Lipoteichoic acid-stimulated p42/p44 MAPK activation via Toll-like receptor 2 in tracheal smooth muscle cells

Chiang-Wen Lee,1 Chin-Sung Chien,1 and Chuen-Mao Yang1,2

1Department of Physiology and Pharmacology, 2Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan, 3332

Submitted 28 April 2003 ; accepted in final form 24 September 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipoteichoic acid (LTA), the principal component of the cell wall of gram-positive bacteria, triggers several inflammatory responses. However, the mechanisms underlying its action on human tracheal smooth muscle cells (HTSMCs) were largely unknown. This study was to investigate the mechanisms underlying LTA-stimulated p42/p44 mitogen-activated protein kinase (MAPK) using Western blotting assay. LTA stimulated phosphorylation of p42/p44 MAPK via a Toll-like receptor 2 (TLR2). Pretreatment with pertussis toxin attenuated the LTA-induced responses. LTA-stimulated phosphorylation of p42/p44 MAPK was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (PLC; D609), phosphatidylinositol (PI)-PLC (U-73122), PKC (staurosporine, Gö-6976, rottlerin, or Ro-318220), MEK1/2 (U-0126), PI 3-kinase (LY-294002 and wortmannin), and an intracellular Ca2+ chelator (BAPTA-AM). LTA directly evoked initial transient peak of [Ca2+]i, supporting the involvement of Ca2+ mobilization in LTA-induced responses. These results suggest that in HTSMCs, LTA-stimulated p42/p44 MAPK phosphorylation is mediated through a TLR2 receptor and involves tyrosine kinase, PLC, PKC, Ca2+, MEK, and PI 3-kinase.

calcium; human; phospholipase C; protein kinase C


BACTERIAL INFECTIONS have been shown to be involved in a number of inflammatory diseases such as lung inflammation. These diseases are characterized by certain inflammatory reactions of the host evoked by pathogens (1, 46). A possible explanation for these findings is due to the secretion of proinflammatory cytokines by host cells triggered by cell wall components released from the bacteria. These responses have been well demonstrated for LPS of gram-negative bacteria and lipoteichoic acid (LTA) of gram-positive bacteria (12, 37). LTA has been shown to be an antigen for increasing the severity of respiratory disorder characterized by inflammatory changes in the lung (26, 27). The inflammation is believed to be initiated by shedding of the epithelial barriers that allows LTA to have relatively easy access to tracheal smooth muscle cells (TSMCs). Moreover, LTA could directly activate inflammation by modulating the function of inflammatory cells such as macrophages and monocytes. LTA can induce the secretion of proinflammatory cytokines, including IL-1{beta}, IL-6, or TNF-{alpha} from these cells (6, 21). These data suggest that LTA might modify specific gene expression in various cell types and thereby augment and possibly initiate tissue inflammation. However, the mechanisms of intracellular signaling pathways involved in LTA-induced gene expression are still unknown. It has been well established that induction of signal transduction cascades that activate transcription factors is essential for the activation of gene expression. Thus identification and characterization of intracellular signaling pathways induced by inflammatory processes are of great importance in the development of new therapeutic strategies targeting these signaling components. Mitogen-activated protein kinases (MAPKs) have been largely characterized as proline-directed serine/threonine kinases and play an important role in the conversion of extracellular signals to intracellular responses through serial phosphorylation cascades (33). There are three MAPK superfamilies, including p42/p44 MAPK, p38 MAPK, and c-Jun NH2-terminal kinase, that have been identified and well characterized in several cell types (23). Once activated, the resultant effects of MAPK activation and phosphorylation depend on their ability to induce the appropriate gene expression to exert as a homeostatic modulator within the cells. Therefore, LTA-initiated inflammatory processes may be mediated through the activation of these signaling pathways.

It has been shown that activation of macrophages caused by LPS is mediated by LPS-binding protein (LBP), which transfers LPS to its cellular receptor consisting of CD14 (a glycosylphosphatidylinositol-anchored protein), Toll-like receptor 4 (TLR4), and the MD-2 molecule in several cell types (5, 41, 45, 50). Binding of LPS to CD14 is enhanced by LBP, which is present in serum (45). Because CD14 lacks a cytoplasmic domain, it is unclear how CD14 transduces signals across the plasma membrane in response to LPS. In addition, LPS apparently initiates signaling via TLR4 (19). It remains controversial, however, whether LTA elicits signaling through TLR2 or TLR4 (27, 40, 43). By interacting with these plasma proteins, LPS can activate several signaling pathways, including activation of phospholipase C (PLC), increase of intracellular Ca2+ concentration ([Ca2+]i) (48), PKC (49), tyrosine kinase (17), p42/p44 MAPK (16, 49), stress-activated protein kinase 1/JNK (15), p38/stress-activated protein kinase 2 (25), and phosphatidylinositol 3-kinase (PI 3-kinase) activity (18). In addition, several studies have demonstrated that responses to LPS in a variety of cell types may involve a pertussis toxin-sensitive G protein (10).

Despite the intensive investigation of LPS, the mechanisms underlying signaling transduction triggered by LTA are largely unknown. LTA has been recognized as a significant virulence factor. It functions as an adhesion molecule to facilitate the binding of bacteria to cells, their colonization, and invasion into tissues (3). Similarly to LPS, LTA could interact with CD14 to induce NF-{kappa}B activation (27, 29, 36, 38), which is involved in proinflammatory cytokine production. LTA also has capacity to activate leukocytes and leads to inflammatory responses (12). Moreover, LTA has been shown to trigger signaling transduction through TLR2 and activate p42/p44 MAPK and p38 MAPK in various cell types (39, 40). Recently, we have demonstrated that LPS causes a rapid phosphorylation of p42/p44 MAPK in canine TSMCs (31). These findings further imply that these components might be implicated in the LTA-induced p42/p44 MAPK in human tracheal smooth muscle cells (HTSMCs).

Although LTA has been shown to activate MAPK pathways in some cell types, little is known about the intracellular signaling pathways leading to p42/p44 MAPK phosphorylation stimulated by LTA in HTSMCs. The aim of this study was to characterize the signaling components involved in p42/p44 MAPK phosphorylation in HTSMCs challenged by LTA. Here, we first show that in HTSMCs, LTA triggers activation of p42/p44 MAPK pathway, mediated through a TLR2 receptor. The stimulatory effects of LTA were modulated by tyrosine kinase, PLC, PKC, Ca2+, MEK, and PI 3-kinase.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. DMEM/Ham's nutrient mixture F-12 (F-12) medium, TRIzol reagent, PLUS-Lipofectamine, and FBS were purchased from GIBCO BRL (Gaithersburg, MD). Hybond C membrane and enhanced chemiluminescence (ECL) Western blot detection system were from Amersham Pharmacia Biotech (Buckinghamshire, UK). PhosphoPlus p42/p44 MAPK and phosphoPlus MEK1/2 antibody kits were from New England Biolabs (Beverly, MA). p42 MAPK antibody was from Santa Cruz (Santa Cruz, CA). Genistein, staurosporine, BAPTA-AM, D609, U-73122, U-0126, Gö-6976, rottlerin, and Ro-318220 were from Biomol (Plymouth Meeting, PA). Fura 2-AM was from Molecular Probes (Eugene, OR). Anti-TLR2 and anti-TLR4 antibodies were from Imgenex (San Diego, CA). Bicinchoninic acid (BCA) protein assay reagent was from Pierce (Rockford, IL). LTA from Staphylococcus aureus (L2515), enzymes, and other chemicals were from Sigma (St. Louis, MO).

Isolation of HTSMCs. HTSMCs were isolated from human trachea during organ transplantation at Chang Gung Memorial Hospital according to guidance by the Institutional Human and Animal Care and Use Committee of Chang Gung University. Tracheal smooth muscle strips were cut into small pieces and placed in six-well culture plates. These explants were grown in DMEM/F-12 containing 10% (vol/vol) FBS and antibiotics (100 U/ml penicillin G, 100 µg/ml streptomycin, and 250 ng/ml fungizone) at 37°C in a humidified 5% CO2 atmosphere as described in a previous study (28). When the cultures reached confluence (14 days), cells were treated with 0.05% (wt/vol) trypsin/0.53 mM EDTA for 5 min at 37°C. The cell suspension was diluted with DMEM/F-12 containing 10% FBS to a concentration of 2 x 105 cells/ml. The cell suspension was plated onto (1 ml/well) 12-well and (2 ml/well) six-well culture plates containing glass coverslips for MAPK and [Ca2+]i measurement, respectively. Culture medium was changed after 24 h and then every 3 days.

To characterize the isolated and cultured HTSMCs and to exclude contamination by epithelial cells and fibroblasts, the cells were identified by an indirect immunofluorescent staining method using a monoclonal antibody of light chain myosin (13). More than 95% of the cell preparation was found to be composed of smooth muscle cells.

Plasmids and transfection. The plasmids encoding human dominant negative mutant TLR2 and wild-type TLR2 were kindly provided by Drs. J. Han and J. D. Li. All plasmids were prepared by using QIAGEN plasmid DNA preparation kits.

HTSMCs were plated at 3 x 105 cells/ml (1 ml/well) in 12-well culture plates for 24 h, reaching ~80% confluence. Cells were washed once with PBS and once with serum-free DMEM/F-12, and 0.4 ml of serum-free DMEM/F-12 medium were added to each well. The DNA PLUS-Lipofectamine reagent complex was prepared according to the manufacturer's instructions (GIBCO-BRL). The amount of plasmid transfected was kept constant (1 µg of pCDNA and TLR2 for each well). The DNA PLUS-Lipofectamine reagent complex (0.1 ml) was added to each well and incubated at 37°C for 3 h, at which time 1 ml of DMEM/F-12 medium containing 10% FBS was added and incubated for 19 h. After 24 h of transfection, the cells were washed twice with PBS and maintained in DMEM/F-12 containing 2% FBS for 24 h before treatment with LTA.

Preparation of cell extracts and Western blot analysis. For experiments, cells were plated in 12-well plates and shifted to DMEM/F-12 containing 2% FBS for 24 h. HTSMCs were incubated with or without LTA at 37°C for various times. When inhibitors were used, they were added 1 h before LTA was applied. After incubation, the cells were rapidly washed with ice-cold PBS, scraped, and collected by centrifugation at 1,000 g for 10 min. The collected cells were lysed with ice-cold lysis buffer containing (in mM except where specified) 25 Tris·HCl, pH 7.4, 25 NaCl, 25 NaF, 25 sodium pyrophosphate, 1 sodium vanadate, 2.5 EDTA, 2.5 EGTA, 0.05% (wt/vol) Triton X-100, 0.5% (wt/vol) SDS, 0.5% (wt/vol) deoxycholate, 0.5% (wt/vol) Nonidet P-40, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 PMSF. The lysates were centrifuged at 45,000 g for 1 h at 4°C to yield the whole cell extract. The protein concentration was determined by the BCA reagents according to the manufacturer's instructions. Samples from these supernatant fractions (30 µg protein) were denatured and subjected to SDS-PAGE using a 10% (wt/vol) running gel. Proteins were transferred to nitrocellulose membrane, and the membrane was incubated successively at room temperature with 5% (wt/vol) BSA in 50 mM Tris·HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5 (TTBS) for 1 h. The phosphorylation of p42/p44 MAPK was identified and quantified by Western blot analysis using phospho-p42/p44 MAPK antibody according to the recommendation of the manufacturer. Briefly, membranes were incubated overnight at 4°C with anti-phospho-p42/p44 MAPK, anti-phospho-MEK1/2, or anti-TLR2 antibody used at a dilution of 1:1,000 in TTBS. Membranes were washed with TTBS four times for 5 min each and incubated with a 1:1,500 dilution of anti-rabbit horseradish peroxidase antibody for 1 h. After being incubated, the membrane was washed extensively with TTBS. The immunoreactive bands detected by ECL reagents were developed by Hyperfilm-ECL (Amersham International).

Total RNA extraction and RT-PCR analysis. Total RNA was isolated from HTSMCs in 10-cm culture dishes with TRIzol (Life Technologies) according to the protocol of the manufacturer. RNA concentration was determined using a spectrophotometer at 260 nm. First strand cDNA synthesis was performed with 2 µg of total RNA using random hexamers as primers in a final volume of 20 µl (5 µg/µl random hexamers, 1 mM dNTPs, 2 U/µl RNasin, and 10 U/µl Moloney murine leukemia virus reverse transcriptase). The reaction was carried out at 37°C for 60 min. cDNAs encoding {beta}-actin, TLR2, and TLR4 were amplified from 3 to 5 µl of the cDNA reaction mixture using specific gene primers. Oligonucleotide primers for {beta}-actin, TLR2, and TLR4 were as follows: {beta}-actin, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' (sense), 5'-CTAGAAGCATTTGCGGTGGACGATG-3' (antisense); TLR2, 5'-GCCAAAGTCTTGATTGATTGG-3' (sense), 5'-TTGAAGTTCTCCAGCTCCTG-3' (antisense); and TLR4, 5'-TGGATACGTTTCCTTATAAG-3' (sense), 5'-GAAATGGAGGCACCCCTTC-3' (antisense).

The amplification profile includes one cycle of initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 1 min, primer annealing at 62°C for 1 min, extension at 72°C for 1 min, and 1 cycle of final extension at 72°C for 5 min. The expression of {beta}-actin was used as an internal control for the assay of a constitutively expressed gene.

Measurement of intracellular Ca2+ level. [Ca2+]i was measured in confluent monolayers with the Ca2+-sensitive dye fura 2-AM as described by Grynkiewicz et al. (14). Upon confluence, the cells were cultured in DMEM/F-12 with 2% FBS for 24 h before measurements were made. When inhibitors were used, they were added 1 h before the application of LTA. The monolayers were covered with 1 ml of DMEM/F-12 with 2% FBS containing 5 µM fura 2-AM and were incubated at 37°C for 45 min. At the end of the period, the coverslips were washed twice with the physiological buffer solution containing (in mM) 125 NaCl, 5 KCl, 1.8 CaCl2, 2 MgCl2, 0.5 NaH2PO4, 5 NaHCO3, 10 HEPES, and 10 glucose, pH 7.4. The cells were incubated in PBS for a further 30 min to complete dye deesterification. The coverslip was inserted into a quartz cuvette at an angle of ~45° to the excitation beam and placed in the temperature-controlled holder of a Hitachi F-4500 spectrofluorometer (Tokyo, Japan). Continuous stirring was achieved with a magnetic stirrer. Fluorescence of Ca2+-bound and -unbound fura 2 was measured by rapidly alternating the dual-excitation wavelengths between 340 and 380 nm and electronically separating the resultant fluorescence signals at an emission wavelength of 510 nm. The autofluorescence of each monolayer was subtracted from the fluorescence data. The ratios of the fluorescence at the two wavelengths were computed and used to calculate changes in [Ca2+]i. The ratios of maximum and minimum fluorescence of fura 2 were determined by adding ionomycin (10 µM) in the presence of PBS containing 5 mM Ca2+ and by adding 5 mM EGTA at pH 8.0 in a Ca2+-free PBS, respectively. The dissociation constant of fura 2 for Ca2+ was assumed to be 224 nM (14).

Analysis of data. Concentration-effect curves were fitted, and EC50 values were estimated using GraphPad Prizm Program (GraphPad, San Diego, CA). Data were expressed as means ± SE and analyzed with a two-tailed Student's t-test at a P < 0.05 level of significance.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
LTA-stimulated phosphorylation of p42/p44 MAPK. MAPK signaling cascades are among the most widespread signaling mechanisms involved in cellular regulation. MAPKs can be classified into at least three subfamilies: JNK/SAPK, p38 MAPK, and p42/p44 MAPK. It is evident that p42/p44 MAPK represents the most extensively studied and best-known MAPK family members implicated in a variety of cellular functions (33). Therefore, we first determined whether LTA stimulated p42/p44 MAPK phosphorylation in HTSMCs. As shown in Fig. 1, treatment of HTSMCs with LTA caused phosphorylation and activation of p42/p44 MAPK in a time-dependent manner (Fig. 1A). LTA rapidly activated p42/p44 MAPK phosphorylation in 3 min, reached a maximal response at 5 min (P < 0.01, n = 4), and then declined close to the basal level within 1 h. In addition, LTA produced a concentration-dependent increase in p42/p44 MAPK phosphorylation (Fig. 1B). The concentration of LTA that produced a maximal effect in p42/p44 MAPK phosphorylation was 0.5 µg/ml. Parallel blots run as controls that used antibody directed against the total p42 MAPK did not show any change (Fig. 1). In contrast, LTA stimulated a minor extent of p38 MAPK phosphorylation and almost no effect on JNK/SAPK phosphorylation by immunoblotting cell lysates with a phosphospecific antibody (data not shown). These results suggest that in HTSMCs, LTA stimulated p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner.



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Fig. 1. Phosphorylation of p42/p44 MAPK stimulated by lipoteichoic acid (LTA) in human tracheal smooth muscle cells (HTSMCs). Cells were grown to confluence on 12-well culture plates and shifted to culture medium containing 2% FBS for 24 h. A: time course; cells were stimulated with LTA (50 µg/ml) for various times. B: concentration dependence; cells were stimulated with various concentrations of LTA for 7 min. After being incubated, the cell lysates were subjected to 12% SDS-PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using an antiserum reactive with anti-phospho-p42/p44 MAPK or total p42 MAPK (as a control) polyclonal antibody. Bands were visualized by an enhanced chemiluminescence (ECL) method and quantified by a densitometer. Data are expressed as means ± SE of 4 independent experiments (bar graphs). *P < 0.05; **P < 0.01 compared with control cells.

 

LTA-stimulated p42/p44 MAPK phosphorylation mediated through a TLR2 receptor. TLRs play an important role for the host to detect and recognize the pathogen and initiate a rapid defensive response (1, 46). We first observed whether TLR2 and TLR4 present in HTSMCs, the expression of TLR receptor genes, was evaluated using RT-PCR. As shown in Fig. 2, both TLR2 and TLR4 receptors were expressed in HTSMCs. To investigate whether the LTA-induced MEK1/2 and p42/p44 MAPK phosphorylation was mediated through these TLR receptors, HTSMCs were pretreated with an antibody of TLR2 (42) or TLR4 (0.5 and 5 µg/ml) for 1 h and then stimulated with 50 µg/ml of LTA for 7 min. Results shown in Fig. 3 indicate that treatment with a TLR2 receptor antibody attenuated the LTA-evoked MEK1/2 and p42/p44 MAPK phosphorylation (P < 0.05, n = 5) in a concentration-dependent manner, but TLR4 receptor antibody had no effect on this response (data not shown). This hypothesis was further supported by the results that transfection of HTSMCs with dominant negative mutant TLR2 significantly attenuated p42/p44 MAPK phosphorylation stimulated by LTA (Fig. 4). These results suggest that the stimulatory effect of LTA was mediated through a TLR2 receptor in HTSMCs.



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Fig. 2. Toll-like receptor 2 (TLR2) and TLR4 expression in cultured HTSMCs. Expression of TLR2 and TLR4 mRNA was measured by RT-PCR. Each lane represents an individual experiment. RT-PCR analysis of {beta}-actin expression was used to as a control. M, marker.

 


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Fig. 3. Involvement of TLR2 receptors in MEK1/2 and p42/p44 MAPK phosphorylation induced by LTA in HTSMCs. Cells were grown to confluence on 12-well culture plates, shifted to culture medium containing 2% FBS for 24 h, preincubated with TLR2 antibody (0.5 and 5 µg/ml) for 1 h, and stimulated with vehicle or LTA (50 µg/ml) for 7 min. After being incubated, the cell lysates were subjected to 12% SDS-PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using an antiserum reactive with an anti-phospho-p42/p44 MAPK, anti-phospho-MEK1/2, or total p42 MAPK (as a control) polyclonal antibody. Bands were visualized by an ECL method as described in Fig. 1. Data are expressed as means ± SE of 5 independent experiments (bar graph). *P < 0.05; **P < 0.01 compared with control cells. Ab, antibody.

 


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Fig. 4. Requirement of TLR2 receptors for LTA-induced activation of p42/p44 MAPK in HTSMCs. Cells were transfected with plasmids encoding pCDNA and human dominant negative (DN) mutant TLR2 for 24 h and then stimulated with 50 µg/ml of LTA for 7 min. Cell lysates were subjected to 10% SDS-PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using an antiserum reactive with an anti-phospho-p42/p44 MAPK or GAPDH (as a control) polyclonal antibody. Bands were visualized by an ECL method as described in Fig. 1. Data are expressed as means ± SE of 3 independent experiments (bar graph). *P < 0.05 compared with control cells.

 

Effects of pertussis toxin and cholera toxin on LTA-stimulated p42/p44 MAPK phosphorylation. LPS has been shown to be linked to a pertussis toxin (PTX)-sensitive G protein (10). To determine whether the effect of LTA on p42/p44 MAPK activation was mediated by a receptor coupled to a PTX-sensitive G protein, the cells were pretreated with 100 ng/ml of PTX for 24 h and then stimulated with 50 µg/ml of LTA for 7 min. Similar to LPS, the LTA-stimulated p42/p44 MAPK activation was significantly attenuated by pretreatment with PTX (Fig. 5; P < 0.05, n = 5), indicating that the effect of LTA was mediated through a PTX-sensitive G protein. To investigate whether PTX-insensitive Gs protein might also play a role in the activation of p42/p44 MAPK pathway, HTSMCs were pretreated with 10 µg/ml of cholera toxin (CTX) for 24 h and then stimulated with 50 µg/ml of LTA for 7 min. As shown in Fig. 5, pretreatment of these cells with CTX had no inhibitory effect on p42/p44 MAPK phosphorylation. The basal levels of p42/p44 MAPK phosphorylation were not altered by incubation with these toxins alone. Because treatment with PTX overnight may reduce cell surface receptor density of TLR2, mRNA and protein of TLR2 receptors were determined in HTSMCs treated with 100 ng/ml of PTX for 24 h using RT-PCR and Western blot analysis. As shown in Fig. 6, both mRNA and protein of TLR2 were not significantly changed when these cells were incubated with PTX, excluding the possible mechanism that reduction of TLR2 receptors by PTX led to an attenuation of p42/p44 MAPK phosphorylation in response to LTA. These results demonstrate that the effect of LTA on these responses may be mediated through a PTX-sensitive G protein in HTSMCs.



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Fig. 5. Involvement of G protein(s) in p42/p44 MAPK phosphorylation induced by LTA in HTSMCs. Cells were grown to confluence on 12-well culture plates, shifted to culture medium containing 2% FBS for 24 h, preincubated with pertussis toxin (PTX; 100 ng/ml) and cholera toxin (CTX; 10 µg/ml) for 24 h, and then stimulated with vehicle or LTA (50 µg/ml) for 7 min. After being incubated, the cell lysates were subjected to 12% SDS-PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using an antiserum reactive with an anti-phospho-p42/p44 MAPK or total p42 MAPK (as a control) polyclonal antibody. Bands were visualized by an ECL method as described in Fig. 1. Data are expressed as means ± SE of 5 independent experiments (bar graph). *P < 0.05; **P < 0.01 compared with control cells.

 


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Fig. 6. mRNA and protein expression of TLR2 in HTSMCs treated with PTX. Cells were grown to confluence on 10-cm culture dishes, shifted to culture medium containing 2% FBS for 24 h, and pretreated with 100 ng/ml of PTX for 24 h. A: expression of TLR2 mRNA was measured by RT-PCR. B: expression of TLR2 protein was determined using Western blot analysis. Data are expressed as means ± SE of 3 independent experiments. C, control.

 

Effect of genistein on LTA-stimulated p42/p44 MAPK phosphorylation. G protein-coupled receptors have been shown to activate several protein tyrosine kinase pathways that, similar to those of growth factors, play important roles in regulation of cellular responses (32). Because LTA-induced responses were sensitive to PTX in HTSMCs, we further determined whether the effect of p42/p44 MAPK phosphorylation was mediated through activation of tyrosine kinases that may be indirectly activated by a PTX-sensitive G protein. The cells were treated with the tyrosine kinase inhibitor genistein (34) for 1 h and then stimulated with 50 µg/ml of LTA for 7 min. As shown in Fig. 7, pretreatment of these cells with genistein inhibited the LTA-stimulated p42/p44 MAPK phosphorylation (P < 0.01, n = 3). Treatment with genistein alone had no significant effect on the basal levels of p42/p44 MAPK phosphorylation. These results suggest the implication of tyrosine kinases in these responses to LTA.



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Fig. 7. Effect of genistein on p42/p44 MAPK phosphorylation induced by LTA in HTSMCs. Cells were grown to confluence on 12-well culture plates, shifted to culture medium containing 2% FBS for 24 h, preincubated with various concentrations of genistein for 1 h, and then stimulated with vehicle or LTA (50 µg/ml) for 7 min. After being incubated, the cell lysates were subjected to 12% SDS-PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using an antiserum reactive with an anti-phospho-p42/p44 MAPK or total p42 MAPK (as a control) polyclonal antibody. Bands were visualized by an ECL method as described in Fig. 1. Data are expressed as means ± SE of 3 independent experiments (bar graph). **P < 0.01 compared with control cells.

 

Effects of D609 and U-73122 on LTA-stimulated p42/p44 MAPK phosphorylation. To investigate whether the effect of LTA on p42/p44 MAPK phosphorylation was mediated through the activation of phosphatidylcholine (PC)-PLC or PI-PLC, HTSMCs were pretreated with the PC-PLC inhibitor D609 (9) and the PI-PLC inhibitor U-73122 (7) for 1 h and then stimulated with 50 µg/ml of LTA for 7 min. As shown in Fig. 8, pretreatment of HTSMCs with either D609 or U-73122 significantly attenuated the LTA-stimulated p42/p44 MAPK phosphorylation (P < 0.05, n = 3). None of these inhibitors alone at the concentrations applied had significant effects on the basal levels of p42/p44 MAPK phosphorylation. These results suggest that LTA-stimulated p42/p44 MAPK phosphorylation is mediated through the activation of PC-PLC or PI-PLC in HTSMCs.



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Fig. 8. Effects of D609 and U-73122 on p42/p44 MAPK phosphorylation induced by LTA in HTSMCs. Cells were grown to confluence on 12-well culture plates, shifted to culture medium containing 2% FBS for 24 h, preincubated with D609 (0.1 µM) or U-73122 (0.3 µM) for 1 h, and then stimulated with vehicle or LTA (50 µg/ml) for 7 min. After being incubated, the cell lysates were subjected to 12% SDS-PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using an antiserum reactive with an anti-phospho-p42/p44 MAPK or total p42 MAPK (as a control) polyclonal antibody. Bands were visualized by an ECL method as described in Fig. 1. Data are expressed as means ± SE of 3 independent experiments (bar graph). *P < 0.05; **P < 0.01 compared with control cells.

 

Involvement of PKC in LTA-stimulated p42/p44 MAPK phosphorylation. The results obtained above demonstrate that LTA-stimulated p42/p44 MAPK phosphorylation was inhibited by D609 and U-73122, implying the involvement of the PC-PLC and PI-PLC pathways in these responses. Activation of these two enzymes increases the generation of diacylglycerol (DAG) and then activates PKC. To determine whether PKC activation was involved in p42/p44 MAPK phosphorylation in response to LTA, HTSMCs were pretreated with the PKC inhibitor staurosporine (20) for 1 h and then stimulated with 50 µg/ml of LTA for 7 min. As shown in Fig. 9A, pretreatment of the cells with staurosporine concentration dependently attenuated p42/44 MAPK phosphorylation (P < 0.05, n = 4) stimulated by LTA. None of the staurosporine at the concentrations applied alone had a significant effect on the basal levels of p42/p44 MAPK phosphorylation. To further confirm the involvement of PKC in these responses, HTSMCs were pretreated with PKC inhibitors (Gö-6976, rottlerin, and Ro-318220) at a concentration of 10 µM for 1 h and stimulated with LTA for 7 min. As shown in Fig. 9B, LTA-stimulated p42/p44 MAPK phosphorylation was almost completely blocked by these three PKC inhibitors. These results suggest that LTA-stimulated p42/p44 MAPK phosphorylation is mediated through the activation of PKC in HTSMCs.



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Fig. 9. Involvement of PKC in p42/p44 MAPK phosphorylation induced by LTA in HTSMCs. Cells were grown to confluence on 12-well culture plates, shifted to culture medium containing 2% FBS for 24 h, preincubated with staurosporine (A) and Gö-6976, rottlerin, or Ro-318220 (B) for 1 h, and then stimulated with vehicle or LTA (50 µg/ml) for 7 min. After being incubated, the cell lysates were subjected to 12% SDS-PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using an antiserum reactive with an anti-phospho-p42/p44 MAPK or total p42 MAPK and GAPDH (as a control) polyclonal antibody. Bands were visualized by an ECL method as described in Fig. 1. Data are expressed as means ± SE of 4 independent experiments (bar graphs). *P < 0.05; **P < 0.01 compared with control cells.

 

Effects of BAPTA on LTA-stimulated p42/p44 MAPK phosphorylation. Several studies have demonstrated that the component of bacterial cell wall, including LTA and LPS, activated PC-PLC and PI-PLC (29) and may lead to an increase in [Ca2+]i (48). To elucidate the involvement of intracellular Ca2+ mobilization in LTA-mediated p42/p44 MAPK phosphorylation, HTSMCs were preincubated with 0.3, 3, or 30 µM BAPTA-AM (a potent intracellular Ca2+ chelator; Ref. 8) plus 1 mM EDTA and then stimulated with 50 µg/ml of LTA for 7 min. Results in Fig. 10 demonstrate that pretreatment of these cells with BAPTA-AM plus EDTA reduced p42/p44 MAPK phosphorylation (P < 0.01, n = 3) exposed to LTA. Treatment with BAPTA-AM alone at the concentrations applied had no significant effect on the basal levels of p42/p44 MAPK phosphorylation. These results demonstrate that Ca2+ mobilization was essential for LTA-mediated p42/p44 MAPK phosphorylation in HTSMCs.



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Fig. 10. Effect of Ca2+ on p42/p44 MAPK phosphorylation induced by LTA in HTSMCs. Cells were grown to confluence on 12-well culture plates, shifted to culture medium containing 2% FBS for 24 h, preincubated with various concentrations of BAPTA-AM plus EGTA (1 mM) for 1 h, and then stimulated with vehicle or LTA (50 µg/ml) for 7 min. After being incubated, the cell lysates were subjected to 12% SDS-PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using an antiserum reactive with an anti-phospho-p42/p44 MAPK or total p42 MAPK (as a control) polyclonal antibody. Bands were visualized by an ECL method as described in Fig. 1. Data are expressed as means ± SE of 3 independent experiments (bar graph). **P < 0.01 compared with control cells.

 

[Ca2+]i response of HTSMCs to LTA. Furthermore, we investigated whether LTA directly induced an increase in intracellular Ca2+ mobilization. The ability of LTA to mobilize Ca2+ was assessed in HTSMCs loaded with fura 2-AM as an indicator. As shown in Fig. 11, LTA (ranging from 50 to 250 µg/ml) caused a concentration-dependent elevation of [Ca2+]i levels. In the presence of extracellular Ca2+, LTA induced in a rapid increase in [Ca2+]i and then declined to the resting level within 1 min. The resting level of [Ca2+]i in HTSMCs ranged from 50 to 150 nM (averaged ~80 nM). To further investigate whether LTA-stimulated increase in [Ca2+]i was mediated through a PTX-sensitive G protein, the cells were pretreated with 100 µg/ml of PTX for 24 h and then exposed to 50 µg/ml of LTA. As shown in Fig. 12, LTA-induced [Ca2+]i decreased from 150 nM (control) to 65 nM (PTX treatment). These results demonstrate that LTA directly stimulated an elevation of [Ca2+]i meditated through a PTX-sensitive G protein in HTSMCs.



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Fig. 11. [Ca2+]i response of HTSMCs to LTA. A: concentration dependence of LTA-stimulated [Ca2+]i change in HTSMCs. Cells grown on glass coverslips coated with collagen were loaded with 5 µM fura 2-AM for 45 min, and fluorescent measurement of [Ca2+]i was carried out in a dual-excitation wavelength spectrofluorometer, with excitation at 340 and 380 nm, when various LTA concentrations [50 (a), 100 (b), 150 (c), 200 (d), and 250 (e) µg/ml] were added. B: data expressed as means ± SE represent an increase in the transient peak above the resting levels from 5 experiments.

 


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Fig. 12. Effect of PTX treatment on LTA-induced [Ca2+]i in HTSMCs. Cells grown on glass coverslips coated with collagen were pretreated with 100 µg/ml of PTX for 24 h, and [Ca2+]i was measured when 50 µg/ml of LTA was added, as described in Fig. 11. Data expressed as the means ± SE represent an increase in the transient peak above the resting levels from 5 experiments. *P < 0.01, as compared with cells exposed to LTA.

 

Effect of U-0126 on LTA-induced p42/p44 MAPK phosphorylation. To ensure that the effect of LTA was mediated through the activation of MAPK pathway, the effect of LTA on p42/p44 MAPK phosphorylation was examined after treatment of HTSMCs with U-0126 (a synthetic inhibitor of MEK1/2 activation; Ref. 11) for 1 h and then stimulated with 50 µg/ml of LTA for 7 min. As shown in Fig. 13, treatment of HTSMCs with 0.01, 0.1, or 1 µM U-0126 caused a significant inhibition of the LTA-induced p42/p44 MAPK phosphorylation (P < 0.01, n = 4). None of the U-0126 at the concentrations applied alone had a significant effect on the basal levels of p42/p44 MAPK phosphorylation. These results suggest that LTA is able to activate p42/p44 MAPK pathway in HTSMCs.



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Fig. 13. Effect of U-0126 on p42/p44 MAPK phosphorylation induced by LTA in HTSMCs. Cells were grown to confluence on 12-well culture plates, shifted to culture medium containing 2% FBS for 24 h, preincubated with various concentrations of U-0126 for 1 h, and then stimulated with vehicle or LTA (50 µg/ml) for 7 min. After being incubated, the cell lysates were subjected to 12% SDS-PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using an antiserum reactive with an anti-phospho-p42/p44 MAPK or total p42 MAPK (as a control) polyclonal antibody. Bands were visualized by an ECL method as described in Fig. 1. Data are expressed as means ± SE of 4 independent experiments (bar graph). **P < 0.01 compared with control cells.

 

Effects of PI 3-kinase inhibitors on LTA-stimulated p42/p44 MAPK phosphorylation. LPS is also capable of activating PI 3-kinase activity (18). To determine whether PI 3-kinase was involved in LTA-induced p42/p44 MAPK phosphorylation, the PI 3-kinase inhibitors wortmannin (4) and LY-294002 (47) were used. As shown in Fig. 14, treatment of these cells with either wortmannin or LY-294002 significantly attenuated p42/p44 MAPK phosphorylation (P < 0.05, n = 4) stimulated by LTA. None of these inhibitors alone at the concentrations applied had a significant effect on the basal levels of p42/p44 MAPK phosphorylation. These results suggest that LTA-stimulated p42/p44 MAPK phosphorylation is mediated through the activation of PI 3-kinase pathway in HTSMCs.



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Fig. 14. Effects of phosphatidylinositol 3-kinase inhibitors on p42/p44 MAPK phosphorylation induced by LTA in HTSMCs. Cells were grown to confluence on 12-well culture plates, shifted to culture medium containing 2% FBS for 24 h, preincubated with various concentrations of LY-294002 (A) or wortmannin (B) for 1 h, and then stimulated with vehicle or LTA (50 µg/ml) for 7 min. After being incubated, the cell lysates were subjected to 12% SDS-PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using an antiserum reactive with an anti-phospho-p42/p44 MAPK or total p42 MAPK (as a control) polyclonal antibody. Bands were visualized by an ECL method as described in Fig. 1. Data are expressed as means ± SE of 4 independent experiments (bar graphs). *P < 0.05; **P < 0.01 compared with control cells.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several lines of evidence have demonstrated that LTA, a component of cell wall released from gram-positive bacteria, plays an important role in the regulation of many gene expressions involved in the inflammatory processes (6, 21). LTA has been shown to be a triggering factor for increasing the severity of respiratory disorder characterized by inflammatory changes in the lung (26, 27). In addition, LTA can activate several intracellular signaling pathways, including p42/p44 MAPK, p38 MAPK, and JNK in various cell types (29, 39). These signaling pathways in turn activate a variety of transcription factors, such as AP-1, which are implicated in the induction of specific genes encoding inflammatory mediators. However, little was known about the molecular mechanisms of LTA implicated in inflammatory responses in HTSMCs. In this study, we have used Western blot analysis and selective pharmacological inhibitors to characterize the mechanisms underlying signaling transductions in primary cultures of HTSMCs treated with LTA. These results demonstrate that in HTSMCs, LTA-stimulated p42/p44 MAPK phosphorylation is mediated through a TLR2 receptor and involves tyrosine kinase, PLC, PKC, Ca2+, MEK, and PI 3-kinase.

During bacterial infection, the mammalian innate immune system can recognize bacteria and their cell wall components through two distinct receptors, CD14 (5, 40, 41) and TLR receptors (1, 42, 46) that initiate inflammatory responses (42, 46). TLR4 is the best-characterized member of the TLR family, which acts as the receptor for LPS (1). TLR2 acts as the receptor for LTA (1, 2). In this study, we have shown the expression of both TLR2 and TLR4 receptor genes on HTSMCs. Pretreatment of these cells with TLR2 antibody significantly attenuated LTA-stimulated p42/p44 MAPK phosphorylation, but TLR4 antibody had no effect on this response, consistent with the results that LTA induced activation of signaling transduction pathways mediated through TLR2 receptors in various cell types (36, 38, 40, 43). This hypothesis was further supported by the results that LTA-stimulated p42/p44 MAPK phosphorylation was attenuated by transfection with human dominant negative mutant of TLR2 plasmid. Moreover, LPS-stimulated cellular responses have been shown to be mediated through a Gi or Gq protein coupling process in several cell types (10, 31). This is confirmed by our observation that the LTA-stimulated p42/p44 MAPK phosphorylation was significantly attenuated by PTX but not by CTX. Pretreatment of the cells with PTX has been shown to inhibit intrinsic GTPase activity of Gi protein by ADP-ribosylation of specific residues. Complete abrogation of PTX-sensitive Gi protein has been revealed by [32P]ADP-ribosylation of cell membranes prepared from TSMCs treated with PTX (51). The inhibitory effect of PTX on p42/p44 MAPK phosphorylation was not due to a reduction of TLR2 receptor number, since TLR2 mRNA and protein expression were not changed during treatment with this toxin for overnight. The results showed that LTA-stimulated p42/p44 MAPK phosphorylation was partially attenuated by PTX treatment, indicating the involvement of a PTX-sensitive G protein in these processes. However, the detailed mechanism of PTX action on LTA-induced responses needs to be further defined. Recently, it is believed that protein tyrosine kinases might be involved in signaling events, including p42/p44 MAPK evoked by G protein-coupled receptor agonists (32). Therefore, we further investigated the implication of protein tyrosine kinases in the p42/p44 MAPK phosphorylation of HTSMCs stimulated by LTA, using the tyrosine kinase inhibitor genistein. The results with the tyrosine kinase inhibitor showed that the LTA-induced p42/p44 MAPK phosphorylation was mediated through the activation of tyrosine kinase.

It has been demonstrated that LTA-triggered activation of PI-PLC and PC-PLC may play an important role in the regulation of cellular functions (29). LTA-stimulated activation of PI-PLC could produce two second messengers: DAG and D-myo-inositol 1,4,5-trisphosphate (IP3) (35). DAG and IP3 activate PKC and release Ca2+ from the intracellular stores, respectively, in several cell types (35). PKC is a predominant component in the kinase cascade, initiating by ligand attachment to both G protein-coupled receptors and receptors containing intrinsic tyrosine kinase activity. In this study, we further investigated the regulatory mechanisms involved in LTA-stimulated p42/p44 MAPK phosphorylation by PKC. These results demonstrated that pretreatment with the PKC inhibitors staurosporine, Gö-6976, rottlerin, or Ro-318220 attenuated the LTA-stimulated p42/p44 MAPK phosphorylation, indicating that PKC activation is required for the LTA-mediated responses in HTSMCs. This is likely to be either Raf-1, which was shown to be phosphorylated by PKC (2), or possibly MEK1/2, which is also believed to be activated in a PKC-dependent manner (24). PKC is activated by DAG, which can be generated by the activation of either PI-PLC or PC-PLC (35). In this study, the PI-PLC and PC-PLC inhibitors U-73122 and D609, respectively, inhibited the LTA-stimulated p42/p44 MAPK phosphorylation, suggesting that LTA may act through the PI-PLC and PC-PLC pathways to activate PKC in HTSMCs. These results are consistent with the findings that LTA activated both PI-PLC and PC-PLC and led to phosphorylation of p42/p44 MAPK in human pulmonary epithelial cells (29).

To assess Ca2+ mobilization that might be implicated in the p42/p44 MAPK phosphorylation in response to LTA, we attempted to analyze some potentially participating pathways. IP3 is known to release Ca2+ from intracellular Ca2+ stores, and Ca2+ release can further induce Ca2+ influx (30, 44). The increase in [Ca2+]i seems to account for the effect of LTA on p42/p44 MAPK phosphorylation. In this study, both [Ca2+]i and p42/p44 MAPK phosphorylation induced by LTA were inhibited by pretreatment with PTX. Our results also showed that the stimulatory effect of LTA on MAPK phosphorylation may be required for the presence of Ca2+ in HTSMCs. This hypothesis was supported by the results that removal of Ca2+ by BAPTA/EDTA significantly attenuated the p42/p44 MAPK phosphorylation induced by LTA in these cells. These results indicate an important role of Ca2+ in mediating LTA-induced p42/p44 MAPK phosphorylation. However, the contribution of intracellular and extracellular Ca2+ resources to LTA-stimulated responses is needed for further investigation.

It has been demonstrated that LTA is a potent stimulator of MAPK pathways, including p42/p44 MAPK in several cell types (39). In our previous study, we showed that LPS induced activation of the p42/p44 MAPK in canine TSMCs (31). Activation of p42/p44 MAPK is known to require both tyrosine and threonine phosphorylations by the dual specificity of MEK1/2. Although activation of MAPK by growth factors has been well characterized, the mechanisms by which LTA activates the components of MAPK pathway are not completely understood in HTSMCs. U-0126, a synthetic and highly specific inhibitor of MEK1/2, has been shown to inhibit the activation of p42/p44 MAPK by several stimuli (11). In the current study, inhibition of MEK1/2 in HTSMCs by U-0126 reduced LTA-stimulated p42/p44 MAPK phosphorylation, indicating a role for MEK-p42/p44 MAPK in an LTA-induced signaling pathway.

Several reports have demonstrated that PI 3-kinase might be implicated in growth factor- and G protein-coupled receptor-induced p42/p44 MAPK activation via several distinct signaling pathways (32). LPS is also capable of activating PI 3-kinase activity (18). This process was demonstrated to involve the association of PI 3-kinase with the Src kinase Lyn and to activate upstream of PKC activation (18). In this study, we have found that the PI 3-kinase inhibitors LY-294002 and wortmannin attenuated the LTA-stimulated p42/p44 MAPK phosphorylation in HTSMCs, suggesting that LTA-stimulated responses are mediated through the activation of a PI 3-kinase pathway.

In conclusion, we report here that LTA appeared to exert its effect on p42/p44 MAPK phosphorylation in HTSMCs. These stimulatory effects of LTA were mediated through a TLR2 receptor and regulated by PTX, tyrosine kinase, PLC, PKC, Ca2+, MEK, and PI 3-kinase. These results raise the possibility that LTA may play an important role in the pathogenesis of airway inflammatory diseases. Studies of cellular signal transduction events elicited by LTA may provide important insights into the regulation of gene expression for induction of proinflammatory cytokines and other mediator production in HTSMCs. Further studies are needed to explain these stimulatory effects of LTA related to inflammatory airway diseases.


    ACKNOWLEDGMENTS
 
We appreciate Dr. Jiahuai Han (Dept. of Immunology, the Scripps Research Institute, La Jolla, CA) and Dr. Jian-Dong Li (Dept. of Cell and Molecular Biology, House Ear Institute, Univ. of Southern California, Los Angeles, CA) for providing human dominant negative mutant TLR2 and wild-type TLR2 constructs.

GRANTS

This work was supported by Chang Gung Medical Research Foundation Grant CMRPD-32043 and National Science Council Grant NSC91-2320-B182-042, Taiwan.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C.-M. Yang, Dept. of Pharmacology, College of Medicine, Chang Gung Univ., 259 Wen-Hwa 1 Road, Kwei-San, Tao-Yuan, Taiwan, 3332 (E-mail: chuenmao{at}mail.cgu.edu.tw).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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