Surfactant Protein A Activation of Atypical Protein Kinase C ζ in IκB-α-Dependent Anti-Inflammatory Immune Regulation
This information is current as Christina Moulakakis, Stefanie Adam, Ulrike Seitzer, Andra of September 29, 2021. B. Schromm, Michael Leitges and Cordula Stamme J Immunol 2007; 179:4480-4491; ; doi: 10.4049/jimmunol.179.7.4480 http://www.jimmunol.org/content/179/7/4480 Downloaded from
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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology
Surfactant Protein A Activation of Atypical Protein Kinase C in IB-␣-Dependent Anti-Inflammatory Immune Regulation1
Christina Moulakakis,* Stefanie Adam,* Ulrike Seitzer,† Andra B. Schromm,‡ Michael Leitges,§ and Cordula Stamme2*¶
The pulmonary collectin surfactant protein (SP)-A has a pivotal role in anti-inflammatory modulation of lung immunity. The mechanisms underlying SP-A-mediated inhibition of LPS-induced NF-B activation in vivo and in vitro are only partially un- derstood. We previously demonstrated that SP-A stabilizes IB-␣, the primary regulator of NF-B, in alveolar macrophages (AM) both constitutively and in the presence of LPS. In this study, we show that in AM and PBMC from IB-␣ knockout/IB- knockin mice, SP-A fails to inhibit LPS-induced TNF-␣ production and p65 nuclear translocation, confirming a critical role for IB-␣ in SP-A-mediated LPS inhibition. We identify atypical (a) protein kinase C (PKC) as a pivotal upstream regulator of SP-A- ؊ ؊ mediated IB-␣/NF-B pathway modulation deduced from blocking experiments and confirmed by using AM from PKC / Downloaded from mice. SP-A transiently triggers aPKCThr410/403 phosphorylation, aPKC kinase activity, and translocation in primary rat AM. Coimmunoprecipitation experiments reveal that SP-A induces aPKC/p65 binding under constitutive conditions. Together the data indicate that anti-inflammatory macrophage activation via IB-␣ by SP-A critically depends on PKC activity, and thus attribute a novel, stimulus-specific signaling function to PKC in SP-A-modulated pulmonary immune response. The Journal of Immu- nology, 2007, 179: 4480–4491. http://www.jimmunol.org/ ulmonary surfactant is a lipid-protein complex that con- Alveolar macrophages (AM), normally accounting for ϳ95% of stitutes the alveolar liquid layer of the lung. The unique airspace leukocytes, are a unique class of professional phagocytes P composition of surfactant facilitates the functional combi- that represent the major effector cells of the pulmonary innate im- nation of different biological effects such as preventing alveolar mune system (6). SP-A directly interacts with AM through binding collapsing at expiration and immunomodulating innate pulmonary to cell surface receptors, resulting in modulation of chemotaxis, host defense responses (1, 2). The latter function is primarily me- phagocytosis, and modified pro- or anti-inflammatory immune re- diated by surfactant proteins (SP)3-A and SP-D belonging to the sponses (7). collectin family. SP-A, the most abundant pulmonary collectin, LPS, the main component of the outer leaflet of the outer by guest on September 29, 2021 binds and aggregates a variety of microorganisms and enhances membrane of Gram-negative bacteria, substantially contributes their phagocytosis and killing both in vivo and in vitro (3). SP-A- to the pathophysiology of the most frequent lung diseases, in- deficient mice exhibit delayed microbial clearance and higher lev- cluding pneumonia, acute lung injury, and acute respiratory dis- els of bronchoalveolar inflammatory mediators after intratracheal tress syndrome (8). Whereas LPS recognition benefits the host inoculation with a variety of clinically relevant pathogens (4), as by sensing bacteria and mobilizing defense mechanisms, an ex- well as to isolated LPS (5). aggerated response to LPS contributes to the development of a local or systemic septic shock syndrome. The interaction of picomolar concentrations of LPS with a receptor complex in- cluding TLR4, MD-2, LPS-binding protein, and CD14 on host *Department of Immunochemistry and Biochemical Microbiology, Division of Cel- cells initiates the sequential activation of multiple signaling lular Pneumology, Research Center Borstel, Leibniz Center for Medicine and Bio- science, Borstel, Germany; †Department of Immunology and Cell Biology, Division pathways, including NF- B, a central regulator of LPS-medi- of Veterinary Infection Biology and Immunology, Research Center Borstel, Leibniz ated cell activation (9–11). ‡ Center for Medicine and Bioscience, Borstel, Germany; Emmy-Noether Group Im- The most prominent NF-B heterodimer p65/p50 is sequestered munobiophysics, Research Center Borstel, Leibniz Center for Medicine and Bio- science, Borstel, Germany; §Biotechnolgy Centre of Oslo, University of Oslo, Oslo, in the cytoplasm by a family of inhibitory proteins, among Norway; and ¶Department of Anesthesiology, University Hospital of Lu¨beck, Lu¨- which IB-␣ is the best characterized and assumed to function beck, Germany as the primary regulator of NF-B in both stimulated and rest- Received for publication February 15, 2007. Accepted for publication July 30, 2007. ing cells (12, 13). In response to many stimuli including LPS, 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 I B- becomes degraded via ubiquitination in an I B kinase with 18 U.S.C. Section 1734 solely to indicate this fact. (IKK)-dependent manner (12, 14, 15). The removal of IB-␣ 1 This work was supported by the Deutsche Forschungsgemeinschaft 609/1-3 and allows the nuclear translocation of NF-B and subsequently the 609/1-4 (to C.S.) and 621/2-2 (to A.B.S.). transcription of downstream target genes, including IB-␣ itself 2 Address correspondence and reprint requests to Dr. Cordula Stamme, Research (16). Secondary clues point to an IB-independent pathway, Center Borstel, Parkallee 22, D-23845 Borstel, Germany. E-mail address: [email protected] and include the phosphorylation of p65 to activate NF- B- dependent gene transcription (17). 3 Abbreviations used in this paper: SP, surfactant protein; AKBI, IB-␣ knockout/ IB- knockin; AM, alveolar macrophage; aPKC, atypical protein kinase C; cPKC, SP-A has been shown to inhibit LPS-induced TNF-␣ production classical protein kinase C; DAG, diacylglycerol; HI, heat inactivated; IKK, IB ki- (18–21), inducible NO synthase protein expression (22), NF-B ac- nase; PKC, protein kinase C; ps, pseudosubstrate; wt, wild type. tivity (20, 23, 24), and, upon diverse stimuli, NADPH oxidase (25) in Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 immunocompetent cells. We recently demonstrated that SP-A exerts www.jimmunol.org The Journal of Immunology 4481
its anti-inflammatory effects on LPS-challenged AM via a mechanism 37°C in a 5% CO2 atmosphere. Then the medium was changed and the involving a SP-A-mediated direct modulation of the basal and LPS- cells were treated with SP-A (20–60 g/ml) and/or LPS (10–100 ng/ml) coupled IB-␣ turnover in these cells (24). In that study, SP-A in- for indicated times at 37°C in the presence of 0.2% heat-inactivated (HI) ␣ FCS. In separate experiments, AM were treated with a panel of kinase creased I B- protein expression in a dose- and time-dependent man- inhibitors to determine their possible effect on IB-␣ protein expression as ner without inducing IB-␣ phosphorylation or IB-␣ mRNA levels follows: Go¨-6976 (5 M), Go¨-6850 (5 nM), chelerythrine chloride (6 and both basal and in the presence of LPS. However, the signaling path- 12 M), or aPKCps peptides (2, 5, and 10 M) were used to inhibit distinct ways controlling the SP-A-mediated attenuation of LPS-induced subsets of cPKC, novel PKC, or aPKCs. Wortmannin (50 nM) was used as PI3K inhibitor, and apigenin (30 M) to inhibit protein kinase CKII. proinflammatory signals via IB-␣ remain unknown. A role for individual members of the protein kinase C (PKC) Nuclear protein extraction ␣ family in both SP-A immune modulation (26) and I B- turnover After treatment, cells were scraped off with 500 l of cold PBS and spun (27) has been suggested previously. PKC family members are ex- at 4,500 ϫ g, 5 min, 4°C. The resulting pellet was resuspended in 400 l
pressed in many different cell types, in which they regulate a wide of ice-cold buffer A (10 mM Tris, 5 mM MgCl2,10mMKCl,1mM variety of cellular processes such as cell differentiation, cytoskel- ethyleneglycol-bis-(2-aminoethyl ether)-N,N,NЈ,NЈ-tetraacetic acid, 0.3 M  etal remodeling, and gene expression in response to diverse stimuli sucrose, 1 mM DTT, 10 mM -glycerol phosphate, 0.5 mM PMSF, and 1.5 l of protease inhibitor mixture (Complete; Roche)) and incubated on ice (28, 29). Based on sequence homology and the mechanism of their for 15 min; 25 l of 10% Nonidet P-40 was then added and vortexed for regulation, individual members of the PKC family are subdivided 10 s. The nuclei were pelleted by centrifugation at 4,500 ϫ g, 5 min, 4°C. ␣   The supernatant was taken to represent the cytosolic fraction. The pellet into three classes as follows: classical PKCs (cPKC: , I, II, and ϩ ␥) are diacylglycerol (DAG) and Ca2 dependent, whereas novel was resuspended in 30 l of buffer B (20 mM Tris, 5 mM MgCl2, 320 mM KCl, 0.2 mM ethyleneglycol-bis-(2-aminoethyl ether)-N,N,NЈ,NЈ-tetraace- ␦ Downloaded from PKCs ( , , , and ) are responsive to DAG, but are insensitive tic acid, 25% glycerol, 1 mM DTT, and the mixture of protease inhibitors 2ϩ to Ca . The atypical (a) PKCs ( and /) do not bind to DAG and mentioned above) and incubated on ice for 15 min, followed by centrifu- are not responsive to Ca2ϩ (30, 31). gation at 16,100 ϫ g, 10 min, 4°C. The supernatant containing the nuclear In the present study, we confirmed the role of IB-␣ in SP-A fraction was transferred to a new vial. Cytosolic cell fractions (30–40 gof ␣ anti-inflammatory effects by using IB-␣ knockout/IB- knockin protein) were immunoblotted for I B- . Nuclear extracts (2 g of protein) of the cells were analyzed by EMSA for NF-B DNA-binding activity. (AKBI) AM and PBMC. We further investigated the role of PKC
in SP-A-specific anti-inflammatory signaling in primary AM and Isolation and stimulation of mouse AM and PBMC http://www.jimmunol.org/ identified the aPKC isoform as a central regulator of SP-A-me- Gene-deficient mice and respective controls were killed by i.p. injection diated IB-␣/NF-B modulation. of pentobarbital, followed by exsanguination by cardiac puncture for PBMC isolation. The lungs were lavaged with 1 ml of PBS containing Materials and Methods 0.2 mM EDTA. AM were plated at a density of 2 ϫ 105/ml in 24-well plates (Nunc) in the presence of 0.2% HI-FCS. PBMC were isolated by Mice and reagents density gradient using Ficoll-Histopaque-1083 (Sigma-Aldrich). Hepa- Primary cells were obtained from pathogen-free male Sprague-Dawley rats rinized blood was mixed with an equal amount of HBSS and centrifuged Ϫ Ϫ ϫ (Charles River Laboratories), from AKBI mice (32), or from PKC / at 420 g. The interphase layer containing the PBMC fraction was mice generated as described before (33). SV129 and CD1 wild-type (wt) collected and washed twice with HBSS and once in serum-free RPMI ϫ 6 control mice were from Charles River Laboratories. Animal care and ex- 1640. Cells were plated at a density of 1 10 /ml in 96-well plates. by guest on September 29, 2021 periments were conducted according to protocols approved by the AM and PBMC were left untreated or treated with SP-A (20–60 g/ml) Schleswig-Holstein Ministry of Environment, Nature, and Forestation. All for 1 h and/or stimulated with LPS (0.1–100 ng/ml) for4hat37°C in mice used were between 6 and 12 wk of age and were maintained at the the presence of 0.2 and 10% HI-FCS, respectively. After stimulation, ϫ Research Center Borstel animal facility under specific pathogen-free cells were centrifuged at 200 g, and cell-free supernatants were col- ␣ conditions. lected for TNF- determination. In separate experiments, AM were left The smooth LPS from Salmonella friedenau strain H909 was extracted untreated or treated with SP-A (40 g/ml) for 1 h, and/or stimulated by the phenol/water method, purified, lyophilized, and transformed into the with LPS (100 ng/ml) for1hat37°C in the presence of 0.2% HI-FCS. triethylamine salt form. RPMI 1640 medium, DMEM, and Dulbecco’s PBS Cytosolic cell fractions (30–40 g of protein/lane) were immunoblot- ␣ were from Invitrogen Life Technologies. Ham’s-F12 medium and FCS ted for I B- . Nuclear extracts (2 g of protein/lane) of the cells were were from BioWhittaker. Poly(dI/dC) was purchased from Pharmacia. analyzed by EMSA for NF- B DNA-binding activity. ␥ 32 [ - P]ATP was supplied by Hartmann Analytics; T4 polynucleotide ki- TNF-␣ ELISA nase was purchased from Roche. Rabbit polyclonal anti-IB-␣, anti-PKC (in the manuscript referred to as anti-aPKC), anti-p65, and HRP-conju- TNF-␣ was determined in pooled cell-free supernatants of stimulated gated goat anti-rabbit IgG were from Santa Cruz Biotechnology; a specific AKBI and wt control cells by sandwich DuoSet ELISA using goat anti- Ab against PKC was generated as described (34); anti-phospho PKC/ mouse TNF-␣ Ab and biotinylated goat anti-mouse TNF-␣ Ab (R&D Sys- (Thr410/403) Ab was from Cell Signaling Technology. Go¨-6850 was from tems), according to the manufacturer’s protocol. Calbiochem; chelerythrine chloride was from Tocris Bioscience; Go¨-6976 was from Alexis Biochemicals; apigenin and wortmannin were from Sig- Immunoprecipitation ma-Aldrich; and aPKC pseudosubstrate (ps) peptide was from BioSource Rat AM (2 ϫ 106/well) were stimulated for the indicated times with SP-A, International. Myelin basic protein and ATP/magnesium mixture were from LPS, or, for some experiments, the complement protein C1q that shares Upstate Biotechnology; C1q was from Advanced Research Technologies. All structural and functional homology with SP-A. After culture, cells were other reagents (except as noted) were obtained from Sigma-Aldrich. lysed on ice for 30 min in 500 l of lysis buffer (50 mM Tris-HCl (pH 8.0), SP-A purification 150 mM NaCl, 1% Nonidet P-40). The lysates were spun at 9300 ϫ g for 15 min, and the supernatants were precleared by adding protein A-agarose Human SP-A was purified from the bronchoalveolar lavage of patients with (50 l) and incubated at 4°C for 45 min, followed by centrifugation at alveolar proteinosis, as described in detail (35). Briefly, the lavage fluid 9300 ϫ g for 10 min. The precleared supernatant was incubated with anti- was treated with butanol to extract SP-A, and the resulting pellet was aPKC Ab, anti-p65 Ab, control IgG, or no Ab for2hat4°C, after which sequentially solubilized in octylglucoside and 5 mM Tris (pH 7.4). SP-A 50 l of protein A-agarose was added for2hat4°Cwith gentle rotation. was treated with polymyxin B agarose beads to reduce endotoxin contam- The immune complexes were collected by centrifugation at 9300 ϫ g for ination. SP-A preparations were tested for the presence of bacterial endo- 5 min at 4°C, washed three times with cold lysis buffer, and released by toxin using a Limulus amebocyte lysate assay (BioWhittaker); all SP-A boiling with 5ϫ sample buffer. Samples were used to determine kinase preparations used contained Ͻ0.2 pg endotoxin/g SP-A. activity or for Western analysis. Stimulation of rat AM Western analysis AM were isolated, as described previously (22). Cells were plated at 0.8 ϫ Western analysis was performed on cytosolic extracts, membrane fractions, 106/500 l in 24-well plates (Nunc) and allowed to attach for 90 min at and immunoprecipitated samples from rat and mouse cells. Experiments 4482 SP-A-ACTIVATED PKC IN IB-␣ STABILIZATION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021
FIGURE 1. SP-A fails to inhibit LPS-induced activation of AKBI cells. Pooled PBMC or AM from either three to four AKBI (A and C) or three wt mice (B and D) were left untreated or treated with 20–60 g/ml SP-A (37°C, 1 h), and then exposed either to medium, or to 0.1–100 ng/ml LPS. Cell-free supernatants were harvested after 4 h for the determination of TNF-␣ by ELISA. The data shown are mean Ϯ SE of three independent p Ͻ 0.01; †, p Ͻ 0.001 (vs ,ءء ;p Ͻ 0.05 ,ء .experiments. Statistical analysis was performed using a two-way ANOVA with a Bonferroni posttest LPS-induced TNF-␣ release in the absence of SP-A). SP-A fails to inhibit LPS-induced p65 nuclear translocation in AKBI AM. E, AM from wt (a–d) or AKBI (e–h) mice were left untreated or treated with 40 g/ml SP-A (37°C, 1 h), exposed to medium, or to 100 ng/ml LPS (1 h), and then analyzed by confocal microscopy. An Ab against NF-B p65 was detected by goat anti-rabbit IgG Ab conjugated to Alexa Fluor 488 (green), and cell nuclei were counterstained with propidium iodide (red). Overlays of single stainings are shown. Images are representative of three independent experiments. p Ͻ 0.001 ,ءء .F, Pixel densitometry of NF-B p65 was quantified and statistically analyzed with one-way ANOVA and Newman-Keuls post hoc test (vs control); #, p Ͻ 0.001 (vs LPS).
using the inhibitors above were performed under four different condi- and transferred to nitrocellulose membrane. The membranes were then tions, as follows: 1) in the absence of SP-A and LPS (basal); 2) in the incubated with anti-IB-␣, anti-aPKC, anti-PKCThr410/403, anti-p65, or presence of SP-A (40 g/ml, 1 h) (constitutive); 3) in the presence of -actin (mouse monoclonal) at a 1/700, 1/200, 1/700, 1/200, and 1/1000 LPS (10 ng/ml, 30 min) (induced); and 4) in the presence of SP-A (40 dilution, respectively. Goat anti-rabbit IgG-HRP or rabbit anti-mouse g/ml, 1 h) before LPS (10 ng/ml, 30 min) (modulated). After treat- IgG-peroxidase conjugate served as secondary Abs. Immunoreactive ment, cytosolic fractions were assayed for protein content by the bicin- proteins were visualized using the ECL Western blotting detection sys- choninic acid reagent (Pierce Biotechnology), separated on SDS-PAGE, tem (Amersham). The Journal of Immunology 4483
FIGURE 2. An aPKC is involved in IB-␣ sta- bilization. A–D, Representative cytosolic IB-␣ and -actin Western blots. A, Primary rat AM were left untreated or treated with 5 MGo¨-6976, 5 nM Go¨- 6850, 6 and 12 M chelerythrine chloride, 50 nM wortmannin (37°C, 30 min), or 30 M apigenin (37°C, 1 h). Equal amounts (30–40 g of protein) of the cytosolic fractions were subjected to SDS- PAGE and immunoblotted for IB-␣ or -actin (A– D). Analysis of band intensities was performed us- ing Optimas software (Media Cybernetics). Data are expressed as arbitrary units (a.u.) (mean Ϯ SE) from four to five independent experiments. B, AM were left untreated or preincubated (37°C, 30 or 60 min) with the inhibitors, as in A, and were then exposed to 40 g/ml SP-A (1 h). Data are expressed as per- centage of IB-␣ protein expression in the absence of SP-A (dotted line, 100%) (mean Ϯ SE) from four to five independent experiments. C, AM were left Downloaded from untreated or treated with the inhibitors, as in A, and were then exposed to 10 ng/ml LPS (30 min). Data are expressed as a.u. (mean Ϯ SE) from four to five independent experiments. D, AM were left un- treated or treated with SP-A, as in B, and were then exposed to 10 ng/ml LPS (30 min). Data are ex- pressed as percentage of IB-␣ protein expression http://www.jimmunol.org/ in the absence of SP-A (dotted line, 100%) (mean Ϯ SE) from four to five independent experiments. Data in A and C were analyzed by one-way ANOVA, followed by Newman-Keuls post hoc test; data in B and D were analyzed by paired Student’s t test when ,ء .expressed as percentage of the control response p Ͻ 0.05 (vs IB-␣ expression in the absence of SP-A). by guest on September 29, 2021
In vitro aPKC kinase assay Total cell lysates were then centrifuged at 100,000 ϫ g for 30 min at 4°C to separate cytosolic from particulate fractions. The resulting pel- Immunoprecipitated aPKC was subjected to the kinase assay in 100 l lets were extracted in 150 l of hypotonic fractionation buffer contain-  of reaction mixture (20 mM MOPS (pH 7.2), 25 mM -glycerol phos- ing 0.5% Triton X-100 and centrifuged at 16,000 ϫ g for 20 min at 4°C. phate, 1 mM DTT, 5 mM EGTA, 1 mM Na3VO4, 75 mM MgCl2, 0.5 The resulting supernatant was taken to represent the membrane fraction. ␥ 32 mM ATP, 100 Ci of [ - P]ATP) and 10 l of myelin basic protein Equal amount of membrane protein for each sample (30–40 g) was substrate at 37°C with gentle shaking. Reaction was stopped by adding separated by SDS-PAGE and blotted with anti-aPKC Ab. 50 l of Laemmli buffer. Immunocomplexes were released from aga- rose beads by boiling and centrifugation. The supernatant was trans- ferred to a new vial and subjected to 12% SDS-PAGE. The gel was analyzed with a PhosphorImager (Amersham Biosciences) to quantitate Immunofluorescence microscopy band intensities. AM were plated at a density of 1 ϫ 105 cells on 8-well Lab Tek II chamber slides (Nunc). Cells were allowed to attach for 90 min at 37°C in a 5% CO 2 NF- B activation assay atmosphere in the presence of 0.2% HI-FCS. After incubation with the Ϫ After exposing the cells to the experimental conditions, nuclear extracts indicated stimuli, cells were fixed with ice-cold ( 20°C) methanol, washed were prepared and analyzed, as described above. The activity of NF-Bin with PBS, and permeabilized with 0.25% Triton X-100. After a repeated the nuclear extracts was determined by EMSA. NF-B oligonucleotides washing, the cells were blocked with 10% BSA/PBS, and incubated with were end labeled with [␥-32P]ATP using T4 kinase. Two micrograms of isoform-specific anti-PKC Ab, anti-p65 Ab, or control IgG at a 1/250, crude nuclear extract was incubated for 20 min in binding buffer containing 1/50, or 1/100 dilution, respectively. Goat anti-rabbit IgG conjugated to 50 g of poly(dI/dC)/ml with 7.5 fmol of the 32P-labeled oligonucleotides Alexa Fluor 488 served as secondary Ab. Cell nuclei were counterstained encoding the consensus NF-B site 5Ј-AGCTCAGAGGGGACTTTC with propidium iodide. Samples were analyzed using a Leica TCS SP CGAGAGAGC-3Ј. Samples were separated by electrophoresis in 5% poly- confocal laser scanning microscope (Leica Microsystems). All images acrylamide gels for2hat180V,after which gels were analyzed with a were acquired under identical settings with Leica TCSNT software and PhosphorImager. assembled using Adobe Photoshop 6.0. An average of 120 (Fig. 1F)or100 (Fig. 5, C and E) cells was counted per condition and experiment, which Assay for aPKC membrane translocation was repeated three times. Rat AM were treated with SP-A (40 g/ml) for the times indicated. Incubation was stopped with ice-cold PBS, and the cells were resus- Statistical analysis pended in 300 l of hypotonic fractionation buffer (10 mM Tris (pH 7.4), 4.5 mM EDTA, 2.5 mM EGTA, 2.3 mM 2-ME, 1 mM PMSF, 10 Data were statistically analyzed, as indicated in the figure legends using g/ml aprotinin, 10 g/ml leupeptin, 10 g/ml pepstatin, and 0.1 mM GraphPad Prism (version 4.0; GraphPad). Values were considered signif- Ͻ Na3VO4), incubated for 20 min at 4°C while rotating, and sonicated. icant when p 0.05. Data are presented as SEM. 4484 SP-A-ACTIVATED PKC IN IB-␣ STABILIZATION
Results SP-A fails to inhibit LPS-induced TNF-␣ release by AKBI cells To confirm our previous findings on the role of IB-␣ in anti- inflammatory activity of SP-A, in this study, we used primary im- mune cells from IB-␣-deficient mice. In AKBI mice, the inte- grated IB- gene is under the control of the IB-␣ promoter, and at the same time a null mutation in IB-␣ is introduced (32). Un- like IB-␣-deficient mice, which are postnatal lethal (36), AKBI mice have a normal phenotype, and NF-B induction by PMA or TNF-␣ in AKBI mouse fibroblasts and thymocytes is identical compared with wt cells (32). In support of this notion, AKBI mice reveal a functional redundancy of IB-␣ and IB- upon LPS challenge in vivo (37). In line with these data, we show in this study that PBMC (Fig. 1A) and AM (Fig. 1C) from AKBI mice released similar amounts of TNF-␣ in response to LPS (0.1–100 ng/ml) compared with wt cells (Fig. 1, B and D). However, whereas SP-A (20–60 g/ml) significantly ( p Ͻ 0.05 to p Ͻ 0.001) and in a dose-dependent manner inhibited LPS-induced Downloaded from TNF-␣ release by wt cells (Fig. 1, B and D), SP-A failed to inhibit LPS-induced TNF-␣ release by PBMC (Fig. 1A) or AM (Fig. 1C) from AKBI mice. These data confirm that SP-A-mediated inhibi- tion of LPS-induced TNF-␣ production in PBMC and AM criti- cally depends on the presence of IB-␣, and that IB- could not compensate the lack of IB-␣ under these conditions. http://www.jimmunol.org/
SP-A fails to inhibit LPS-induced p65 nuclear translocation in AKBI AM Because p65 nuclear translocation is tightly linked to NF-B- dependent TNF-␣ gene induction (15), we next investigated the effect of SP-A on LPS-induced p65 localization in AKBI and wt AM by confocal microscopy. LPS-induced p65 nuclear accu- FIGURE 3. aPKC inhibition suppresses SP-A-mediated IB-␣ stabili- mulation in wt AM (Fig. 1Ec) was almost completely prevented zation. A–D, Representative cytosolic IB-␣ and actin Western blots. A, (60 Ϯ 5%) by pretreatment of the cells with SP-A (Fig. 1, Ed Primary rat AM were left untreated or treated with 2–10 M aPKCps by guest on September 29, 2021 and F). In contrast LPS-induced p65 nuclear accumulation in (37°C, 1 h) (control). Cytosolic cell fractions were immunoblotted for IB-␣ or -actin. Densitometric results are in arbitrary units (a.u.) (mean Ϯ AKBI AM (Fig. 1Eg) was not inhibitable by SP-A (Fig. 1, Eh SE) from five independent experiments. B, AM were left untreated or and F). As expected, comparable amounts of nuclear p65 are treated as in A and were then exposed to 40 g/ml SP-A (1 h). Densito- seen under resting or SP-A conditions in wt and AKBI AM. metric results are expressed as percentage of IB-␣ protein expression in Together the data suggest that SP-A-mediated inhibition of the absence of SP-A (control, dotted line) (mean Ϯ SE) from five inde- LPS-induced p65 nuclear translocation critically depends on the pendent experiments. C, AM were left untreated or treated as in A and were presence of IB-␣. then exposed to 10 ng/ml LPS (30 min). Densitometric results are in a.u. (mean Ϯ SE) from five independent experiments. D, AM were left un- An aPKC is involved in IB-␣ stabilization treated or treated with SP-A as in B and were then exposed to 10 ng/ml LPS (30 min). Data are expressed as percentage of IB-␣ protein expression in To elucidate the underlying molecular mechanisms of SP-A-me- the absence of SP-A (control, dotted line) (mean Ϯ SE) from five inde- diated immune protection, we focused on identifying the upstream pendent experiments. Data in A and C were analyzed by one-way ANOVA, regulators involved in the stabilization of IB-␣ by SP-A. Because followed by Newman-Keuls post hoc test; data in B and D were analyzed members of the PKC family have been shown to be involved in by paired Student’s t test when expressed as percentage of the control .(p Ͻ 0.02 (vs control ,ءء ;p Ͻ 0.05 ,ء .IB-␣ turnover (27) and SP-A immune functions (26), we used a response panel of PKC inhibitors with different specificity to test the role of PKC in SP-A-mediated IB-␣ stabilization in primary rat AM. Experiments were performed under four different conditions as follows: 1) distinct inhibitors alone (basal, Fig. 2A); 2) inhibitors Go¨-6976. The effect of SP-A on IB-␣ stabilization was still sig- plus SP-A (40 g/ml) (constitutive, Fig. 2B); 3) inhibitors plus nificant (149 Ϯ 12% of control) in the presence of Go¨-6976 (Fig. LPS (10 ng/ml) (induced, Fig. 2C); and 4) inhibitors plus SP-A (40 2B), but was abolished in the presence of LPS (Fig. 2D). Whereas g/ml), followed by LPS (10 ng/ml) (modulated, Fig. 2D). Cyto- treatment of AM with Go¨-6850, which inhibits the cPKCs ␣ and ␥ solic IB-␣ protein expression was determined by Western as well as the DAG-sensitive but Ca2ϩ-insensitive novel PKC iso- analysis. forms ␦ and , did not inhibit the SP-A-mediated stabilization of Neither of the inhibitors had a significant effect on IB-␣ protein IB-␣ (Fig. 2B), it inhibited SP-A’s effect in the presence of LPS expression under basal conditions (Fig. 2A). In line with our pre- (Fig. 2D). In sharp contrast, treatment of the cells with cheleryth- vious data (24), SP-A significantly increased IB-␣ protein ex- rine chloride, an inhibitor of Ca2ϩ- and DAG-dependent and pression by 210 Ϯ 44% ( p Ͻ 0.05) under constitutive conditions DAG-independent PKCs including the DAG-insensitive atypical (Fig. 2B) and by 137 Ϯ 10% ( p Ͻ 0.05) in the presence of LPS isoforms and /, abolished the stabilizing effect of SP-A on ␣   ␥ ␣ (Fig. 2D). cPKC isoforms ( , I, II, ), which are diacylglycerol I B- both constitutively and in the presence of LPS, suggesting (DAG) sensitive and Ca2ϩ responsive, are specifically inhibited by that aPKCs might be involved in SP-A effects (Fig. 2, B and D). The Journal of Immunology 4485 Downloaded from
FIGURE 4. SP-A stimulates aPKCThr410/403 phosphorylation and kinase activity. A, AM were incubated with 20 g/ml SP-A at 37°C for 1, 3, 5, or 10 min. aPKC was immunoprecipitated from whole cell lysates. The presence of aPKCThr410/403 was detected by immunoblotting with the corresponding Ab. Anti-aPKC Ab was used to evaluate the aPKC loads. Anti-rabbit IgG Ab was used as a control to show specificity for aPKC. Blots are representative of
three independent experiments. B, AM were treated with 20 g/ml SP-A for 0, 5, 10, 30, or 60 min. aPKC was immunoprecipitated from whole cell lysates. http://www.jimmunol.org/ The presence of aPKCThr410/403 was detected by immunoblotting with the corresponding Ab. Densitometric results (mean Ϯ SE) are given as percentage of the zero time point (control) from four independent experiments. C, AM were left untreated or treated with 20 g/ml SP-A for the times indicated. aPKC was immunoprecipitated from cell lysates and subjected to an in vitro kinase assay using myelin basic protein as a substrate. aPKC kinase activity was determined, as described in Materials and Methods. The phosphorylated proteins were run on a 12% SDS-PAGE gel. D, Densitometrical analysis of four independent experiments. The SP-A structural homologue C1q (20 g, 1 h) was included as control protein. Data are expressed as percentage of aPKC activity at the corresponding time point (control). E, AM were left untreated (lane 1), or treated with 40 g/ml SP-A (lane 2) or 100 ng/ml LPS (lane 3), or pretreated with 10 M aPKCps peptides before the addition of 40 g/ml SP-A (lane 4). Cells were subjected to an in vitro aPKC kinase assay. The gel is representative of three independent experiments. Data were analyzed by a one-way ANOVA with a Dunnett’s post hoc test, or with paired Student’s t .(p Ͻ 0.05 (vs control ,ء .test by guest on September 29, 2021
Both SP-A and LPS can activate PI3K in human macrophages 3D). Treatment of the cells with aPKCps at 10 M resulted in (38) and in THP-1 cells (39), respectively. In the present study, a significant inhibition of IB-␣ by SP-A (Fig. 3B). Interest- the PI3K inhibitor wortmannin significantly reduced IB-␣ ex- ingly, in the presence of LPS, aPKCps pretreatment at any con- pression in the presence of LPS (Fig. 2C). Wortmannin abro- centration resulted in a significant inhibition ( p Ͻ 0.02) of gated SP-A’s effect on IB-␣ stabilization both constitutively IB-␣ protein levels by SP-A (Fig. 3D) when compared with and in the presence of LPS (Fig. 2, B and D). In mouse em- the corresponding aPKCps concentration in the absence of bryonic fibroblasts, CKII is critically involved in the basal turn- SP-A. As expected, LPS-induced NF-B activity in AM was over of IB-␣ (12). Apigenin, a selective CKII inhibitor, in- abolished after pretreatment with aPKCps (data not shown). hibited IB-␣ stabilization by SP-A constitutively and in the Taken together, these results suggest that an aPKC isoform is presence of LPS (Fig. 2, B and D). involved in IB-␣ stabilization by SP-A both constitutively and Because, among aPKCs, PKC has been shown to modulate in the presence of LPS. IB-␣ turnover (27) and IKK/NF-B activation in both nuclear and whole lung extracts (33), we wanted to elucidate the role of 410/403 aPKC isoforms in SP-A’s AM immunomodulation more precisely. SP-A stimulates aPKCThr phosphorylation and kinase Therefore, we used the isoform-specific cell-permeable inhibi- activity tory myristoylated peptide derived from the ps motif of aPKCs. To investigate whether SP-A can stimulate aPKC activation, the aPKCps mimics the substrate and maintains aPKC in its non- phosphorylation status of aPKC was determined. To activate active form. Of note, the ps peptides are not totally specific, aPKC, phosphorylation of the activation loop consensus threo- because the ps sequences (SIYRRGARRWRKL) are identical nine residue Thr410 by PI3K-dependent PDK1 is substantial in both isoforms PKC and PKC/ (40). (41). In fact, treatment of AM with SP-A significantly increased aPKCThr410/403 phosphorylation after 1–10 min (Fig. 4A)of ␣ aPKC inhibition suppresses SP-A-mediated I B- stabilization incubation and then declined to baseline (Fig. 4B). Again, cytosolic IB-␣ protein in AM was determined under We then investigated whether the observed SP-A-mediated in- four different conditions (basal, in the presence of SP-A, or crease in aPKCThr410/403 phosphorylation correlates with an in- LPS, or both). Treatment of the cells with aPKCps (2–10 M) creased kinase activity. SP-A, but not C1q, a structural homologue did not significantly affect IB-␣ protein level under basal (Fig. of SP-A, stimulates aPKC activity in a time-dependent manner, 3A) or LPS (Fig. 3C) conditions. Compared with basal condi- reaching a maximum at1h(p Ͻ 0.05) (Fig. 4, C and D). SP-A- tions, SP-A significantly increased IB-␣ protein expression induced aPKC activity (Fig. 4E, lane 2) was comparable to that both constitutively (Fig. 3B) and in the presence of LPS (Fig. induced by LPS (Fig. 4E, lane 3), used as a positive control (42). 4486 SP-A-ACTIVATED PKC IN IB-␣ STABILIZATION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021
FIGURE 5. SP-A favors PKC plasma membrane translocation. A, AM were left untreated or treated with 40 g/ml SP-A for the times indicated. Cell membrane isolation was performed, as described in Materials and Methods. Cytosolic and membrane fractions were subjected to SDS-PAGE and immunoblotted for aPKC. Data shown are representative of three experiments. B, AM adhered to chamber slides at 1 ϫ 105 cells/well and were left untreated and then exposed to medium or treated with 40 g/ml SP-A (1 h), and were then further either exposed to 100 ng/ml LPS (1 h) or treated with 100 ng/ml LPS (1 h) alone. Slides were blocked and incubated with an isotype-specific Ab (control IgG; a), or a specific Ab against PKC (b–e) that was detected by goat anti-rabbit IgG Ab conjugated to Alexa Fluor 488. f–j, Cell nuclei were counterstained with propidium iodide (PI). Cells were visualized by confocal microscopy. k–o, Overlay of single stainings. Images shown are representative of three independent experiments with similar results. C, Pixel density of cell-membrane PKC was quantified from total PKC and statistically analyzed by one-way p Ͻ 0.001 (vs control); #, p Ͻ 0.001 (vs LPS). D, AM were left untreated or treated with 40 g/ml ,ءء .ANOVA and Newman-Keuls post hoc test SP-A (1 h), and then exposed to medium, or to 100 ng/ml LPS (1 h) and further treated as described for B. a–e, Cells were incubated with an isotype-specific Ab (control IgG; a), or an Ab against NF-B p65 (b–e) that was detected by goat anti-rabbit IgG Ab conjugated to Alexa Fluor 488. f–j, Cell nuclei were counterstained with PI; k–o, overlay of single stainings. Images shown are representative of three independent experiments. E, Pixel density of cell membrane was quantified from total NF-B p65 and statistically analyzed by one-way ANOVA and Newman-Keuls post hoc p Ͻ 0.001 (vs control); #, p Ͻ 0.001 (vs LPS). SP-A enhances PKC/p65 coimmunoprecipitation under resting conditions. F, AM were left ,ءء .test untreated or treated with 40 or 60 g/ml SP-A (1 h). NF-B p65 was immunoprecipitated from whole cell lysates, as described in Materials and Methods. Anti-rabbit IgG or no Ab was used as a control. aPKC was detected by immunoblotting with the corresponding Ab. Anti-p65 Abs were used to evaluate p65 loads. G, Densitometric results of three independent experiments. Data are expressed as arbitrary units (a.u.) (mean Ϯ SE). Data .(p Ͻ 0.05 (vs untreated ,ء .were analyzed by one-way ANOVA with DunnettЈs posttest
Incubation of AM with aPKCps peptides (Fig. 4E, lane 4) abol- translocation by two approaches, i.e., membrane fractionation and ished the aPKC activity in the presence of SP-A. Together, the confocal microscopy. results support the idea that SP-A mediates IB-␣ stabilization in primary AM via activation of aPKC. SP-A favors the accumulation of PKC in the plasma membrane Even though phosphorylation at Thr410 facilitates kinase activ- Cell fractionation demonstrated that aPKC membrane translo- ity, it is not sufficient to provide full activity. A rapid autophos- cation is stimulated by SP-A in a time-dependent manner, phorylation is necessary for a concomitant translocation of PKC reaching a maximum at 1 h (Fig. 5A). Using confocal micros- (43, 44). Therefore, we examined the effect of SP-A on aPKC copy, we confirmed that SP-A, compared with basal conditions, The Journal of Immunology 4487 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021
FIGURE 5. (continued)
favors a translocation of PKC to the plasma membrane in pri- ence of LPS, favored both PKC and p65 membrane mary AM, as detected by an isoform-specific Ab (Fig. 5B, l and localization, the effect of SP-A on PKC/p65 interaction was m). In contrast, LPS stimulation of AM induced a translocation examined next. SP-A, significantly and in a concentration-de- of PKC (Fig. 5Bn) toward the nucleus. Pretreatment of the pendent manner, increased PKC/p65 coimmunoprecipitation cells with SP-A, however, largely reduced LPS-mediated nu- under constitutive conditions (Fig. 5, F and G). Because the clear translocation of PKC (Fig. 5Bo). SP-A-enhanced interaction obviously has no NF-B trans-ac- tivating potency, we hypothesized that this effect of SP-A di- SP-A modulates p65 localization and p65/PKC rectly or indirectly prevents p65 nuclear translocation. coimmunoprecipitation ␣ Stimulus-induced PKC associates with and phosphorylates PKC is essential for SP-A-mediated I B- stabilization and trans-activating p65 (33, 45). We next asked whether SP-A- inhibition of LPS-induced NF- B activity favored PKC membrane localization is associated with the dis- To establish the selective role of PKC in mediating the SP-A tribution of p65. We confirmed our initial observation in wt effect described, we used mice deficient for the PKC isoform (33). mouse AM (Fig. 1E) and in rat AM (Fig. 5D), and indeed re- Whereas PKC/Ϫ/Ϫ mice are embryonic lethal (46), PKCϪ/Ϫ vealed that p65 distribution strongly parallels that of PKC mice are grossly normal, but exhibit an impairment of IKK (Fig. 5B) under SP-A or LPS conditions (Fig. 5, B, Dc, Dd, and activation in whole lung extracts after LPS challenge (33). In E). Furthermore, as in wt mouse AM, pretreatment of the cells freshly isolated AM from PKCϪ/Ϫ mice, however, NF-Bac- with SP-A almost abolished LPS-induced p65 nuclear translo- tivation was apparent both basal and LPS induced. SP-A failed cation and induced an accumulation of p65 at the plasma mem- to inhibit basal and only slightly reduced LPS-induced NF-B brane (Fig. 5, De and E). Because SP-A, alone or in the pres- DNA binding compared with wt AM (Fig. 6, A and B, lower 4488 SP-A-ACTIVATED PKC IN IB-␣ STABILIZATION
FIGURE 6. PKC is vital for SP- A-mediated IB-␣ stabilization and inhibition of LPS-induced NF-B ac- tivity. A and B, Pooled AM from ei- ther four wt (A) or four PKC-defi- cient mice (B) were left untreated or treated with 40 g/ml SP-A (1 h), and then exposed to either medium or 100 ng/ml LPS (1 h). Cytosolic cell frac- tions were immunoblotted for IB-␣ (upper panel). Nuclear extracts of the cells were analyzed by EMSA for NF-B DNA-binding activity Downloaded from (middle panel). Data shown are rep- resentative of three independent ex- periments. Densitometric results are expressed as arbitrary units (a.u.). Data were analyzed by one-way ANOVA with a post hoc Newman- /p Ͻ http://www.jimmunol.org ,ءء ;p Ͻ 0.01 ,ء .Keuls test 0,001 (vs control); #, p Ͻ 0.01; ##, p Ͻ 0.001 (vs LPS). by guest on September 29, 2021
panel). Compared with wt AM, SP-A-mediated IB-␣ stabili- B-dependent gene expression in the basal state of cell activation zation was almost abolished in PKCϪ/ϪAM (Fig. 6, A and B, and for making p65/IB complexes responsive to diverse stimuli. upper panel). Surprisingly, in the presence of LPS, IB-␣ was We previously suggested that SP-A inhibits LPS-induced NF-B enhanced in PKCϪ/Ϫ AM compared with wt AM, and this activation by posttranscriptionally slowing the basal turnover of effect was abolished when cells had been pretreated with SP-A IB-␣ in primary AM (24). However, the in vivo proof and the (Fig. 6B). Taken together, these data indicate that PKC, con- intracellular signaling pathways involved have not yet been stitutively and in the presence of LPS is required for SP-A- defined. mediated anti-inflammatory signaling in AM. The present study confirms that IB-␣ is critically involved in SP-A anti-inflammatory effects. In AKBI mice, the integrated Discussion IB- gene is under the control of the IB-␣ promoter, and at the Emerging evidence demonstrates a pivotal role of the pulmonary same time a null mutation in IB-␣ is introduced (32). AKBI mice collectin SP-A in anti-inflammatory immunomodulation of innate have a normal phenotype and reveal a functional redundancy of immune responses of the lung to LPS (1, 2). Airway inflammation IB-␣ and IB- upon LPS challenge in vivo (37). In line with associated with local or systemic LPS release from Gram-negative this, we found that LPS-induced TNF-␣ release by AKBI cells was bacteria is still a major cause of life-threatening pulmonary dis- comparable to that by wt cells, confirming the described in vivo eases (8). Efficient negative signaling cascades to prevent auto- redundancy of IB-␣ and IB- in response to LPS (37). How- toxic mediator release by AM, the major effector cell of the pul- ever, whereas SP-A significantly inhibited LPS-induced TNF-␣ monary innate immune system, in response to LPS, and, in release (Fig. 1, B and D) and p65 nuclear translocation in wt cells particular, the modulation of NF-B activation threshold by SP-A (Fig. 1Ed), it failed to inhibit TNF-␣ production (Fig. 1, A and C) have been studied intensively in recent years. Inhibition of LPS- as well as p65 nuclear accumulation in AKBI cells (Fig. 1Eh). induced NF-B activity by SP-A has been suggested to occur via These data strongly suggest that IB- could not compensate the direct interaction of SP-A with components of the LPS receptor lack of IB-␣ in SP-A-mediated inhibition of LPS-induced cell complex, including LPS-binding protein, CD14, TLR-4, and activation. MD-2, but also independently of LPS-specific signal transduction The present study further identifies aPKC as an essential up- pathways (19–21, 23, 24, 47, 48). Intracellularly, the pivotal role stream mediator of IB-␣/NF-B regulation by SP-A in primary of IB-␣ in NF-B regulation has recently been highlighted by cells. In PKCϪ/Ϫ AM, SP-A-mediated IB-␣ stabilization was Tergaonkar et al. (13), demonstrating that IB-␣ (as well as  and almost completely abrogated (Fig. 6B), strongly supporting our ) is essential for both preventing NF-B DNA binding and NF- experiments using the inhibitory aPKCps peptides in rat AM The Journal of Immunology 4489
FIGURE 7. A model implicating PKC in SP-A- mediated IB-␣ stabilization, leading to inhibited NF-B-induced cell activation. A, In resting AM, SP-A-activated PKC is implicated as a p65-inter- acting kinase that reduces macrophage-inflamma- tory responsiveness by promoting IB-␣ stabiliza- tion via retarded degradation (broken arrow), subsequently altering the threshold for NF-B acti- vation. B, Classical LPS-induced NF-B activation pathway. LPS-induced PKC leads to IB-␣ phos- phorylation, ubiquitination, and degradation (bold arrow) via IKK, allowing p65 nuclear translocation and NF-B activation. Downloaded from
(Fig. 3, A and B). The combined data confirm that the PKC iso- SP-A, directly or indirectly, prevents p65 nuclear translocation.
form is critically involved in SP-A-mediated IB-␣ stabilization Confocal and cell fractionation analysis of p65 localization in rat http://www.jimmunol.org/ and suggest that the inhibition or the lack of PKC potentiates the AM revealed a substantial inhibition of LPS-induced p65 nuclear rate of IB-␣ turnover in resting AM. translocation by SP-A. Based on the combined results, we propose In contrast to the well-characterized IB-␣ turnover under stim- a model in which SP-A-mediated PKC activation stabilizes IB-␣ ulus-induced conditions, little is known about the mechanisms that by preventing p65 nuclear translocation in primary AM. regulate the basal turnover of IB-␣. In mouse embryonic fibro- A previous study (33) showed that NF-B DNA-binding activ- blasts, an efficient basal turnover of IB-␣ requires the C-terminal ity is reduced in nuclear lung extracts of PKCϪ/Ϫ mice treated CKII phosphorylation (Ser293), whereas IKK phosphorylation with LPS or IL-1 i.p. In whole lung extracts, the lack of PKC (Ser32 and Ser36) plays no role under basal conditions (12). Even resulted in an impairment of IKK activation upon LPS stimulation, though IB-␣ is not a direct substrate of PKC (49), PKC-asso- suggesting a substantial role of PKC in the control of stimulus- by guest on September 29, 2021 ciated CKII preferentially phosphorylates Ser293 compared with induced IKK/NF-B signaling cascade in the pulmonary compart- nonassociated CKII in transfected NIH3T3 cells, thereby poten- ment (33). In the present study, IB-␣ expression in response to tially accelerating the basal turnover of IB-␣ (27). In the present LPS was decreased in wt AM (Fig. 6A), as expected. Surprisingly, study, however, the inhibition or the lack of PKC accelerated LPS enhanced IB-␣ in PKCϪ/Ϫ AM (Fig. 6B). Possible expla- IB-␣ turnover under basal conditions in AM. In addition, apige- nations might include that the lack of PKC impairs the LPS- nin, a selective CKII inhibitor, abolished the SP-A-mediated sta- induced IKK activation and the subsequent IB-␣ degradation, as bilization of IB-␣, suggesting that in AM both PKC and CKII previously suggested (33). Alternatively, the IB-␣ increase ob- activity contribute to this process. served in PKCϪ/Ϫ AM could result from de novo transcription One central route controlling IB-␣ turnover is the proteasome- and might be referred to a compensatory increase of PKC/. How- mediated degradation of ubiquitinated IB-␣. PKC has been ever, Western blot analysis of lung extracts from wt and PKC- shown to influence proteasome-mediated protein degradation (50, deficient mice for PKC and PKC/ protein expression with spe- 51). However, when we investigated the effect of SP-A on IB-␣ cific Abs revealed that the level of PKC/ is not affected by the ubiquitination in AM, SP-A enhanced the presence of ubiquitin- loss of PKC (55), rendering the latter explanation unlikely. The conjugated IB-␣ (and other proteins) alone and in combination LPS-mediated IB-␣ increase in PKCϪ/Ϫ AM was abolished by with a proteasome inhibitor and/or aPKCps, suggesting that SP- pretreatment of the cells with SP-A, proposing that SP-A not only A-activated PKC does not stabilize IB-␣ by inhibiting its deg- fails to stabilize IB-␣, but provides a signal that triggers IB-␣ radation via the ubiquitin-dependent pathway (data not shown). degradation, presumably by activating IKK in a PKC-indepen- The finding that this PKC isoenzyme is involved in SP-A-me- dent way. Of note, in PKCϪ/Ϫ AM, SP-A failed to inhibit basal diated anti-inflammation was initially surprising because LPS- or and LPS-induced NF-B activation compared with wt cells, sug- cytokine-induced PKC activity was previously shown to induce gesting that the down-modulation of constitutive and LPS-induced NF-B activation by distinct mechanisms, as follows: first, NF-B activity by SP-A is dependent on PKC (Fig. 6B). through its activation of IKK (52, 53), and second, by phosphor- The mechanisms underlying PKC activation are not completely ylating p65 (45, 54). Besides PKC, kinases implicated in p65 understood and display substantial differences for different cell phosphorylation include CKII, protein kinase A, IKK2, Akt, p38, types, stimuli, and incubation conditions (30). PKC can be acti- p42, and p44 (17). Among these kinases, only PKC has been vated in vitro by phosphatidylinositol-3,4,5-triphosphat (42, 56), shown to directly interact with p65 (33) and phosphorylates p65 at phosphatidic acid (57), ceramide, and arachidonic acid (58, 59), Ser311 in TNF-␣-stimulated mouse fibroblasts (45). In the present and/or by direct interaction with binding proteins (60). To activate study, SP-A enhanced the interaction of PKC with p65 in primary PKC, phosphorylation of the activation loop consensus threonine AM under constitutive conditions (Fig. 5D). Because SP-A alone residue Thr410 by PI3K-induced phosphoinositide-dependent ki- has no NF-B trans-activating potency, we thus hypothesized that nase 1 is substantial (41). Besides different activation mechanisms, 4490 SP-A-ACTIVATED PKC IN IB-␣ STABILIZATION
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