Am J Physiol Lung Cell Mol Physiol 305: L175–L184, 2013. First published May 17, 2013; doi:10.1152/ajplung.00295.2012.
Induction of nitric oxide synthase expression by lipopolysaccharide is mediated by calcium-dependent PKC␣-1 in alveolar epithelial cells Émilie Boncoeur,1* Guillaume F. Bouvet,1,6* Francis Migneault,1,6* Valérie Tardif,1 Pasquale Ferraro,3 Danuta Radzioch,4 Juan B. de Sanctis,7 David Eidelman,5 Karuthapillai Govindaraju,5 André Dagenais,1,2,6 and Yves Berthiaume1,2,6 1
Centre de recherche, Centre hospitalier de l’Université de Montréal (CR-CHUM)-Hôtel-Dieu 2Département de médecine, Université de Montréal, Montreal, Quebec, Canada; 3Division of Thoracic Surgery, Departement of Surgery, Centre hospitalier de l’Université de Montréal (CR-CHUM), Université de Montréal, Montreal, Quebec, Canada; 4Departments of Medicine and Human Genetics, McGill University, Montreal, Quebec, Canada; 5Meakins-Christie Laboratories and Respiratory Division, Department of Medicine, McGill University, Montreal, Quebec, Canada; and 6Institut de recherches cliniques de Montréal, Research Division of Cardiovascular and Metabolic Diseases, Montreal, Quebec, Canada; 7Institute of Immunology, Universidad Central de Venezuela at Caracas, Caracas, Venezuela Submitted 29 August 2012; accepted in final form 9 May 2013
Boncoeur É, Bouvet GF, Migneault F, Tardif V, Ferraro P, Radzioch D, de Sanctis JB, Eidelman D, Govindaraju K, AD, Berthiaume Y. Induction of nitric oxide synthase expression by lipopolysaccharide is mediated by calcium-dependent PKC␣-1 in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 305: L175–L184, 2013. First published May 17, 2013; doi:10.1152/ajplung.00295.2012.— Nitric oxide (NO) plays an important role in innate host defense and inflammation. In response to infection, NO is generated by inducible nitric oxide synthase (iNOS), a gene product whose expression is highly modulated by different stimuli, including lipopolysaccharide (LPS) from gram-negative bacteria. We reported recently that LPS from Pseudomonas aeruginosa altered Na⫹ transport in alveolar epithelial cells via a suramin-dependent process, indicating that LPS activated a purinergic response in these cells. To further study this question, in the present work, we tested whether iNOS mRNA and protein expression were modulated in response to LPS in alveolar epithelial cells. We found that LPS induced a 12-fold increase in iNOS mRNA expression via a transcription-dependent process in these cells. iNOS protein, NO, and nitrotyrosine were also significantly elevated in LPS-treated cells. Ca2⫹ chelation and protein kinase C (PKC␣-1) inhibition suppressed iNOS mRNA induction by LPS, implicating Ca2⫹-dependent PKC signaling in this process. LPS evoked a significant increase of extracellular ATP. Because PKC activation is one of the signaling pathways known to mediate purinergic signaling, we evaluated the hypothesis that iNOS induction was ATP dependent. Although high suramin concentration inhibited iNOS mRNA induction, the process was not ATP dependent, since specific purinergic receptor antagonists could not inhibit the process. Altogether, these findings demonstrate that iNOS expression is highly modulated in alveolar epithelial cells by LPS via a Ca2⫹/PKC␣-1 pathway independent of ATP signaling. ATP; epithelial cells; iNOS; PKC, purinergic system THE LUNG EPITHELIUM, covering the largest surface of the body at interface between host and environment (60), is continuously exposed to a wide variety of inhaled environmental toxins and airborne pathogens. The ability to maintain epithelial homeostasis and barrier integrity depends on innate and adaptive immunity by the lung epithelial cells and specialized hemo-
*E. Boncoeur, G. Bouvet, and F. Migneault contributed equally to this work. Address for reprint requests and other correspondence: Y. Berthiaume, CRCHUM-Hôtel-Dieu, 3840 St. Urbain, Montréal, Québec, Canada H2W 1T7 (e-mail:
[email protected]). http://www.ajplung.org
poietic cells such as alveolar macrophages, neutrophils, and dendritic cells (17). Recent data have shown that alveolar epithelial cells not only are a passive barrier to pathogen but play an active role by secreting an array of proinflammatory molecules in response to pathogen (2, 4, 11, 17, 43, 58). Nitric oxide (NO) production is one of the critical components of innate host defense against pathogens (55). However, because NO has been found to have beneficial microbial, antiviral, and antiparasitic effects (31), aberrant or excessive NO formation seems to be involved in the pathophysiology of asthma (45, 52), multiple organ failure, and acute lung injury, owing to its participation in the generation of pathological inflammation (15, 40). In the lungs, NO production has been shown to be markedly augmented in many cell types by suitable agents, such as bacterial lipopolysaccharide (LPS), cytokines, and other compounds (45). This augmented NO generation results from increased expression of inducible nitric oxide synthase (iNOS; NOS2 gene). The mechanism of the signal transduction cascade involved in iNOS induction in response to LPS is an active area of investigation and depends on cell type. Classically, NOS2 gene expression is regulated at the transcription level by the activation of transcription factors, such as activator protein-1 (AP-1), nuclear factor-B (NF-B), or signal transducer and activator of transcription-1 (STAT-1) (31). However, some authors have postulated protein kinase C (PKC) pathway involvement in the regulation of NOS induction in RAW 264.7 macrophages by LPS (9, 16, 44). PKC was first characterized as a Ca2⫹- and phospholipid-dependent protein serine/threonine kinase that requires diacylglycerol (DAG) for its activity. PKC comprises a family of at least 12 different members divided into two classes: Ca2⫹ dependent (PKC␣, PKC) and Ca2⫹ independent (others). PKC members may exert specific functions as a consequence of many differences in their structure, activation, subcellular localization and substrate specificity (51). Furthermore, extracellular adenosine triphosphate (ATP) and activation of purinergic receptors have been reported to induce iNOS expression in mice (24, 39). Surprisingly, because ATP has been found to be released by LPS in RAW 264.7 cells (48), it has also been demonstrated to potentiate or enhance NOS expression induced by LPS in these cells (53, 54). Purinergic receptors of the P2Y family are G proteincoupled receptors (GPCR) that binds ATP and activate PKC
1040-0605/13 Copyright © 2013 the American Physiological Society
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through phospholipase C breakdown of PIP2 and the generation of inositol 1,4,5-trisphosphate (IP3) and DAG (18). It has not been established yet whether purinergic stimulation and PKC could modulate iNOS mRNA induction. Although neutrophils and macrophages are known to highly express iNOS upon LPS stimulation (40), several evidences suggest that NO production by alveolar epithelial cells could be associated to the pathophysiology of lung injury. In an animal model, the selective depletion of iNOS expression by ⫺/⫺ bone marrow cells transplanted in iNOS ⫹/⫹ irradiated mice where hemopoietic cells cannot generate NO has shown that parenchymal cells are a major contributor to NO production in the lung after intraperitoneal injection of LPS (57) or nebulization of LPS in the lung (3). These studies suggest that alveolar epithelial cells, not macrophages, could play an important role in NO synthesis in response to LPS. We reported previously that LPS downregulates the activity of the epithelial Na channel (ENaC) in alveolar epithelial cells via a PKC- and suramin-sensitive pathway (6), suggesting that PKC activation, via purinergic signaling, could be an important proinflammatory pathway triggered by LPS in alveolar epithelial cells. In the present study, we investigated whether iNOS expression can be modulated by LPS in alveolar epithelial cells and whether PKC and purinergic signaling could play a role in this modulation. We found that iNOS expression is highly modulated in these cells by LPS via a Ca2⫹/PKC␣-1-dependent pathway that is not triggered by ATP signaling. MATERIALS AND METHODS
Materials. Minimum essential medium (MEM) and fetal bovine serum (FBS) were purchased from Invitrogen Canada (Burlington, ON, Canada). Porcine pancreatic elastase was obtained from Worthington Biochemical (Lakewood, NJ). LPS from Pseudomonas aeruginosa, actinomycin D (Act D), cycloheximide (CCX), suramin, brilliant blue G (BBG), 2-methylthioadenosine-5=-O-triphosphate (2-MeSATP), adenosine-5=-O-(3-thiotriphosphate) (ATP␥S) and monoclonal antibody against -actin (Ac-74) were procured from Sigma-Aldrich (Oakville, ON, Canada). pan-PKC and primary antibodies against iNOS were supplied by Abcam (Cambridge, MA); pan-phospho-PKC (P-PKC) came from Cell Signaling Technology (Beverly, MA). Mouse monoclonal and polyclonal antibodies against nitrotyrosine, and polyclonal goat antirabbit IgG-peroxidase were from Upstate Biotechnology (Lake Placid, NY). PKC inhibitors (RO 318220, GO 6976), mitogen-activated protein kinase (MAPK) inhibitors (PD 98059, SP 600125), phosphatidylinositol 3 kinase (PI3K) pathway inhibitors (LY294002, wortmannin, and rapamycin), and NF-B inhibitors (BAY-117082, SN50) were supplied by Calbiochem (Gibbstown, NJ). 1,2-Bis(2-aminophenoxy)ethane-N,N,N=,N=-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) came from Molecular Probes (Invitrogen, Carlsbad, CA). 2-Aminoethyl diphenylborinate (2-APB), verapamil, and nifepidine were purchased from Sigma-Aldrich (Oakville, ON, Canada). Alveolar epithelial cell isolation and experimental conditions. Alveolar epithelial cells were isolated from male Sprague-Dawley rats (Charles River Laboratories, Senneville, QC, Canada), as described previously (7, 12, 20), and according to a procedure approved by our institutional animal care committee. Perfused lungs were digested with elastase, and the cells were purified by a differential adherence technique on bacteriological plastic plates coated with rat immunoglobulin G. The cells were maintained in MEM containing 10% FBS, 0.08 mg/l gentamicin, 0.2% NaHCO3, 0.01 M HEPES, and 2 mM L-glutamine. They were plated at 1 ⫻ 106 cells/cm2 density in 35-mm plastic dishes or polycarbonate filters (Costar Transwell, Toronto, ON, Canada) and cultured at 37°C under 5% CO2 in a humidified incuba-
tor. The medium was supplemented with Septra (3 g/ml trimethoprim and 17 g/ml sulfamethoxazole) for the first 3 days. The medium was replaced thereafter, and the cells were cultured without Septra. RNA purification and semiquantitative RT-PCR. Total RNA from rat alveolar epithelial cells cultured with or without LPS 15 g/ml for 4 h was purified with TRIzol reagent according to the manufacturer’s instructions (Life Technologies, Burlington, ON, Canada). Three micrograms of total RNA were reverse transcribed to cDNA with Moloney murine leukemia virus reverse transcriptase (Life Technologies) in the presence of oligo(dT) primers (Roche Diagnostics, Laval, QC, Canada). cDNAs were amplified with Taq polymerase (Life Technologies) by using specific primers designed for rat iNOS or -actin. The PCR primer pairs for iNOS [sense: 5= CTT GTG TCA GCC CTC AGA GT 3= and antisense: 5= TCT GTG CTG AGA GTC ATG GA 3= (1 M final concentration of each)] were amplifying a 427-bp amplicon between exon 21 and 24 whereas the -actin primers [sense: 5= CTA AGG CCA ACC GTG AAA AG 3= and antisense: 5= GCC ATC TCT TGC TCG AAG TC 3= (0.25 M final concentration of each)] were amplifying a 350-bp amplicon between exon 4 and 5, respectively. Semiquantitative RT-PCR amplification was undertaken according to a well-established laboratory protocol (13). To remain in the linear phase of amplification, the iNOS product was amplified for 23 PCR cycles, whereas -actin amplification was stopped after 15 cycles. The amplification products were separated on agarose gels, stained with Syber Safe (Life Technologies), and analyzed by Typhoon Gel Imager. For semiquantitative evaluation of iNOS cDNA in rat alveolar epithelial cells, the signals were normalized to -actin amplification of the same cDNA sample. RT-qPCR. RT-quantitative PCR (RT-qPCR) analysis of iNOS and -actin was undertaken as described below: cDNA synthesis was performed with Quantitect Reverse Transcription kit (Qiagen- Canada, Toronto, ON, Canada), according to the manufacturer’s instructions. PCR amplification was carried out with SYBR Green PCR Master Mix (Life Technologies), following the manufacturer’s instructions, in Rotor-Gene Q real time cycler (Qiagen Canada, Toronto, ON, Canada). Reactions were performed in triplicate for each sample, 2 l of template cDNA was mixed with 2⫻ Universal PCR Master Mix with 200 nM of iNOS primers or 100 nM of -actin primers in a final volume of 15 l. The iNOS primers (sense: 5= AGT GAG GAG CAG GTT GAG GA 3= and antisense: 5= TGG GTG TCA GAG TCT TGT GC 3=) were amplifying a 144 bp amplicon between exon 26 and 27 while the -actin primers (sense: 5= ACC GTG AAA AGA TGA CCC AGA T 3= and antisense: 5= CAC AGC CTG GAT GGC TAC GT 3=) were amplifying a 74-bp amplicon between exon 3 and 4. Relative iNOS mRNA quantity was estimated according to the comparative threshold cycle (CT) method (Rotor-gene 6000 software). The cDNA ratio was calculated on the basis of CT values standardized to the amount of -actin gene expression. Protein extraction and immunoblotting. Alveolar epithelial cells treated or not with 15 g/ml LPS were washed twice with PBS and incubated for 1 h at 4°C with agitation in lysis buffer (1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.5) supplemented with protease inhibitor cocktail [1 mM phenylmethylsulfonyl fluoride (PMSF), 10 g/ml aprotinin, 500 ng/ml leupeptin]. The cells were subsequently scraped with a rubber policeman, collected, and centrifuged at 12,000 g for 5 min. Protein concentration of the supernatant was evaluated by the Bradford method (Bio-Rad Life Science, Mississauga, ON, Canada). Fifty micrograms of total proteins were solubilized in sample buffer [50 mM Tris·HCl, 2% sodium dodecyl sulfate (SDS), 0.1% bromophenol blue, 10% glycerol, and 125 mM DTT], subjected to SDS-PAGE, and transferred electrophoretically onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were blocked for 16 h at 4°C with 5% wt/vol skim milk in TBS-Tween buffer (500 mM NaCl, 20 mM Tris·HCl, and 0.1% Tween 20, pH 7.4) before incubation in the same buffer for 2 h at room temperature with 1:1,000 dilution of iNOS
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RESULTS
Exposure of alveolar epithelial cells to LPS induces iNOS expression. Alveolar epithelial cells were cultured from 1 to 4 h with 15 g/ml LPS, and iNOS transcript modulation was estimated by RT-PCR. As illustrated in Fig. 1, strong induction of iNOS expression was detected after 2 and 4 h of incubation. Although smaller doses of LPS (0.5 g/ml) promoted iNOS mRNA induction (data not included), the effect was dose dependent and was highest at 15 g/ml LPS concentration. Therefore, the 15 g/ml LPS concentration was chosen to further study the modulation of iNOS expression following a 4-h treatment. In addition to the iNOS transcript, LPS induced a significant increase of iNOS protein expression detected by immunoblotting (Fig. 2A). Although NO is a very labile substance, it was found to be significantly elevated in extracts of alveolar epithelial cells treated with LPS (Fig. 2B). LPS also
* *
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antibody (Abcam), 1/1,000 dilution of anti-PKC␣ (phospho S657 ⫹ Y658) antibody (Abcam). The membranes were then washed in TBS-Tween and incubated for 1 h at room temperature with 1:2,500 dilution of horseradish peroxidase-linked secondary antibody (Cell Signaling Technology). After 3 ⫻ 5 min washes with TBS-Tween, the membranes were incubated with ECL (GE Healthcare Life Sciences, Baie d’Urfe, QC, Canada) for 1 min before the luminescent signals were recorded on Kodak X-Omat BT films (Kodak, Rochester, NY), which were then scanned, and band densitometry was quantified by MultiGauge software (FujiFilm, Mississauga, ON, Canada). Equal loading was evaluated by incubation of the same membrane with a 1/5,000 dilution of -actin antibody (Ac-74; Sigma-Aldrich, Oakville, ON, Canada). Western blotting was repeated with lysates extracted from cells purified from different animals. PKC kinase activity assay. Cell lysate was prepared from alveolar epithelial cells incubated in six-well plates. PKC kinase activity from 0.05 g of protein extract was measured in presence or absence of suramin by using the PKC kinase activity kit according to the manufacturer’s instructions (Enzo Life Science; Farmingdale, NY). Measurement of total NO. Total NO was measured, as described by ˙ O-free Govindaraju et al. (23). Briefly, cell lysates were diluted with N HPLC grade water (Fisher Scientific, Fair Lawn, NJ). Thirty microliters of diluted samples were injected by Hamilton gas-tight microsyringe through the rubber septum into a purging glass vessel containing saturated VCl3 (in 1 N HCl) and 1% potassium iodide (KI) in glacial acetic acid solution at 90°C, with continuous purging through helium ˙ O was released immediately from the gas. Under these conditions, N samples and passed onto the NO analyzer through an ice-cold trap ˙ O was quantified with containing 1 N NaOH and 100 mM cysteine. N integration of digitally recorded signals by software from Sievers ˙ O analyzer was calibrated with Instrument (Boulder, CO). The N standard nitrate solutions (0.3–50 M). Each sample was analyzed in duplicate or triplicate. Nitrotyrosine measurement. The total amount of 3-nitrotyrosine was determined by ELISA as previously described by Montes de Oca et al. (41) using antibodies to nitrotyrosine (59). As reported previously (36), the antibodies (mouse IgG monoclonal and polyclonal antibodies against nitrotyrosine and polyclonal goat anti-rabbit IgGperoxidase) were from Upstate Biotechnology. The quantification of nitrotyrosine was performed by using a standard curve with known concentrations of nitrotyrosine from chemically modified bovine serum albumin. The sensitivity of the assay was 50 pg/ml. Statistical analysis. The data, presented as means ⫾ SE, were analyzed by the Mann-Whitney test, unpaired t-test, Wilcoxon signedrank test, or 1-way analysis of variance (ANOVA). The level of significance was defined as P ⬍ 0.05. The experiments were repeated at least three times, and n refers to alveolar epithelial cells isolated from different animals.
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Fig. 1. Impact of lipopolysaccharide (LPS) exposure on inducible nitric oxide synthase (iNOS) mRNA expression. Alveolar epithelial cells were cultured for 1 h, 2 h or 4 h with 15 g/ml LPS. Total mRNA was extracted and iNOS transcripts were estimated by semiquantitative RT-PCR. iNOS expression was quantified by ImageQuant software, and the signal was normalized to -actin expression. RT-PCR signals are expressed as the iNOS/-actin arbitrary units (A.U.) ratio. The iNOS/-actin transcript level estimated by RT-PCR is shown along with a typical gel for the 1-h to 4-h time course. Ctrl, untreated control. *P ⬍ 0.05 by the Mann-Whitney test compared with Ctrl (n ⫽ 6).
significantly augmented intracellular nitrosylated protein level as a consequence of NO oxidative stress (Fig. 2C). Act D, a transcription inhibitor, and CCX, a translation inhibitor, were tested to determine whether gene transcription, mRNA stability, and/or de novo protein synthesis were involved in iNOS mRNA induction. Act D abrogated iNOS mRNA induction by LPS (Fig. 3A). However, there was no difference in the degradation profile of iNOS mRNA between control and LPStreated cells (Fig. 3A) since the half-life of the transcript was ⬃2 h in both cases. De novo protein synthesis was not required for iNOS mRNA induction by LPS since CCX had no impact on this process (Fig. 3B). Altogether, these results show that LPS increases iNOS mRNA transcription in alveolar epithelial cells. Inhibition of calcium-dependent PKC reverses the impact of LPS on iNOS expression. The signaling pathways involved in iNOS transcript elevation by LPS were investigated. Inhibitors of major pathways known to be activated by LPS were tested for their ability to abolish the effect of LPS on iNOS induction (Fig. 4). MAPK inhibition with 20 M PD 98059 (an ERK1/2 inhibitor) or 10 M SP 600125 (a JNK inhibitor) could not prevent iNOS increment by LPS (Fig. 4). Inhibition of the PI3K cascade with 100 nM LY294002 and 100 nM wortmannin, both Akt inhibitors, or with 5 ng/ml rapamycin [a mammalian target of rapamycin (mTOR) inhibitor] was also ineffective (Fig. 4). NF-B pathway suppression with 50 g/ml SN50, an inhibitor of P50 nuclear translocation, and 5 M BAY-117082, which prevents the IB phosphorylation needed for NF-B activation, were also ineffective in abrogating the effect of LPS (Fig. 4). PKC activation has been shown to be involved in the induction of iNOS expression by LPS in macrophages (9, 16, 44). For this
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Fig. 2. Impact of LPS on iNOS protein expression, intracellular NO, and nitrotyrosine. Alveolar epithelial cells were treated or not with 15 g/ml LPS for 4 h. A: densitometric analysis of iNOS protein detected by immunoblotting is shown. iNOS level was normalized to -actin and expressed as the percentage of untreated cells (Ctrl). *P ⬍ 0.05 by the Wilcoxon signed-rank test compared with untreated controls (n ⫽ 6). B: intracellular NO level was estimated in alveolar epithelial cells treated or not with 15 g/ml LPS for 4 h. NO concentration was normalized to protein amount and expressed as nmol/mg of protein. *P ⬍ 0.05 by the Mann-Whitney test compared with untreated controls (n ⱖ 4). C: nitrotyrosine level as a consequence of NO oxidative stress was estimated in alveolar epithelial cells treated or not with 15 g/ml LPS for 4 h. Nitrotyrosine concentration was normalized to protein amount and expressed in ng/mg of protein. *P ⬍ 0.05 by unpaired t-test compared with untreated controls (n ⫽ 3).
reason, we tested whether PKC inhibitors could blunt iNOS expression in response to LPS. As seen in Fig. 5A, pretreatment of alveolar epithelial cells with GO 6976, an inhibitor of classical PKC isoenzymes (PKC␣ and PKC), or RO 318220, an inhibitor of PKC, PKC␥, and PKCε, prevented iNOS mRNA induction by LPS (Fig. 5A). In addition, GO 6976 also prevented iNOS protein induction by LPS (Fig. 5B). Immunoblotting with a specific anti-PKC␣ (phospho S657 ⫹ Y658) antibody confirmed that the PKC␣-1 isoforms were activated by phosphorylation,
with maximum activation occurring 45 min after LPS stimulation (P ⬍ 0.05; Fig. 5C). It has been reported that classical Ca2⫹-dependent PKC is associated with iNOS mRNA induction (47). To further test this hypothesis, the cells were incubated with BAPTA-AM to chelate intracellular Ca2⫹. As shown in Fig. 6A, BAPTA-AM pretreatment inhibited iNOS mRNA induction by LPS in alveolar epithelial cells. Inhibition of the L-type calcium channel with verapamil or nifedipine as well as the IP3 receptor in the endoplasmic reticulum (ER) did not abolish LPS-induced iNOS expression (Fig. 6B). Purinergic signaling in the induction of iNOS expression by LPS. ATP released by treatment with LPS has been demonstrated to increase NO production via P2X7 receptors in response to LPS in macrophages (48). For this reason, we investigated whether purinergic stimulation could induce iNOS expression in alveolar epithelial cells. Two agonists of purinergic receptors, ATP␥S and 2MeSATP, were tested for their ability to modify iNOS expression. As depicted in Fig. 7, both treatments significantly modulated iNOS mRNA expression (P ⬍ 0.05). Suramin, but not P2 receptor antagonists, inhibits iNOS induction by LPS. To explore purinergic signaling in the modulation of iNOS expression by LPS, cells were treated with PPADS (pyridoxalphosphate-6-azophenyl-2=,4=-disulfonic acid), BBG, or suramin, antagonists of different P2 purinergic receptors. As shown in Fig. 8A, only suramin blocked iNOS mRNA induction by LPS. At the fairly high concentration used (500 M), it also abrogated the impact of LPS on NOS2 protein expression (Fig. 8B). Because suramin is pharmacologically potent against a wide range of targets, it was tested at different concentrations to determine whether P2 receptor inhibition was involved in this process. As illustrated in Fig. 8C, while 500 and 200 M suramin significantly decreased iNOS induction by LPS (*P ⬍ 0.05), at 100 M, a concentration specific to several P2 receptors (P2Y1, P2Y2, and P2X1) (27), iNOS expression was not significantly reduced compared with LPS-treated cells. In addition to inhibit purinergic receptors, suramin has been shown to inhibit PKC␣, , and ␥ activity (38). For this reason, we tested the hypothesis that suramin could inhibit PKC kinase activity in alveolar epithelial cells. We found that suramin treatments (500, 100, and 10 M) inhibit PKC activity in vitro (Fig. 9). Whereas PKC activity was completely abolished at 500 and 100 M of suramin, a 10 M concentration was still able to significantly decrease PKC activity (Fig. 9). DISCUSSION
NO generated from L-arginine by iNOS is a major factor in innate host defense against pathogens because of its antiviral, antiparasitic, and immunomodulatory effects (31). In the present work, we report that LPS induced iNOS mRNA and protein expression in alveolar epithelial cells via a PKC- and calciumdependent process. Although high suramin concentrations could inhibit this induction, the process was not ATP dependent. A specific signaling pathway is involved in iNOS mRNA induction in alveolar epithelial cells exposed to LPS. Treatment of rat alveolar epithelial cells with LPS from P. aeruginosa induced a time-dependent increase in iNOS mRNA expression (Fig. 1). iNOS protein level was also significantly elevated in these cells as well as intracellular NO and nitrotyrosine. iNOS mRNA has
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Fig. 3. Effect of actinomycin D (Act D) and cycloheximide (CCX) on iNOS mRNA regulation by LPS. A: alveolar epithelial cells were cultured for 1, 2, or 4 h in the presence or absence of 15 g/ml LPS. The kinetics of iNOS transcript degradation were estimated in the presence of 5 g/ml Act D, a transcription inhibitor added 30 min before LPS treatment. Percentages of the iNOS/-actin ratio compared with the controls for each series are depicted along with a typical RT-PCR result for the 1-h to 4-h time course. No significant differences were apparent with the Wilcoxon signed-rank test in iNOS expression level after Act D treatment in the presence or absence of LPS for a given time point (n ⫽ 4). B: the kinetics of iNOS transcript induction by 15 g/ml LPS were estimated in the presence or absence of 10 g/ml CCX, a protein synthesis inhibitor. Percentages of the iNOS/-actin ratio compared with the controls for each series are depicted at the bottom along with a typical RT-PCR result for the 1-h to 4-h time course. Although 1-way ANOVA shows that LPS treatment induced a significant change in iNOS expression level with time (P ⬍ 0.05), there were no significant differences with the Wilcoxon signed-rank test in iNOS expression level after LPS treatment in the presence or absence of CCX for a given time point (n ⫽ 4).
been observed to be highly expressed in the lungs after endotoxic shock (29), but the effect of LPS on iNOS expression gave conflicting results in immortalized A549 alveolar epithelial cells (28, 33). Our results confirm the work of Rose and colleagues (46), disclosing that apical LPS highly modulates iNOS mRNA expression in rat alveolar epithelial cells in primary cultures. In addition, the present report for the first time documents that iNOS protein expression also alters intracellular NO production and nitrotyrosine, a consequence of NO
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oxidative activity in these cells. Our results indicate that LPS elicits strong NO induction in alveolar epithelial cells. iNOS gene expression is known to be highly regulated at the transcriptional level by binding sites for cytokine-responsive elements and redox-sensitive transcription factors, such as AP-1, NF-B, NFAT, and STAT-1␣ (31), in the 5= flanking region of the gene. In addition, iNOS mRNA translation has been shown to be inhibited in human cardiomyocytes by factors interacting in the 5= and/or 3=-UTR sequence of iNOS mRNA (37). Posttranscrip-
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Fig. 4. Signaling pathways potentially implicated in iNOS induction by LPS. Alveolar epithelial cells were treated with MAPK, phosphatidylinositol 3 kinase (PI3K), or NF-B inhibitor pretreatment before LPS stimulation to determine the nature of the signaling pathways involved in the induction of iNOS transcripts after 4-h treatment with 15 g/ml LPS. The iNOS/-actin transcript level estimated by RT-PCR is shown for cells treated (solid bars) or not (open bars) with LPS, along with a typical gel. Sixty-minute pretreatment with 20 M PD98059 (an ERK1/2 inhibitor) and 30-min pretreatment with 10 M SP600125 (a JNK inhibitor) were tested to inhibit MAPK pathways. Sixty-minute pretreatment with 100 nM LY294002 (an Akt inhibitor), 30-min pretreatment with 100 nM wortmannin (an Akt inhibitor), and 30-min pretreatment with 5 ng/ml rapamycin (a mTOR inhibitor) were tested to inhibit the PI3K pathway. Sixty-minute pretreatment with 5 M BAY-117082 (an IB phosphorylation inhibitor) and 60-min pretreatment with 50 g/ml SN50 (an inhibitor of P50 nuclear translocation) were tested to inhibit the NF-B pathway.
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P-PKC α β−actin Fig. 5. Involvement of PKC in iNOS transcript induction by LPS. A: 10 M RO 318220 and 2.5 M GO 6976 pretreatments were tested to determine whether the PKC pathway is implicated in iNOS transcript induction after 4-h treatment with 15 g/ml LPS. The iNOS/-actin transcript level estimated by RT-PCR is shown for cells treated (solid bars) or not (open bars) with LPS, along with a typical gel. *P ⬍ 0.05 by the Mann-Whitney test for PKC inhibitors ⫹ LPS compared with LPS alone (n ⱖ 4). B: the relative amount of intracellular iNOS protein was estimated by immunoblotting in alveolar epithelial cells pretreated for 30 min with 2.5 M GO 6976 prior to 4-h 15 g/ml LPS treatment. iNOS level was normalized to -actin and expressed as the percentage of untreated cells (Ctrl). *P ⬍ 0.05 by the Wilcoxon signedrank test compared with untreated controls (n ⫽ 6). C: PKC signaling pathway activation after 15 g/ml LPS treatment was assessed by immunoblotting. Phosphorylated PKC␣-1 protein (PKC␣-1) level was normalized to -actin and expressed as a percentage of untreated controls (Ctrl). A representative chemiluminescent reaction is shown at the bottom. *P ⬍ 0.05 by the MannWhitney test compared with untreated controls (n ⫽ 5).
Fig. 6. Involvement of Ca2⫹ in iNOS transcript induction by LPS. A: alveolar epithelial cells were treated with 50 M BAPTA-AM to test whether intracellular Ca2⫹ was implicated in iNOS transcript induction after 4-h stimulation with 15 g/ml LPS. The cells were pretreated for 10 min prior to LPS stimulation. The iNOS/-actin transcript level ratio is depicted for the different treatments, along with a typical RT-PCR gel. *P ⬍ 0.05 by the Mann-Whitney test compared with matched untreated controls (n ⱖ 5). B: 100 M verapamil, 100 M nifepidine, L-type Ca2⫹ channel blockers, and 100 M 2-aminoethyl diphenylborinate (2-APB), an inositol 1,4,5-trisphosphate (IP3) receptor antagonist involved in Ca2⫹-store release, were tested in iNOS transcript induction after 4-h stimulation with 15 g/ml LPS. The cells were pretreated for 10 min before LPS stimulation. The iNOS/-actin transcript level ratio is depicted for the different treatments. *P ⬍ 0.05 by the Mann-Whitney test compared with matched controls not treated with LPS. Verapamil, nifepidine (n ⫽ 6); 2-APB (n ⫽ 3).
tional mechanisms, such as kalirin in humans and NAP110 in mice, are also critically involved in the regulation of iNOS expression as they interact with iNOS and control its activity (31). Furthermore, the inhibition of iNOS expression by different agents, such as transforming growth factor-1 and dexamethasone, has been found to result in iNOS mRNA and protein destabilization (32). To determine whether iNOS mRNA induction after LPS treatment was the consequence of increased mRNA stability or iNOS transcription, alveolar epithelial cells were tested with Act D, a transcription inhibitor, which completely inhibited
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A *
1.5
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Fig. 7. Purinergic agonists on iNOS transcript induction in alveolar epithelial cells. Alveolar epithelial cells were treated for 4 h with 100 M adenosine5=-O-(3-thiotriphosphate) (ATP␥S) or 100 M 2-methylthioadenosine-5=-Otriphosphate (2MeSATP) to test whether purinergic stimulation could induce iNOS transcript expression. The iNOS/-actin transcript level ratio is depicted for the different treatments. *P ⬍ 0.05 by the Mann-Whitney test compared with untreated controls (n ⫽ 5).
iNOS mRNA induction by LPS. In addition, the half-life of iNOS mRNA was similar in the presence or absence of LPS, clearly showing that transcription, and not mRNA stability, was involved in the increment of iNOS transcripts. Because a 2-h lag before iNOS induction was detected, we also tested whether de novo protein synthesis inhibition with CCX would affect iNOS expression. As depicted in Fig. 3, CCX pretreatment did not modulate iNOS mRNA induction by LPS. This result indicates that translation of a cofactor is not required for the induction of iNOS expression in response to LPS. LPS-induced iNOS expression via Ca2⫹-dependent PKC in pulmonary epithelial cells. iNOS mRNA expression is known to be modulated by numerous signaling pathways such as MAPK, PI3K, PKC, and NF-B (31). For this reason, different inhibitors selected to interfere with these pathways were tested for their ability to inhibit iNOS mRNA induction after LPS treatment in alveolar epithelial cells. Inhibition of MAPK (ERK, P38), PI3K (Akt, mTOR), and NF-B pathways had no impact on iNOS expression. GO 6976 and RO 318220, inhibitors of the PKC pathway, abolished or very significantly reduced iNOS mRNA and protein expression. The PKC pathway has been reported to induce, inhibit, or have no effect on iNOS expression (31), depending on cell type. These discrepancies could be explained by the nature of the PKC isoforms expressed in different cells. In rat mesangial cells, IL-1 induction of iNOS expression was inhibited by PKCε activation by phorbol esters (42). In rat vascular smooth muscle cells and human DLD1 cells, PKC activation or inhibition had no effect on the induction of iNOS expression by cytokines (31). In RAW 264.7 murine macrophages overexpressing the dominant negative PKC␣ isoform, LPS induction of iNOS expression was decreased (49). In activated macrophages, PKC has been found to upregulate iNOS expression in a NF-B-independent manner (47). In this study, the isoforms PKC␣ and 1 are specifically involved and activated by a Ca2⫹-dependent mechanism. The inhibition of iNOS induction after chelation of intracellular Ca2⫹ with BAPTA-AM further indicates that Ca2⫹-dependent PKC is involved in the modulation of iNOS
B NOS2/β-actin (% of Ctrl)
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Fig. 8. 500 M suramin inhibits iNOS gene product induction after LPS treatment. A: different inhibitors were tested to determine whether purinergic signaling was involved in iNOS transcript induction by LPS. Alveolar epithelial cells were treated with 100 M pyridoxalphosphate-6-azophenyl-2=,4=disulfonic acid (PPADS), 10 M brilliant blue G (BBG), or 500 M suramin (Sur 500) 15 min prior to 4-h stimulation with 15 g/ml LPS. The iNOS/actin transcript level ratio estimated by RT-PCR is depicted for the different treatments in the presence (solid bars) or absence (open bars) of LPS. *P ⬍ 0.05 by unpaired t-test compared with matched controls not treated with LPS (n ⫽ 3). B: densitometric analysis of NOS2 protein expression detected by immunoblotting in alveolar epithelial cells. Alveolar epithelial cells were treated with 500 M suramin 15 min prior to 4-h stimulation with 15 g/ml LPS. NOS2 level was normalized to -actin and expressed as the percentage of untreated cells (Ctrl). *P ⬍ 0.05 by the Wilcoxon signed-rank test compared with untreated controls (n ⫽ 6). C: suramin at 100 M (Sur 100), 200 M (Sur 200), and 500 M (Sur 500) was tested for its ability to inhibit iNOS transcript induction by LPS. Alveolar epithelial cells were treated with suramin for 15 min prior to 4-h stimulation with 15 g/ml LPS. The relative level of iNOS transcript was evaluated by quantitative PCR and expressed as iNOS/-actin fold change compared with untreated controls. *P ⬍ 0.05 by 1-way ANOVA compared with LPS (n ⫽ 3).
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Relative PKC activity
20 15 10 5 0
Suramin (µM)
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500
100
10
Fig. 9. PKC kinase activity after suramin treatments in alveolar epithelial cells. Different suramin concentrations were tested to determine whether the drug could inhibit PKC kinase activity. Cellular extracts from alveolar epithelial cells were treated with 500, 100, or 10 M suramin and PKC activity was determined with the PKC kinase activity kit from Enzo Life Sciences. The results in triplicate for each sample tested show a direct and spontaneous inhibition of PKC activity with 500 and 100 M suramin and a decrease of PKC activity with 10 M suramin. *P ⬍ 0.05 by 1-way ANOVA compared with control (n ⫽ 4).
mRNA by LPS in our cell system. Several PKC isoforms, including PKC␣, have been shown to be directly involved in TLR signaling (35). LPS from Pseudomonas are TLR 4 and 2 ligand (19, 26). What could be the link between PKC and TLR signaling? Myristoylated alanine-rich C kinase substrate (MARCKS) is one of the major proteins phosphorylated by PKC. Recent data show that MARCKS inhibition with an inhibitory peptide decreases the expression of several proinflammatory cytokines (CXCL1, IL-8, TNF-␣) by LPS in canine neutrophils (34). This suggests a cross talk between PKC and TLR signaling via MARCKS in the inflammation response. It is not clear so far what is the source of Ca2⫹ for PKC activation. L-type calcium channel inhibition with verapamil or nifedipine did not abolish LPS-induced iNOS expression. 2-APB, an inhibitor of the IP3 receptor that is important for Ca2⫹ mobilization from the ER pool, also failed to reduce iNOS induction as well as U73122, a phospholipase C inhibitor (data not shown). It is possible that an intracellular Ca2⫹ pool, independent of the IP3 receptor pathway, is activated after LPS treatment. Thapsigargin, an inhibitor of SERCA, the ER Ca2⫹-ATPase pump that replenishes the ER calcium pool, has been shown to suppress iNOS protein expression, in part, in response to LPS in RAW 264.7 macrophages (9) and to potentiate the response to LPS in peritoneal macrophages (8). In our study, we failed to demonstrate an inhibitory impact of thapsigargin on iNOS induction by LPS (data not shown). Other studies should be conducted to find the Ca2⫹ source that activates iNOS regulation. An orphan GPCR could be involved in the iNOS induction pathway. LPS have been shown to promote ATP secretion in RAW 264.7 macrophages whereas ATP increases NO production in these cells (48). In addition, ATP and UTP mono- and dinucleotide metabolites have been reported to modulate cytoplasmic Ca2⫹ increment and iNOS protein expression in A549 alveolar epithelial cells (33). For these reasons, the roles of ATP and purinergic signaling were investigated in the modulation of iNOS expression elicited by LPS in our cell model. Different activators or antagonists of the purinergic pathway were tested to identify whether purinergic receptors were
involved in iNOS expression modulation by LPS. The P2 agonists ATP␥S and 2MeSATP significantly increased iNOS mRNA expression, suggesting that purinergic signaling was able to modulate iNOS expression in these cells. However, the data presented here clearly show that selective inhibitors of P2Y (PPADS) and P2X (BBG) receptors were ineffective in inhibiting the effect of LPS on iNOS expression. Furthermore, at 100 and 200 M suramin concentrations, which are known to suppress P2 receptors, iNOS expression was only partially inhibited. A high suramin concentration (500 M) was the only pretreatment that could completely prevent the effect of LPS on iNOS expression. At this concentration, suramin not only is a P2-purinoceptor antagonist but also inhibits the binding of platelet-derived growth factor, epidermal growth factor, basic fibroblast growth factor, transforming growth factor-3, and insulin-like growth factor to their specific receptors (5). Suramin is also known to disrupt the coupling of GPCR to the G subunit in rat lung plasma membranes (10) and to inhibit PKC activity (14, 25, 30). The results presented in Fig. 9 show that suramin can directly inhibit PKC activity in vitro. Several reports show that suramin can be internalized in different cell type (1, 21, 50). Although it is difficult to estimate the cytoplasmic concentration of suramin in the experimental conditions used, the very high concentration (500 M) needed to fully inhibit iNOS expression suggests that a certain amount of suramin could have been present in alveolar epithelial cells. The effects of suramin on PKC activity could well explain the inhibition of iNOS expression that is reported here. Altogether, these results show that purinergic signaling does not modulate LPS-mediated iNOS expression in rat alveolar epithelial cells. This was also confirmed by the data presented above where 2-APB could not inhibit LPS-induced iNOS expression, since purinergic signaling, which is known to elevate cytoplasmic Ca2⫹, is elicited via IP3 receptor activation. Could other GPCRs, besides P2, be involved in the induction of iNOS expression by LPS? The relationship between GPCR in iNOS and chemokine regulation by LPS in mouse microglial cells has been reported (22). Bosentan and tezosentan, antagonists of G protein-endothelin receptor, have been shown to block iNOS expression and limit liver injury in endotoxinchallenged cirrhotic rats (56). Because endothelin receptors are expressed in rat alveolar epithelial cells, pretreatment with these inhibitors was tested but had no significant effect on the modulation of iNOS expression by LPS (data not reported). Alveolar epithelial cells are clearly different from hepatic cells. Although we found that suramin can directly inhibit PKC activity, it is not possible at the moment to reject the hypothesis that a suramin-sensitive GPCR or orphan receptor could also be involved in the modification of iNOS expression by LPS in rat alveolar epithelial cells. In summary, our data show that LPS from P. aeruginosa increased iNOS mRNA and protein expression in rat alveolar epithelial cells through a suramin/PKC␣-1/Ca2⫹-sensitive pathway. Intracellular NO and nitrotyrosine elevation suggests that NO synthesis is part of the innate defense mechanism elicited by alveolar epithelial cells in response to endotoxin. Although the pathways upstream and downstream of Ca2⫹-PKC are not known, our data show that a direct inhibition of PKC activity by suramin or inhibition of GPCR(s) could be involved in the induction of iNOS transcript by LPS. Our data show that PKC plays a central
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PKC-DEPENDENT iNOS INDUCTION BY LPS IN ALVEOLAR EPITHELIAL CELLS
role in the triggering of the innate response to LPS in the distal lung. ACKNOWLEDGMENTS The authors acknowledge the editorial work done on this manuscript by Ovid Da Silva. GRANTS E. Boncoeur was supported by a fellowship from the Canadian Thoracic Society and the Canadian Cystic Fibrosis Foundation. This work was funded in part by the Canadian Cystic Fibrosis Foundation and the Canadian Institutes of Health Research. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS E.B. and Y.B. conception and design of research; E.B., G.B., F.M., V.T., J.B.d.S., K.G., and A.D. performed experiments; E.B., G.B., F.M., V.T., D.R., J.B.d.S., D.H.E., K.G., A.D., and Y.B. analyzed data; E.B., G.B., F.M., D.R., J.B.d.S., D.H.E., K.G., A.D., and Y.B. interpreted results of experiments; E.B., G.B., and A.D. prepared figures; E.B. and G.B. drafted manuscript; E.B., P.F., D.R., A.D., and Y.B. edited and revised manuscript; E.B., P.F., D.R., J.B.d.S., D.H.E., K.G., A.D., and Y.B. approved final version of manuscript. REFERENCES 1. Baghdiguian S, Boudier JL, Boudier JA, Fantini J. Intracellular localisation of suramin, an anticancer drug, in human colon adenocarcinoma cells: a study by quantitative autoradiography. Eur J Cancer 32A: 525– 532, 1996. 2. Bals R, Hiemstra PS. Innate immunity in the lung: how epithelial cells fight against respiratory pathogens. Eur Respir J 23: 327–333, 2004. 3. Baron RM, Carvajal IM, Fredenburgh LE, Liu X, Porrata Y, Cullivan ML, Haley KJ, Sonna LA, De Sanctis GT, Ingenito EP, Perrella MA. Nitric oxide synthase-2 down-regulates surfactant protein-B expression and enhances endotoxin-induced lung injury in mice. FASEB J 18: 1276 –1278, 2004. 4. Berthiaume Y, Voisin G, Dagenais A. The alveolar type I cells: the new knight of the alveolus? J Physiol 572: 309 –310, 2006. 5. Bojanowski K, Lelievre S, Markovits J, Couprie J, Jacquemin-Sablon A, Larsen AK. Suramin is an inhibitor of DNA topoisomerase II in vitro and in Chinese hamster fibrosarcoma cells. Proc Natl Acad Sci USA 89: 3025–3029, 1992. 6. Boncoeur E, Tardif V, Tessier MC, Morneau F, Lavoie J, GendreauBerthiaume E, Grygorczyk R, Dagenais A, Berthiaume Y. Modulation of epithelial sodium channel activity by lipopolysaccharide in alveolar type II cells: involvement of purinergic signaling. Am J Physiol Lung Cell Mol Physiol 298: L417–L426, 2010. 7. Brochiero E, Dagenais A, Prive A, Berthiaume Y, Grygorczyk R. Evidence of a functional CFTR Cl⫺ channel in adult alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 287: L382–L392, 2004. 8. Chen BC, Hsieh SL, Lin WW. Involvement of protein kinases in the potentiation of lipopolysaccharide-induced inflammatory mediator formation by thapsigargin in peritoneal macrophages. J Leukoc Biol 69: 280 – 288, 2001. 9. Chen YJ, Hsu KW, Tsai JN, Hung CH, Kuo TC, Chen YL. Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages. Int J Biochem Cell Biol 37: 2574 –2585, 2005. 10. Chung WC, Kermode JC. Suramin disrupts receptor-G protein coupling by blocking association of G protein alpha and betagamma subunits. J Pharmacol Exp Ther 313: 191–198, 2005. 11. Chuquimia OD, Petursdottir DH, Rahman MJ, Hartl K, Singh M, Fernandez C. The role of alveolar epithelial cells in initiating and shaping pulmonary immune responses: communication between innate and adaptive immune systems. PloS One 7: e32125, 2012. 12. Dagenais A, Frechette R, Clermont ME, Masse C, Prive A, Brochiero E, Berthiaume Y. Dexamethasone inhibits the action of TNF on ENaC expression and activity. Am J Physiol Lung Cell Mol Physiol 291: L1220 –L1231, 2006.
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