac6 cells, which were pretreated with PS-PKC (0.3-3 M) (Fig. 3B). Interestingly, treatment with CSE, the reactive aldehydes, and LPS significantly induced KC release and PKC phosphorylation (Thr 410 ) in mouse alveolar macrophages, which was decreased by the PKC inhibitor (Fig.  4, A and B). These results suggest that CS-and LPS-induced pro-inflammatory mediator release is mediated by activation of PKC, and aldehyde components are crucial for CS-induced PKC activation and inflammatory response in monocytes/macrophages. Phosphorylated, Acetylated, and Total Levels of RelA/p65 Are Decreased in Lung Nuclear Proteins of PKC Ϫ/Ϫ Mice Exposed to CS and LPS-NF-B is shown to be regulated by PKC through the phosphorylation of RelA/p65 in embryo fibroblasts treated with tumor necrosis factor-␣ (19). However, it is not known whether PKC modulates NF-B activation in lungs in response to CS exposure or LPS aerosolization. Hence, immunoblotting was performed to determine the phosphorylation and acetylation of RelA/p65 in lung nuclear proteins of WT and PKC Ϫ/Ϫ mice exposed to CS and LPS. Both CS and LPS exposures led to increased levels of phosphorylated (Ser 311 ) and acetylated (Lys 310 ) RelA/p65 in WT mice, which was attenu-ated in PKC Ϫ/Ϫ mice (Fig. 5). However, the phosphorylated levels of RelA/p65 at Ser 276 were not altered in lungs of PKC Ϫ/Ϫ mice as compared with WT mice exposed to CS and LPS (Fig. 5). Interestingly, the total level of RelA/p65 was also decreased in lung nuclear proteins of PKC Ϫ/Ϫ mice compared with WT mice exposed to CS and LPS (Fig. 5). These results suggest that PKC activates NF-B by translocating RelA/p65 into the nucleus as well as inducing phosphorylation and subsequent acetylation of RelA/p65 in response to CS and LPS exposures.

Increased Interaction of CBP with PKC in Response to CS and LPS Exposures in WT Mice and Disruption of PKC Decrease CS-and LPS-mediated Interaction of CBP with RelA/ p65 in Lung
Tissues and BAL Cells-Given the decreased phosphorylation of RelA/p65 (Ser 311 ) in PKC Ϫ/Ϫ mice in response to CS and LPS, and enhanced binding of RelA/p65 with CBP upon RelA/p65 phosphorylation (20,21), we proposed that PKC induces the interaction of RelA/p65 with CBP in response to CS and LPS exposures. Immunoprecipitation was performed to determine the interaction of CBP with RelA/p65 in lung tissues and BAL cells of WT and PKC Ϫ/Ϫ mice by CS and LPS. Both CS and LPS exposures increased the interaction of CBP with RelA/p65 in lung tissues and BAL cells of WT mice that was ameliorated in PKC Ϫ/Ϫ mice (Fig. 6, A and B). It is interesting to note that the interaction of CBP with PKC was also increased in lungs and BAL cells of WT mice in response to CS and LPS exposures (Fig. 6, A and B). These results suggest that PKC recruits the essential components of transcriptional machinery, such as CBP, and thereby controls the activation of NF-B in lungs in response to CS and LPS exposures.

Pretreatment with PS-PKC Attenuates the Phosphorylation (Ser 311 ) and Acetylation (Lys 310 ) of RelA/p65 and the Interaction of CBP with PKC and RelA/p65 by CSE and LPS in
Monocytes/Macrophages-In light of the increased interaction of CBP with PKC and RelA/p65 in mouse lungs and BAL cells in response to CS and LPS exposures, we determined whether inhibition of PKC attenuated these interactions in MonoMac6 cells in response to CSE and LPS treatments. As shown in Fig. 7, CSE-and LPS-induced interaction of CBP with PKC and RelA/p65 was significantly decreased in response to pretreatment with PS-PKC in MonoMac6 cells. Furthermore, PS-PKC (1 and 3 M) significantly reduced CSE-and LPSmediated the phosphorylation (Ser 311 ) and acetylation (Lys 310 ) of RelA/p65 in nuclear proteins of MonoMac6 cells (Fig. 3B). Similarly, the phosphorylation of RelA/p65 (Ser 311 ) in primary mouse alveolar macrophages was induced by CSE/aldehydes and LPS, which was significantly decreased by PS-PKC (1 M) (Fig. 4B). These data indicate that PKC functions as a master regulator and facilitates the assembly of RelA/p65 with coactivator CBP in transcriptional machinery, thereby increasing NF-B-dependent inflammation in monocytes/macrophages by CS and LPS exposures.

CBP Is Required for PKC-mediated RelA/p65 Acetylation and Pro-inflammatory Cytokine Release in MonoMac6 Cells in
Response to CSE, Acrolein, and LPS Treatments-To further study whether CBP is required for the regulation of NF-B acti-  , acetylated (Lys 310 ), and total RelA/p65 in lung nuclear proteins were analyzed by immunoblotting. Lamin B was used as a nuclear marker, and no bands of ␣-tubulin were observed in nuclear fractions. Levels of phosphorylated RelA/ p65 on Ser 311 was decreased, whereas no alteration in phosphorylated RelA/ p65 on Ser 276 was observed in lungs of PKC-deficient mice as compared with WT mice exposed to CS and LPS at 2 and 24 h of post-last exposures. Knockout of PKC decreased the levels of acetylated (Lys 310 ) and total RelA/p65 in lung nuclear protein of mice exposed to CS and LPS. Each lane represents results of three independent experiments. p-p65 (ser276), phosphorylated RelA/p65 on Ser 276 ; p-p65 (ser311), phosphorylated RelA/p65 on Ser 311 ; Ac-p65 (lys310), acetylated RelA/p65 at Lys 310 ; p65, RelA/p65. vation and pro-inflammatory cytokine release by PKC in response to CSE, acrolein, and LPS, MonoMac6 cells were transfected with wild-type CBP mutant or CBP mutated with Leu 1690 / Cys 1691 , which lacked its HAT activity (20,32). Pretreatment with PKC inhibitor attenuated the acetylation of RelA/p65 and IL-8 release induced by CSE, acrolein, and LPS in MonoMac6 cells (Fig.  3, A and B, and Fig. 8). This response to PKC inhibitor pretreatment was significantly ameliorated when MonoMac6 cells were transfected with WT CBP mutant and treated with CSE, acrolein, and LPS (Fig. 8, A and B). However, transfection of cells with CBP mutant without HAT activity further augmented the response to PKC inhibitor pretreatment in MonoMac6 cells by CSE, acrolein, and LPS exposures (Fig. 8, A and B). These results suggest that HAT activity of CBP is required for PKC-mediated NF-B activation and inflammatory response.

PKC Recruits CBP and RelA/p65 on Pro-inflammatory Gene Promoters in Mouse Lungs in Response to CS and LPS
Exposures-It is known that both CS and LPS cause chromatin remodeling on the promoters of pro-inflammatory genes (1,43,44). Based on this, we hypothesized that CS and LPS induce PKC-mediated chromatin modifications, thereby promoting the binding of CBP and RelA/p65 on pro-inflammatory gene promoters in mouse lungs. ChIP assay was performed in lung homogenates using antibodies against PKC, acetylated RelA/ p65 (Lys 310 ), CBP, phosphorylated/acetylated (Ser 10 /Lys 9 ) histone H3, and acetylated histone H4 so as to determine the chromatin remodeling on the promoters of mip-2 and il-6. We found that PKC was recruited on the promoters of mip-2 and il-6 in lungs of WT mice exposed to CS and LPS (Fig. 9, A and   . PKC recruited CBP and RelA/p65 on pro-inflammatory gene promoters in mouse lungs in response to CS and LPS exposures. The recruitment of PKC, acetylated RelA/p65 (Lys 310 ), CBP, phosphorylated/ acetylated histone H3 (Ser 10 /Lys 9 ), and acetylated histone H4 on the promoters mip-2 and il-6 in mouse lung was analyzed by ChIP assay. Lung homogenates were cross-linked and immunoprecipitated with relevant antibodies against the aforementioned proteins, and then chromatin modification on the promoter regions of mip-2 (A) and il-6 (B) was detected by PCR. Both CS and LPS exposures caused the recruitment of acetylated RelA/p65 (Lys 310 ), CBP, phosphorylated/acetylated histone H3 (Ser 10 /Lys 9 ), and acetylated histone H4 on the promoters mip-2 and il-6 in mouse lung, which was attenuated in PKC-deficient mice at 2 h of post-last exposures. Furthermore, PKC was also recruited on the promoters of mip-2 and il-6 in lungs of WT mice, but not in PKC deficient mice exposed to CS and LPS. IgG was used as a negative control. Gel pictures shown are representative of at least three separate experiments. Ac-p65 (lys310), acetylated RelA/p65 on Lys 310 ; p-Ac-H3, phosphorylated/acetylated histone H3 at Ser 10 /Lys 9 ; Ac-H4, acetylated histone H4.
These results indicate that PKC increases the pro-inflammatory gene transcription in lungs by recruitment of RelA/p65 and transcriptional coactivator CBP, and thus increases histone H3 and H4 acetylation on the promoters of pro-inflammatory genes in response to CS and LPS exposure.
Disruption of PKC Attenuates the Phosphorylation/Acetylation of Histone H3 and Acetylation of Histone H4 in Mouse Lung Exposed to CS and LPS-To determine the effect of PKC on histone acetylation, immunoblotting was performed in acidextracted histone proteins of WT and PKC Ϫ/Ϫ mice in response to CS and LPS exposures. Both CS and LPS exposures increased the phosphorylation on Ser 10 and acetylation on Lys 9 of histone H3 in lungs of WT mice, which was decreased in PKC Ϫ/Ϫ mice (Fig. 10). Similarly, deficiency of PKC also attenuated the acetylation of histone H4 in lungs in response to CS and LPS exposures (Fig. 10). These data suggest that both CS and LPS exposures-mediated histone modifications, such as phosphorylation and acetylation, are dependent on PKC activation.

DISCUSSION
Cigarette smoke-induced oxidative stress, reactive aldehydes, and endotoxin present in tobacco smoke are important risk factors in the development of COPD/emphysema by increasing the release of pro-inflammatory mediators and recruiting inflammatory cells into the lungs (23)(24)(25)(45)(46)(47)(48)(49)(50)(51). Previous studies have shown that PKC is activated in lungs of patients with COPD, and PKC participates in inflammatory response through NF-B activation (15,16,18,19,22). However, the role of endogenous PKC in lung inflammation via chromatin modifications on pro-inflammatory genes by CS exposure or LPS aerosolization or any other environmental agents is not known. We hypothesized that down-regulation of PKC would dampen lung inflammatory response to CS, its reactive aldehydes, and LPS exposure by attenuating chromatin modifications on pro-inflammatory gene promoters. Consistent with its pro-inflammatory effect of endogenous PKC seen in vitro and in vivo (14 -17), the inflammatory response, such as inflammatory cell influx and pro-inflammatory mediator release, in lungs was attenuated in PKC Ϫ/Ϫ mice as compared with WT mice exposed to CS and LPS exposures suggesting that CS-and LPS-mediated lung inflammatory response is dependent on PKC activation. This is confirmed by our results showing that the activation of PKC, which is reflected by increased phosphorylation of PKC on Thr 410 , in WT mouse lung exposed to CS and LPS, and in monocytes/macrophages treated with CSE, LPS, and reactive aldehydes (i.e. acrolein and acetaldehyde). Similarly, the activation of PKC is also observed in lungs of patients with COPD, and in human adenocarcinoma A549 cells treated with nicotine (22,52). The mechanism underlying the activation of PKC may be associated with activation of phosphoinositide 3-kinase and increased ceramide, which can be induced by CS and LPS (53)(54)(55)(56)(57).
Post-translational modifications of NF-B, such as phosphorylation and acetylation, play an important role in regulation of its transcriptional activity (20,21). It has been shown that PKC activates NF-B by phosphorylating RelA/p65 at Ser 311 (19). Hence, we determined whether reduced inflammatory response in PKC Ϫ/Ϫ mice is associated with decreased NF-B activation in lungs in response to CS and LPS exposures. As expected, the levels of phosphorylated RelA/p65 on Ser 311 were decreased in lungs of PKC-deficient mice as compared with WT mice exposed to CS and LPS. However, the phosphorylated levels of RelA/p65 on serine 276 were not altered in lungs of PKC-deficient mice compared with WT mice in response to CS and LPS. This is consistent with the previous observation where serine 311, but not serine 276, accounts for the phosphorylation of RelA/p65 by PKC, although the mechanism underlying these discrepancies is not clear (19). Furthermore, the levels of total RelA/p65 were also decreased in lung nuclear proteins of PKC-deficient mice as compared with WT mice exposed to CS and LPS implicating the involvement of PKC in activation/translocation of RelA/p65 into the nucleus. The mechanism might be associated with activation of IB kinase ␤ FIGURE 10. Disruption of PKC decreased the phosphorylation/acetylation of histone H3 and acetylation of histone H4 in mouse lung exposed to CS and LPS. Acid-extracted histone proteins in lungs of WT and PKC-deficient mice were used for immunoblotting against anti-phosphorylated/acetylated (Ser 10 /Lys 9 ) histone H3 and acetylated histone H4. Levels of phosphorylated/acetylated (Ser 10 /Lys 9 ) histone H3 and acetylated histone H4 were decreased in lungs of PKC Ϫ/Ϫ mice as compared with WT mice exposed to CS and LPS at 2 h of post-last exposure. Gel pictures shown are representative of at least three separate experiments. *, p Ͻ 0.05; ***, p Ͻ 0.001, significant compared with respective air-or saline-exposed groups; ϩ, p Ͻ 0.05; ϩϩ, p Ͻ 0.01; ϩϩϩ, p Ͻ 0.001, significant compared with respective CS-and LPS-exposed WT mice. p-Ac-H3, phosphorylated/acetylated histone H3 on Ser 10 /Lys 9 ; H3, histone H3; Ac-H4, acetylated histone H4; H4, histone H4. by PKC because the levels of phosphorylated IB kinase ␤ were decreased in PKC-deficient mice in response to CS and LPS exposures. 3 This contention is in agreement with the previous studies showing that PKC was able to phosphorylate and activate IB kinase ␤ leading to NF-B activation by inducing IB degradation (17, 58 -60). Nevertheless, these results suggest that PKC regulates CS-and LPS-mediated NF-B activation by inducing the phosphorylation (Ser 311 ) and translocation of RelA/p65, leading to increased transcription of pro-inflammatory genes in lungs, although certain pro-inflammatory cytokines (e.g. granulocyte-macrophage colony-stimulating factor and interferon-␥) determined in this study can also be transcriptionally regulated by other transcription factors, such as SP-1, AP-1, and nuclear factor of activated T-cells in addition to NF-B (61)(62)(63)(64).
Phosphorylation of RelA/p65 (Ser 311 ) is shown to enhance its interaction with CBP resulting in its acetylation, which is required for full activation of NF-B (19,65). Previous studies have shown that LPS increased the interaction of CBP with RelA/p65, and increased acetylation of RelA/p65 occurs in lungs in response to CS (3,66). Most importantly, we show that the phosphorylation of RelA/p65 on Ser 311 was regulated in lungs by PKC. In the light of these observations, we hypothesized that acetylation of RelA/p65 and its association with CBP was altered in lungs of PKC-deficient mice in response to CS and LPS exposures. As expected, disruption/inhibition of PKC attenuated the CS-and LPS-induced interaction of CBP with RelA/p65 and acetylation of RelA/p65 on Lys 310 in lungs and MonoMac6 cells. This observation corroborates our previous study showing that the interaction of RelA/p65 with CBP is decreased in PKC-deficient embryonic fibroblasts treated with tumor necrosis factor-␣ (19). It is interesting to note that both CS and LPS exposures also increased the association of CBP with PKC in WT mouse lungs and MonoMac6 cells suggesting that PKC does translocate into the nucleus and forms a complex with CBP and RelA/p65. This is confirmed by our results showing the increased levels of phosphorylated and total PKC by CS and LPS in lung nuclear proteins of WT mice. Furthermore, HAT activity of CBP is required for PKC-mediated RelA/p65 acetylation and inflammatory response to CS, aldehyde, and LPS exposures. Therefore, we propose that PKC is a master regulator and facilitates the assembly of RelA/p65 with coactivator CBP in transcriptional machinery, thereby increasing acetylation of RelA/p65, subsequently enhancing the transcription of pro-inflammatory genes in lungs in response to CS and LPS exposures (Fig. 11).
CS and LPS can induce phosphorylation or acetylation of histones H3 and H4, and it is known that chromatin modification plays an important role in CS-and LPS-mediated transcription of pro-inflammatory genes (1, 43, 44, 66 -68). Furthermore, it has been recently recognized that alterations in chromatin remodeling on pro-inflammatory gene promoters are the key mechanisms for underlying abnormal inflammation seen in lungs of patients with COPD. Interestingly, PKC is shown to regulate IL-6 expression by altering the acetylation of histone H4 on its promoter (69). Our data showed that the acetylation of RelA/p65 (Lys 310 ), histone H3 (Lys 9 ), and histone H4 on promoters of il-6 and mip-2 genes was decreased in lungs of PKC-deficient mice as compared with WT mice in response to CS and LPS exposures. This is associated with decreased recruitment of CBP, which is known to acetylate histone or non-histone proteins, on the promoters of pro-inflammatory genes in PKC Ϫ/Ϫ mice. Furthermore, the phosphorylation/ acetylation of histone H3 (Ser 10 /Lys 9 ) and acetylation of histone H4 were decreased in lungs of PKC-deficient mice exposed to CS and LPS. These results suggest that once PKC is activated, it forms a complex with CBP and RelA/p65 on the promoters of pro-inflammatory genes leading to acetylation of RelA/p65 and histones H3 and H4. Indeed, both CS and LPS exposures were able to recruit PKC on the promoters of il-6 and mip-2 genes in lungs of WT mice as detected by ChIP assay. Therefore, acetylated RelA/p65 interacts with PKC and CBP forming a coactivator complex on the promoters of pro-inflammatory genes, thereby leading to sustained transcription of proinflammatory mediators in lungs.
Inflammatory cells, such as macrophages, are the main orchestrators and amplifiers in chronic inflammatory and injurious responses in lungs of smokers and patients with COPD (70 -73). Previous studies have shown that PKC is essential for activation and function of macrophages and neutrophils (16, 56, 74 -76). In this study, we showed the enhanced activation of PKC in BAL cells, which is mainly composed of macrophages (neutrophils will migrate into BAL fluids in the context of lung inflammation), in WT mice exposed to CS and LPS. Importantly, the phosphorylation of PKC was also significantly increased by CSE and LPS treatments in MonoMac6 cells and primary mouse alveolar macrophages. Furthermore, inhibition 3 H. Yao and I. Rahman, unpublished observations. FIGURE 11. Summarized schematic model of the regulation of PKC in CS/aldehyde-and LPS-induced lung inflammatory response and chromatin remodeling. PKC was activated and translocated into the nucleus in response to CS and LPS exposures. Activated PKC interacted and phosphorylated RelA/p65 on Ser 311 , leading to increased NF-B transactivation. Furthermore, PKC functions as a master regulator and facilitates the assembly of RelA/p65 with coactivator CBP in transcriptional machinery, thereby increasing acetylation of RelA/p65 and histones on the promoters of pro-inflammatory genes in response to CS and LPS exposures in lungs.
of PKC by PS-PKC reduced CSE-and LPS-mediated release of pro-inflammatory cytokines from both MonoMac6 cells and mouse alveolar macrophages suggesting that PKC in macrophages plays an important role in CS-and LPS-induced lung inflammation. Interestingly, both acrolein and acetaldehyde, the reactive components of CS, significantly induced pro-inflammatory mediator release in primary mouse alveolar macrophages and MonoMac6 cells. This observation is corroborated by a previous report that shows that acrolein treatment increases IL-8 release in human alveolar macrophages (45). Importantly, these aldehyde-induced inflammatory responses were significantly decreased in the presence of the PKC inhibitor. Hence, it may be surmised that the reactive aldehydes play a critical role in CS-mediated sustained macrophage activation and lung inflammation through PKC signaling pathway. It is tempting to mention here that 80 -85% aldehyde/acrolein is retained in the respiratory tract of animals exposed to CS at a dose of 400 -600 mg/m 3 total particulate matter according to the 1992 World Health Organization/International Programme on Chemical Safety report. One cigarette yields ϳ500 g of acrolein and ϳ1000 g of acetaldehyde depending on cigarette type and determination methods (31,(33)(34)(35). Hence, CS at a dose of ϳ300 mg/m 3 total particulate matter (corresponding to human consumption of 1-1.5 packs per day (28)) used in our study certainly relates to human exposure. Moreover, we used CSE (0.25-1%) to treat cells, which is equivalent to ϳ10 -40 M aldehydes present in CS (31) and compares the in vivo exposure conditions and the elicited pro-inflammatory response. However, CSE differs from the gaseous phase of smoke, which makes it difficult to determine and compare which dose and exposure time are best to mimic the effects of CS in the lung. Moreover, individual components are unlikely to produce the same biological effects akin to whole CS to which humans are exposed. Nevertheless, our data obtained in lung in vivo and monocytes/macrophages in vitro suggest the importance of PKC in cigarette smoke/aldehydes-induced lung inflammation via its effects on pro-inflammatory gene promoters by chromatin modifications.
In conclusion, we have demonstrated a novel role of PKC in CS/aldehyde-and LPS-mediated inflammatory response and chromatin modifications in the lungs. Both CS and LPS exposures lead to activation and translocation of PKC into the nucleus where it forms a complex with CBP and RelA/p65 on the promoters of pro-inflammatory genes thereby resulting in sustained transcription of pro-inflammatory mediators in lungs, which is the hallmark of abnormal inflammation seen in lungs of patients with COPD (Fig. 11). These findings not only unravel the importance of PKC-mediated histone modifications in CS/aldehyde-and LPS-induced abnormal lung inflammation but also provide new insights into the molecular mechanisms underlying the pathogenesis of chronic lung diseases.