Mol Cell Biochem DOI 10.1007/s11010-014-2266-1
Studies on mitogen-activated protein kinase signaling pathway in the alveolar macrophages of chronic bronchitis rats Yan Huang • Xiao-Ming Meng • Guo-Lin Jiang Ya-Ru Yang • Juan Liu • Xiong-Wen Lv • Jun Li
•
Received: 19 July 2014 / Accepted: 29 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Lipopolysaccharide (LPS), a potent stimulator of inflammatory responses in alveolar macrophages (AMs), activates several intracellular signaling pathways, including mitogen-activated protein kinases (MAPK). In the present study, we investigated the MAPK pathway in AMs of chronic bronchitis (CB) rats. CB was induced by endotracheal instillation of LPS followed by Bacillus Calmette Guerin injection through the caudal vein 1 week later. Specific inhibitors were used and protein phosphorylations were detected by Western blot. We found that Genistein (PTK inhibitor) could inhibit protein kinase C (PKC), phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt or PKB) MAPK signaling pathway with different degrees, LY294002 (PI3K inhibitor) could not only inhibit phospho-PI3K/Akt expression, but also inhibit p38 and c-Jun NH2-terminal kinases (JNK) phosphorylation. Calphostin C (PKC inhibitor) could inhibit phospho-PKC
Y. Huang X.-M. Meng G.-L. Jiang Y.-R. Yang J. Liu X.-W. Lv J. Li (&) School of Pharmacy, Anhui Medical University, Tunxi West Road, Hefei 230032, Anhui, China e-mail: [email protected] X.-M. Meng e-mail: [email protected]
expression and exerted significant effects on extracellular signal-regulated kinases (ERK) phosphorylation, however, it had no impact on p38 and JNK phosphorylation. These results demonstrated that the LPS mediated signaling pathway of MAPK in AMs of CB rats could be described as follows: PTK-PI3K-Akt-JNK/p38 or PTK-PI3K-PKCERK, and PI3K may have a negative regulation on the activation of downstream proteins. Keywords Alveolar macrophages Chronic bronchitis Mitogen-activated protein kinase signaling pathway Abbreviations AMs Alveolar macrophages CB Chronic bronchitis LPS Lipopolysaccharide BCG Bacillus Calmette Guerin MAPK Mitogen-activated protein kinases PKC Protein kinase C PKA Protein kinase A ERK Extracellular signal-regulated kinases JNK c-Jun NH2-terminal kinases FBS Fetal bovine serum CMC Carboxymethylcellulose BALF Bronchoalveolar lavage fluid PI3K Phosphatidylinositol-3 kinase PTK Receptor tyrosine kinase
G.-L. Jiang e-mail: [email protected] Y.-R. Yang e-mail: [email protected]
Introduction
J. Liu e-mail: [email protected]
Chronic bronchitis (CB) is ventable disease [1]. Its include calcium overload, bolic abnormalities, and
X.-W. Lv e-mail: [email protected]
a frequently occurring yet prepathophysiological processes free radical production, metainflammatory reaction, etc.
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Chronic inflammation is a key feature and is tightly associated with alveolar macrophages (AMs) in CB. AMs are regarded as one of the most important sources of inflammatory cytokines and mediators, growth factors, and free oxygen radicals. It also has roles in defense, protection, regulatory effects and can interact with other cells within airways especially alveoli. These interactions result in intracellular signaling cascade activation, leading to various effects or functions that make up the inflammatory response in the lung [2–4], but the exact signaling transduction pathway remains unclear. Lipopolysaccharide (LPS) may activate many intracellular signaling pathways by binding to receptors on cell membrane, such as G protein, protein kinase C (PKC), protein kinase A (PKA), calcium signal systems, and particularly mitogen-activated protein kinases (MAPK) [5–8]. MAPK are Ser–Thr kinases comprised of three principal family members: extracellular signal-regulated kinases (ERK), p38, and c-Jun NH2-terminal kinases (JNK). Previous studies suggest that the three members of MAPK cascade are involved in the critical LPS-induced intracellular signaling transduction in macrophages [9–12]. Results from our group have revealed that AMs were significantly increased in CB rats. In disease conditions, the balance of proliferation and apoptosis was disrupted. AMs played an important role in CB through synthesis and release of cytokines (such as TNF-a, IL-8, IL-1) and inflammatory mediators (such as PGE2, LTB4 and NO) [13–15]. Since either synthesis of cytokines and inflammatory mediators, or AMs proliferation and apoptosis has a close relationship with MAPK pathway, activation of MAPK pathway in AMs may affect the pathology process of CB. The MAPK signaling pathway in AMs of CB rats induced by LPS and Bacillus Calmette Guerin (BCG) is uncertain. Therefore, in the present study, we focused on the role of MAPKs in AMs of CB rats using specific inhibitors of the MAPKs pathway to further clarify the possible molecule mechanism of CB and to develop new target drugs.
inhibitor), p38 MAP Kinase antibody;Phospho-p38 MAPK (Thr180/Tyr182) Mouse mAb; p44/42 MAP Kinase antibody;Phospho-p44/42 MAP Kinase (Thr202/Tyr204) antibody; SAPK/JNK antibody;Phospho-SAPK/JNK (Thr183/ Tyr185) Mouse mAb; Akt antibody; Phospho-Akt (Ser473) antibody;PKC-delta antibody; Phospho-PKCdelta (Tyr311) antibody; PI3 Kinase p85 antibody; Phospho-PI3K p85 (Tyr458)/p55 (Tyr199) antibody were purchased from Cell Signaling. SuperSignal West Femto Maximum Sensitivity Substrate 34094 was purchased from Pierce. Animals and chronic bronchitis model Thirty-five male SD rats weighing 180 ± 20 g were randomly divided into normal and model groups. Animals were provided by the Experimental Animal Center of Anhui Medical University. The animal experimental protocol was approved by the University Animal Care and Use Committee. Rats of model group were administered 5 mg/ kg BCG by tail vein injection, then injected LPS 200 ll (1 g/l) through trachea 1 week later [1, 13]. Bronchoalveolar lavage and cell counts Three weeks after LPS injection, the rats were anesthetized with 10 % chloral hydrate (0.3 ml/100 g i.c.). Bronchoalveolar lavage (BAL) was performed by infusing 20 ml normal saline (NS) through the tracheal cannula. BAL fluid (BALF) was pooled and centrifuged at 1,5009g for 10 min at 4 °C, then washed once with D-Hank’s balanced salt solution (HBSS). The deposition was resuspended with RPMI-1640 containing 10 % fetal bovine serum (FBS), 50 U penicillin G, and 50 lg/ml streptomycin at a density of 2 9 106 cells/ml. Cell suspenstion was then transferred to 24-well dish (1 ml per well) and incubated at 37 °C in 5 % CO2 for 2 h to allow adherence of AMs. Isolated adherent cells were stained with a rabbit anti-CD68 antibody. More than 95 % cells were CD68 positive cells, which confirmed the feasibility of the method to separate and purify AMs [13, 14]. Then, the AMs were cultured with LPS in vitro [16].
Reagents
Relationship between different concentrations of LPS and MAPK signaling pathway activation in AMs of chronic bronchitis rats
RPMI-1640, fetal bovine serum (FBS), LPS, carboxymethylcellulose (CMC), and Chelerythrine Chloride (PKC inhibitor) were purchased from Sigma. PD98059 (ERK inhibitor), SB203580 (p38 inhibitor) were purchased from Promega. Curcumin (JNK inhibitor), Genistein (PTK inhibitor), Triciribine KC-4T001 (Akt inhibitor) was purchased from Kang Chen company. LY294002 (PI3K
Rat model of CB was induced as mentioned above. After purifying, AMs were cultured with different concentration of LPS (10, 100 ng/ml, 1, 10 lg/ml) for 30 min. Then the phosphorylation level of ERK in AMs from different LPS stimulated groups was detected in order to determine the appropriate LPS concentration on MAPK signaling transduction pathway activation.
Materials and methods
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Dynamic changes of MAPK signaling pathway activation induced by LPS in AMs of CB rats
ECLTM. The relative density was analyzed by the ratio of phosphorylated and appropriate total protein.
AMs were cultured with appropriate concentration of LPS for different times as follows: 0, 15, 30, 60, 120 min, respectively, then ERK and PI3K phosphorylation were observed in order to determine the best time point for MAPK phosphorylation stimulated by LPS in the AMs.
Statistical analysis Measurement data were expressed as mean ± standard deviation (SD). Statistical analysis was done with SPSS 11.0 software to assess statistical significance between drug groups and various related control groups.
LPS induced MAPK signaling pathway activation in AMs of normal rats and CB rats Results AMs seperated from normal and model rats were cultured with or without LPS using an optimal concentration of 6 lg/ml. After 30 min (the best time point), the activation of MAPK signaling proteins was detected in AMs of each group. Cell extracts preparation Single-cell suspension of AMs from model rats was plated in the six-well plates (1.0 9 106 cells per well). After 24 h incubation, different reagents were separately added into the culture medium and cells were divided into the following groups: model (nothing added), DMSO, Genistein (30 lM), LY294002 (20 lM), Triciribine KC-4T001 (1 lM), Chelerythrine Chloride (10 lM). After 2 h incubation, cells in each well were cultured in the presence of 6 lg/ml LPS for 15 or 30 min. Then the cells were washed twice with phosphate-buffered saline (PBS) and lysed with 100 ll/106 cells of ice-cold lysis buffer (200 ll, 100 lg/ml phenylmethylsulfonyl fluoride (PMSF) for 30 min at 4 °C [17]. Cell lysates were centrifuged at 12,0009g for 10 min at 4 °C. Supernatants were stored at -80 °C until further analysis. Protein content was assessed by Lowry’s method with a commercially available kit. Western blot analysis Cell extracts (30 lg protein per lane) were separated via electrophoresis on 10 % sodium dodecyl sulfate–polyacrylamide gels, and proteins were electroblotted to nitrocellulose membrane at 200 mA for 0.5 h as described previously [18]. Blots were then blocked overnight at 4 °C with 5 % nonfat dry milk in 50 mM Tris–HCl (pH 7.5), 200 mM NaCl, Tris-buffered saline, 0.05 % Tween 20 (TBST). After three 10-min washes with TBST, membranes were incubated for 2 h with different antibodies diluted 1:1,000. After subsequent washes with TBST, HRP-IgG goat anti-rabbit secondary antibody, diluted 1:1,000, was incubated for 2 h. After washing, membranes were developed with an enhanced chemiluminescence (ECL) reagent followed by exposure to HyperfilmTM
Time and dose-dependent effect of LPS on MAPK phosphorylation in AMs of CB rats As shown in Fig. 1, ERK MAPK phosphorylation in AMs increased when the dose of LPS increased after 30 min culture and reached peak at 1 lg/ml LPS. When LPS dose increased to 10 lg/ml, ERK phosphorylation did not further increase. In order to maintain consistency with our previous paper [14, 15], we used 6 lg/ml as the maximal stimulus concentration of LPS in the following experiments. As shown in Fig. 2, AMs were stimulated by 6 lg/ml LPS for different durations, then ERK and PI3K phosphorylation in AMs were detected. The results indicated that both ERK and PI3K phosphorylation demonstrated time-dependence. After 15 min, ERK phosphorylation increased significantly, peaked at 30 min, and declined significantly after 60 min. PI3K phosphorylation peaked at 15 min, and then decreased gradually. Effect of LPS on MAPK phosphorylation in AMs of normal and CB rats Compared with the nomal group, MAPK phosphorylation increased significantly in the model group (Fig. 3). After treatment with LPS, MAPK phosphorylation in the normal and model groups both increased, and MAPK phosphorylation in the model ? LPS group increased the most. MAPK phosphorylation in the normal group without LPS had weak expression. This showed that there was a basal amount of ERK/JNK/p38 phosphorylation in AMs of normal rats. Investigation on MAPK signaling transduct pathway in AMs of CB rats Effect of MAPK signaling transduct protein inhibitors on p38 MAPK phosphorylation Compared with DMSO group, Genistein (PTK inhibitor) could inhibit p38 MAPK phosphorylation in AMs of CB
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**#
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Fig. 1 Dose-dependence of MAPK phosphorylation induced by LPS in AMs of CB rats. AMs were cultured with different concentrations of LPS (10, 100 ng/ml, 1, 10 lg/ml) for 30 min. Then, the
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phosphorylation level of ERK in AMs from different LPS stimulated groups was detected. *P \ 0.05, **P \ 0.01 versus LPS 10 ng/ml; # P \ 0.05 versus LPS 100 ng/ml
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Fig. 2 Time-dependence of MAPK phosphorylation induced by LPS in AMs of CB rats. AMs were cultured with 6 lg/ml LPS for different times as follows: 0,15,30,60,120 min, respectively. Then, ERK and PI3K phosphorylation were observed in order to determine the best
time point of MAPK phosphorylation stimulated by LPS in the AMs. *P \ 0.05, **P \ 0.01 versus 0 min; #P \ 0.05, ##P \ 0.01 versus 15 min (A) or 30 min (B)
rats, while LY294002 (PI3K inhibitor) and Triciribine (Akt inhibitor) could further increase p38 MAPK phosphorylation (P \ 0.01). In contrast, Calphostin C (PKC inhibitor) had no such effect on p38 MAPK phosphorylation (Fig. 4).
Effect of MAPK inhibitors on JNK MAPK phosphorylation
Effect of MAPK inhibitors on ERK MAPK phosphorylation Compared with DMSO group, Genistein (PTK inhibitor) and Calphostin C (PKC inhibitor) could inhibit ERK MAPK phosphorylation inordinately in AMs of CB rats (P \ 0.05). In contrast, LY294002 (PI3K inhibitor) and Triciribine (Akt inhibitor) had no such effect on ERK MAPK phosphorylation (Fig. 5).
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Compared with DMSO group, Genistein (PTK inhibitor) could inhibit JNK MAPK phosphorylation in AMs of CB rats. In contrast, LY294002 (PI3K inhibitor) and Triciribine (Akt inhibitor) could further increase JNK MAPK phosphorylation (P \ 0.01). There is no difference of JNK MAPK phosphorylation in model group and Calphostin C (PKC inhibitor) group (Fig. 6). Effect of MAPK inhibitors on PKC MAPK phosphorylation Compared with DMSO group, both Genistein (PTK inhibitor) and Calphostin C (PKC inhibitor) could significantly
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Fig. 3 Effect of LPS on MAPK phosphorylation in AMs from normal and CB rats. AMs separated from normal and model rats were cultured with or without LPS using an optimal concentration of 6 lg/
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Fig. 4 Effect of MAPK inhibitors on p38 MAPK phosphorylation. 1 model; 2 DMSO; 3 genistein (PTK inhibitor); 4 LY294002 (PI3K inhibitor); 5 triciribine (Akt inhibitor); 6 Calphostin C (PKC inhibitor). *P \ 0.05, **P \ 0.01 versus DMSO group
inhibit PKC MAPK phosphorylation in AMs of CB rats (P \ 0.01). LY294002 (PI3K inhibitor) could slightly increase PKC MAPK phosphorylation (P \ 0.05) (Fig. 7).
rats, while Triciribine and Calphostin C had no effect on PI3K phosphorylation (P \ 0.01) (Fig. 8). Effect of MAPK inhibitors on Akt MAPK phosphorylation
Effect of MAPK inhibitors on PI3K MAPK phosphorylation Compared with DMSO group, both Genistein (PTK inhibitor) and LY294002 (PI3K inhibitor) could significantly inhibit PI3K MAPK phosphorylation in AMs of CB
As shown in Fig. 9, Genistein, as well as LY294002 and Triciribine could inhibit Akt MAPK phosphorylation in AMs of CB rats with different degree (P \ 0.01). Calphostin C had no such effect.
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Fig. 5 Effect of MAPK inhibitors on ERK MAPK phosphorylation. 1 model; 2 DMSO; 3 genistein (PTK inhibitor); 4 LY294002 (PI3K inhibitor); 5 triciribine (Akt inhibitor); 6 Calphostin C (PKC inhibitor). *P \ 0.05 versus DMSO group
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Fig. 6 Effect of MAPK inhibitors on JNK MAPK phosphorylation. 1 model; 2 DMSO; 3 genistein (PTK inhibitor); 4 LY294002 (PI3K inhibitor); 5 triciribine (Akt inhibitor); 6 Calphostin C (PKC inhibitor). **P \ 0.01 versus DMSO group
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Fig. 7 Effect of MAPK inhibitors on PKC MAPK phosphorylation. 1 model; 2 DMSO; 3 genistein (PTK inhibitor); 4 LY294002 (PI3K inhibitor); 5 triciribine (Akt inhibitor); 6 Calphostin C (PKC inhibitor). *P \ 0.05, **P \ 0.01 versus DMSO group
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Fig. 8 Effect of MAPK inhibitors on PI3K MAPK phosphorylation. 1 model; 2 DMSO; 3 genistein (PTK inhibitor); 4 LY294002 (PI3K inhibitor); 5 triciribine (Akt inhibitor); 6 Calphostin C (PKC inhibitor). **P \ 0.01 versus DMSO group
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Fig. 9 Effect of MAPK inhibitors on Akt MAPK phosphorylation. 1 model; 2 DMSO; 3 genistein (PTK inhibitor); 4 LY294002 (PI3K inhibitor); 5 triciribine (Akt inhibitor); 6 Calphostin C (PKC inhibitor). **P \ 0.01 versus DMSO group
Discussion The present study has examined for the first time the effect of different signaling transduct protein inhibitors of MAPK on PTK, PI3K, PKC, ERK, JNK, p38 MAPK phosphorylation in AMs of CB rats in order to investigate the possible MAPK signaling transduct pathway in AMs of CB rats. Many cellular responses, including cell-cycle progression, differentiation, and apoptosis are regulated by MAPK signaling transduct pathway [19]. It has been reported that members of MAPK family including ERK, JNK, p38 are involved in inflammatory mediator production in response to a wide variety of microbial stimuli [20, 21]. A number of laboratories have found that MAPK signaling transduct pathway is involved in macrophage activation by LPS exposure [22, 23]. Activated MAPK signaling transduct pathway proteins will then phosphorylate downstream effectors, such as membrane proteins, cytoplasmic proteins, and nuclear proteins [24, 25]. Small molecule kinases inhibitors of the MAPK pathway have been developed to treat various diseases, including inflammatory diseases [26], ischemic heart disease [27], and cancer [28, 29]. Phosphatidylinositol-3-kinase (PI3K) is a lipid kinase comprised of P85 and P110 subunits. It is phosphorylated and then activated when growth factors bind to their receptor tyrosine kinase (RTK) or G protein-coupled receptors (GPCR). Activated PI3K converts phosphatidylinositol (4,5)-phosphate [PI(4,5)P2] into phosphatidylinositol (3,4,5)-phosphate[PI(3,4,5)P3], which results in membrane localization of phosphotidylinositol-dependent kinase-1 (PDK1) via its pleckstrin homology (PH) domain. As a downstream kinase of PI3K, Akt can mediate PI3Kinitiated signaling. Akt translocates to the cell membrane and interacts with PI(3,4,5)P3 via its PH domain, being phosphorylated by phosphoinositide-dependent kinase (PDK) 1, PDK2, integrin-linked kinase (ILK) and unidentified kinase, respectively [30].
LPS+CD14
+ PTK
+ PI3K
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+ p38 MAPK
C-Jun, NF-
TNF-
ERK MAPK
B, ATF-2,ELK (transcription factor)
iNOS
HO-1
PGE2
TGF-
Fig. 10 Mitogen-Activated protein kinase signaling pathway in the alveolar macrophages of chronic bronchitis rats
In the present study, we found that PI3K, Akt, PKC phosphorylation reached its peak after 15 min treatment of LPS, then slowly declined, whereas p-ERK, p-p38, and p-JNK reached peak after 30 min treatment of LPS. Compared with normal group, MAPK phosphorylation increased significantly in the model group. After treatment of LPS, MAPK phosphorylation in the normal and model group both increased. MAPK phosphorylation in the LPS induced model group was the most significant. Genistein (PTK inhibitor) could inhibit PKC, PI3K/Akt MAPK
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phosphorylation, suggesting that activation of PTK was involved in MAPK phosphorylation. PI3K/Akt was activated in AMs of CB rats induced by LPS, which could inhibit activation of p38/JNK MAPK. LY294002 (PI3K inhibitor) could inhibit PI3K/Akt phosphorylation, and increase p-p38, p-JNK expression. Meanwhile, Calphostin C (PKC inhibitor) could inhibit PKC phosphorylation, but had no effect on p38 and JNK phosphorylation. Thus, our hypothesis, the signaling transduct pathway of MAPK, may be as follows: PTK-PI3K-Akt-JNK/p38 or PTK-PI3KPKC-ERK (As shown in Fig. 10). PI3K may have a negative regulation on the activation of downstream protein. It is well known that Ras is a small G protein, which can independently regulate many different pathways [31]. Raf is a serine/threonine (Ser/Thr) protein kinase (also known as MAPKK). When Ras binds to the N-terminal domain of Raf, Raf is activated and then activates MAPKK. MAPKK activates MAPK by phosporylating the threonine and serine residues [25]. Therefore, Ras ? MAPK pathway is a classic MAPK signaling pathway [32], which plays an important role in the control of a wide variety of cellular processes, including growth, survival and differentiation [33–35]. Our next study is to find out the relationship and interaction between PI3K ? Akt/PKC ? MAPK and Ras ? MAPK,JAK-STAT in AMs of CB rats.
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Conclusion The current study demonstrated an increased expressions of activated PTK, PI3K, PKC, ERK, JNK, p38 MAPK proteins in AMs of CB rats, suggesting that activation of MAPK signaling pathway is a crucial step in the pathogenesis of CB. These findings support the relevance of MAPK as a potential molecular target for the development of effective therapeutic strategies for CB. Acknowledgments This work was supported by the National Science Foundations of China (No. 30572355), Provincial Science Foundation of Anhui (No. 090413134), Outstanding Young Teachers Foundation in Higher Education Institution (No. 2009SQRZ049ZD).
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Studies on mitogen-activated protein kinase signaling pathway in the alveolar macrophages of chronic bronchitis rats Yan Huang • Xiao-Ming Meng • Guo-Lin Jiang Ya-Ru Yang • Juan Liu • Xiong-Wen Lv • Jun Li
•
Received: 19 July 2014 / Accepted: 29 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Lipopolysaccharide (LPS), a potent stimulator of inflammatory responses in alveolar macrophages (AMs), activates several intracellular signaling pathways, including mitogen-activated protein kinases (MAPK). In the present study, we investigated the MAPK pathway in AMs of chronic bronchitis (CB) rats. CB was induced by endotracheal instillation of LPS followed by Bacillus Calmette Guerin injection through the caudal vein 1 week later. Specific inhibitors were used and protein phosphorylations were detected by Western blot. We found that Genistein (PTK inhibitor) could inhibit protein kinase C (PKC), phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt or PKB) MAPK signaling pathway with different degrees, LY294002 (PI3K inhibitor) could not only inhibit phospho-PI3K/Akt expression, but also inhibit p38 and c-Jun NH2-terminal kinases (JNK) phosphorylation. Calphostin C (PKC inhibitor) could inhibit phospho-PKC
Y. Huang X.-M. Meng G.-L. Jiang Y.-R. Yang J. Liu X.-W. Lv J. Li (&) School of Pharmacy, Anhui Medical University, Tunxi West Road, Hefei 230032, Anhui, China e-mail: [email protected] X.-M. Meng e-mail: [email protected]
expression and exerted significant effects on extracellular signal-regulated kinases (ERK) phosphorylation, however, it had no impact on p38 and JNK phosphorylation. These results demonstrated that the LPS mediated signaling pathway of MAPK in AMs of CB rats could be described as follows: PTK-PI3K-Akt-JNK/p38 or PTK-PI3K-PKCERK, and PI3K may have a negative regulation on the activation of downstream proteins. Keywords Alveolar macrophages Chronic bronchitis Mitogen-activated protein kinase signaling pathway Abbreviations AMs Alveolar macrophages CB Chronic bronchitis LPS Lipopolysaccharide BCG Bacillus Calmette Guerin MAPK Mitogen-activated protein kinases PKC Protein kinase C PKA Protein kinase A ERK Extracellular signal-regulated kinases JNK c-Jun NH2-terminal kinases FBS Fetal bovine serum CMC Carboxymethylcellulose BALF Bronchoalveolar lavage fluid PI3K Phosphatidylinositol-3 kinase PTK Receptor tyrosine kinase
G.-L. Jiang e-mail: [email protected] Y.-R. Yang e-mail: [email protected]
Introduction
J. Liu e-mail: [email protected]
Chronic bronchitis (CB) is ventable disease [1]. Its include calcium overload, bolic abnormalities, and
X.-W. Lv e-mail: [email protected]
a frequently occurring yet prepathophysiological processes free radical production, metainflammatory reaction, etc.
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Chronic inflammation is a key feature and is tightly associated with alveolar macrophages (AMs) in CB. AMs are regarded as one of the most important sources of inflammatory cytokines and mediators, growth factors, and free oxygen radicals. It also has roles in defense, protection, regulatory effects and can interact with other cells within airways especially alveoli. These interactions result in intracellular signaling cascade activation, leading to various effects or functions that make up the inflammatory response in the lung [2–4], but the exact signaling transduction pathway remains unclear. Lipopolysaccharide (LPS) may activate many intracellular signaling pathways by binding to receptors on cell membrane, such as G protein, protein kinase C (PKC), protein kinase A (PKA), calcium signal systems, and particularly mitogen-activated protein kinases (MAPK) [5–8]. MAPK are Ser–Thr kinases comprised of three principal family members: extracellular signal-regulated kinases (ERK), p38, and c-Jun NH2-terminal kinases (JNK). Previous studies suggest that the three members of MAPK cascade are involved in the critical LPS-induced intracellular signaling transduction in macrophages [9–12]. Results from our group have revealed that AMs were significantly increased in CB rats. In disease conditions, the balance of proliferation and apoptosis was disrupted. AMs played an important role in CB through synthesis and release of cytokines (such as TNF-a, IL-8, IL-1) and inflammatory mediators (such as PGE2, LTB4 and NO) [13–15]. Since either synthesis of cytokines and inflammatory mediators, or AMs proliferation and apoptosis has a close relationship with MAPK pathway, activation of MAPK pathway in AMs may affect the pathology process of CB. The MAPK signaling pathway in AMs of CB rats induced by LPS and Bacillus Calmette Guerin (BCG) is uncertain. Therefore, in the present study, we focused on the role of MAPKs in AMs of CB rats using specific inhibitors of the MAPKs pathway to further clarify the possible molecule mechanism of CB and to develop new target drugs.
inhibitor), p38 MAP Kinase antibody;Phospho-p38 MAPK (Thr180/Tyr182) Mouse mAb; p44/42 MAP Kinase antibody;Phospho-p44/42 MAP Kinase (Thr202/Tyr204) antibody; SAPK/JNK antibody;Phospho-SAPK/JNK (Thr183/ Tyr185) Mouse mAb; Akt antibody; Phospho-Akt (Ser473) antibody;PKC-delta antibody; Phospho-PKCdelta (Tyr311) antibody; PI3 Kinase p85 antibody; Phospho-PI3K p85 (Tyr458)/p55 (Tyr199) antibody were purchased from Cell Signaling. SuperSignal West Femto Maximum Sensitivity Substrate 34094 was purchased from Pierce. Animals and chronic bronchitis model Thirty-five male SD rats weighing 180 ± 20 g were randomly divided into normal and model groups. Animals were provided by the Experimental Animal Center of Anhui Medical University. The animal experimental protocol was approved by the University Animal Care and Use Committee. Rats of model group were administered 5 mg/ kg BCG by tail vein injection, then injected LPS 200 ll (1 g/l) through trachea 1 week later [1, 13]. Bronchoalveolar lavage and cell counts Three weeks after LPS injection, the rats were anesthetized with 10 % chloral hydrate (0.3 ml/100 g i.c.). Bronchoalveolar lavage (BAL) was performed by infusing 20 ml normal saline (NS) through the tracheal cannula. BAL fluid (BALF) was pooled and centrifuged at 1,5009g for 10 min at 4 °C, then washed once with D-Hank’s balanced salt solution (HBSS). The deposition was resuspended with RPMI-1640 containing 10 % fetal bovine serum (FBS), 50 U penicillin G, and 50 lg/ml streptomycin at a density of 2 9 106 cells/ml. Cell suspenstion was then transferred to 24-well dish (1 ml per well) and incubated at 37 °C in 5 % CO2 for 2 h to allow adherence of AMs. Isolated adherent cells were stained with a rabbit anti-CD68 antibody. More than 95 % cells were CD68 positive cells, which confirmed the feasibility of the method to separate and purify AMs [13, 14]. Then, the AMs were cultured with LPS in vitro [16].
Reagents
Relationship between different concentrations of LPS and MAPK signaling pathway activation in AMs of chronic bronchitis rats
RPMI-1640, fetal bovine serum (FBS), LPS, carboxymethylcellulose (CMC), and Chelerythrine Chloride (PKC inhibitor) were purchased from Sigma. PD98059 (ERK inhibitor), SB203580 (p38 inhibitor) were purchased from Promega. Curcumin (JNK inhibitor), Genistein (PTK inhibitor), Triciribine KC-4T001 (Akt inhibitor) was purchased from Kang Chen company. LY294002 (PI3K
Rat model of CB was induced as mentioned above. After purifying, AMs were cultured with different concentration of LPS (10, 100 ng/ml, 1, 10 lg/ml) for 30 min. Then the phosphorylation level of ERK in AMs from different LPS stimulated groups was detected in order to determine the appropriate LPS concentration on MAPK signaling transduction pathway activation.
Materials and methods
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Dynamic changes of MAPK signaling pathway activation induced by LPS in AMs of CB rats
ECLTM. The relative density was analyzed by the ratio of phosphorylated and appropriate total protein.
AMs were cultured with appropriate concentration of LPS for different times as follows: 0, 15, 30, 60, 120 min, respectively, then ERK and PI3K phosphorylation were observed in order to determine the best time point for MAPK phosphorylation stimulated by LPS in the AMs.
Statistical analysis Measurement data were expressed as mean ± standard deviation (SD). Statistical analysis was done with SPSS 11.0 software to assess statistical significance between drug groups and various related control groups.
LPS induced MAPK signaling pathway activation in AMs of normal rats and CB rats Results AMs seperated from normal and model rats were cultured with or without LPS using an optimal concentration of 6 lg/ml. After 30 min (the best time point), the activation of MAPK signaling proteins was detected in AMs of each group. Cell extracts preparation Single-cell suspension of AMs from model rats was plated in the six-well plates (1.0 9 106 cells per well). After 24 h incubation, different reagents were separately added into the culture medium and cells were divided into the following groups: model (nothing added), DMSO, Genistein (30 lM), LY294002 (20 lM), Triciribine KC-4T001 (1 lM), Chelerythrine Chloride (10 lM). After 2 h incubation, cells in each well were cultured in the presence of 6 lg/ml LPS for 15 or 30 min. Then the cells were washed twice with phosphate-buffered saline (PBS) and lysed with 100 ll/106 cells of ice-cold lysis buffer (200 ll, 100 lg/ml phenylmethylsulfonyl fluoride (PMSF) for 30 min at 4 °C [17]. Cell lysates were centrifuged at 12,0009g for 10 min at 4 °C. Supernatants were stored at -80 °C until further analysis. Protein content was assessed by Lowry’s method with a commercially available kit. Western blot analysis Cell extracts (30 lg protein per lane) were separated via electrophoresis on 10 % sodium dodecyl sulfate–polyacrylamide gels, and proteins were electroblotted to nitrocellulose membrane at 200 mA for 0.5 h as described previously [18]. Blots were then blocked overnight at 4 °C with 5 % nonfat dry milk in 50 mM Tris–HCl (pH 7.5), 200 mM NaCl, Tris-buffered saline, 0.05 % Tween 20 (TBST). After three 10-min washes with TBST, membranes were incubated for 2 h with different antibodies diluted 1:1,000. After subsequent washes with TBST, HRP-IgG goat anti-rabbit secondary antibody, diluted 1:1,000, was incubated for 2 h. After washing, membranes were developed with an enhanced chemiluminescence (ECL) reagent followed by exposure to HyperfilmTM
Time and dose-dependent effect of LPS on MAPK phosphorylation in AMs of CB rats As shown in Fig. 1, ERK MAPK phosphorylation in AMs increased when the dose of LPS increased after 30 min culture and reached peak at 1 lg/ml LPS. When LPS dose increased to 10 lg/ml, ERK phosphorylation did not further increase. In order to maintain consistency with our previous paper [14, 15], we used 6 lg/ml as the maximal stimulus concentration of LPS in the following experiments. As shown in Fig. 2, AMs were stimulated by 6 lg/ml LPS for different durations, then ERK and PI3K phosphorylation in AMs were detected. The results indicated that both ERK and PI3K phosphorylation demonstrated time-dependence. After 15 min, ERK phosphorylation increased significantly, peaked at 30 min, and declined significantly after 60 min. PI3K phosphorylation peaked at 15 min, and then decreased gradually. Effect of LPS on MAPK phosphorylation in AMs of normal and CB rats Compared with the nomal group, MAPK phosphorylation increased significantly in the model group (Fig. 3). After treatment with LPS, MAPK phosphorylation in the normal and model groups both increased, and MAPK phosphorylation in the model ? LPS group increased the most. MAPK phosphorylation in the normal group without LPS had weak expression. This showed that there was a basal amount of ERK/JNK/p38 phosphorylation in AMs of normal rats. Investigation on MAPK signaling transduct pathway in AMs of CB rats Effect of MAPK signaling transduct protein inhibitors on p38 MAPK phosphorylation Compared with DMSO group, Genistein (PTK inhibitor) could inhibit p38 MAPK phosphorylation in AMs of CB
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**#
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Fig. 1 Dose-dependence of MAPK phosphorylation induced by LPS in AMs of CB rats. AMs were cultured with different concentrations of LPS (10, 100 ng/ml, 1, 10 lg/ml) for 30 min. Then, the
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Fig. 2 Time-dependence of MAPK phosphorylation induced by LPS in AMs of CB rats. AMs were cultured with 6 lg/ml LPS for different times as follows: 0,15,30,60,120 min, respectively. Then, ERK and PI3K phosphorylation were observed in order to determine the best
time point of MAPK phosphorylation stimulated by LPS in the AMs. *P \ 0.05, **P \ 0.01 versus 0 min; #P \ 0.05, ##P \ 0.01 versus 15 min (A) or 30 min (B)
rats, while LY294002 (PI3K inhibitor) and Triciribine (Akt inhibitor) could further increase p38 MAPK phosphorylation (P \ 0.01). In contrast, Calphostin C (PKC inhibitor) had no such effect on p38 MAPK phosphorylation (Fig. 4).
Effect of MAPK inhibitors on JNK MAPK phosphorylation
Effect of MAPK inhibitors on ERK MAPK phosphorylation Compared with DMSO group, Genistein (PTK inhibitor) and Calphostin C (PKC inhibitor) could inhibit ERK MAPK phosphorylation inordinately in AMs of CB rats (P \ 0.05). In contrast, LY294002 (PI3K inhibitor) and Triciribine (Akt inhibitor) had no such effect on ERK MAPK phosphorylation (Fig. 5).
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Compared with DMSO group, Genistein (PTK inhibitor) could inhibit JNK MAPK phosphorylation in AMs of CB rats. In contrast, LY294002 (PI3K inhibitor) and Triciribine (Akt inhibitor) could further increase JNK MAPK phosphorylation (P \ 0.01). There is no difference of JNK MAPK phosphorylation in model group and Calphostin C (PKC inhibitor) group (Fig. 6). Effect of MAPK inhibitors on PKC MAPK phosphorylation Compared with DMSO group, both Genistein (PTK inhibitor) and Calphostin C (PKC inhibitor) could significantly
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Fig. 3 Effect of LPS on MAPK phosphorylation in AMs from normal and CB rats. AMs separated from normal and model rats were cultured with or without LPS using an optimal concentration of 6 lg/
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inhibit PKC MAPK phosphorylation in AMs of CB rats (P \ 0.01). LY294002 (PI3K inhibitor) could slightly increase PKC MAPK phosphorylation (P \ 0.05) (Fig. 7).
rats, while Triciribine and Calphostin C had no effect on PI3K phosphorylation (P \ 0.01) (Fig. 8). Effect of MAPK inhibitors on Akt MAPK phosphorylation
Effect of MAPK inhibitors on PI3K MAPK phosphorylation Compared with DMSO group, both Genistein (PTK inhibitor) and LY294002 (PI3K inhibitor) could significantly inhibit PI3K MAPK phosphorylation in AMs of CB
As shown in Fig. 9, Genistein, as well as LY294002 and Triciribine could inhibit Akt MAPK phosphorylation in AMs of CB rats with different degree (P \ 0.01). Calphostin C had no such effect.
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Fig. 5 Effect of MAPK inhibitors on ERK MAPK phosphorylation. 1 model; 2 DMSO; 3 genistein (PTK inhibitor); 4 LY294002 (PI3K inhibitor); 5 triciribine (Akt inhibitor); 6 Calphostin C (PKC inhibitor). *P \ 0.05 versus DMSO group
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Fig. 6 Effect of MAPK inhibitors on JNK MAPK phosphorylation. 1 model; 2 DMSO; 3 genistein (PTK inhibitor); 4 LY294002 (PI3K inhibitor); 5 triciribine (Akt inhibitor); 6 Calphostin C (PKC inhibitor). **P \ 0.01 versus DMSO group
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Fig. 8 Effect of MAPK inhibitors on PI3K MAPK phosphorylation. 1 model; 2 DMSO; 3 genistein (PTK inhibitor); 4 LY294002 (PI3K inhibitor); 5 triciribine (Akt inhibitor); 6 Calphostin C (PKC inhibitor). **P \ 0.01 versus DMSO group
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Discussion The present study has examined for the first time the effect of different signaling transduct protein inhibitors of MAPK on PTK, PI3K, PKC, ERK, JNK, p38 MAPK phosphorylation in AMs of CB rats in order to investigate the possible MAPK signaling transduct pathway in AMs of CB rats. Many cellular responses, including cell-cycle progression, differentiation, and apoptosis are regulated by MAPK signaling transduct pathway [19]. It has been reported that members of MAPK family including ERK, JNK, p38 are involved in inflammatory mediator production in response to a wide variety of microbial stimuli [20, 21]. A number of laboratories have found that MAPK signaling transduct pathway is involved in macrophage activation by LPS exposure [22, 23]. Activated MAPK signaling transduct pathway proteins will then phosphorylate downstream effectors, such as membrane proteins, cytoplasmic proteins, and nuclear proteins [24, 25]. Small molecule kinases inhibitors of the MAPK pathway have been developed to treat various diseases, including inflammatory diseases [26], ischemic heart disease [27], and cancer [28, 29]. Phosphatidylinositol-3-kinase (PI3K) is a lipid kinase comprised of P85 and P110 subunits. It is phosphorylated and then activated when growth factors bind to their receptor tyrosine kinase (RTK) or G protein-coupled receptors (GPCR). Activated PI3K converts phosphatidylinositol (4,5)-phosphate [PI(4,5)P2] into phosphatidylinositol (3,4,5)-phosphate[PI(3,4,5)P3], which results in membrane localization of phosphotidylinositol-dependent kinase-1 (PDK1) via its pleckstrin homology (PH) domain. As a downstream kinase of PI3K, Akt can mediate PI3Kinitiated signaling. Akt translocates to the cell membrane and interacts with PI(3,4,5)P3 via its PH domain, being phosphorylated by phosphoinositide-dependent kinase (PDK) 1, PDK2, integrin-linked kinase (ILK) and unidentified kinase, respectively [30].
LPS+CD14
+ PTK
+ PI3K
+
-
Akt
PKC
JNK MAPK
+ p38 MAPK
C-Jun, NF-
TNF-
ERK MAPK
B, ATF-2,ELK (transcription factor)
iNOS
HO-1
PGE2
TGF-
Fig. 10 Mitogen-Activated protein kinase signaling pathway in the alveolar macrophages of chronic bronchitis rats
In the present study, we found that PI3K, Akt, PKC phosphorylation reached its peak after 15 min treatment of LPS, then slowly declined, whereas p-ERK, p-p38, and p-JNK reached peak after 30 min treatment of LPS. Compared with normal group, MAPK phosphorylation increased significantly in the model group. After treatment of LPS, MAPK phosphorylation in the normal and model group both increased. MAPK phosphorylation in the LPS induced model group was the most significant. Genistein (PTK inhibitor) could inhibit PKC, PI3K/Akt MAPK
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phosphorylation, suggesting that activation of PTK was involved in MAPK phosphorylation. PI3K/Akt was activated in AMs of CB rats induced by LPS, which could inhibit activation of p38/JNK MAPK. LY294002 (PI3K inhibitor) could inhibit PI3K/Akt phosphorylation, and increase p-p38, p-JNK expression. Meanwhile, Calphostin C (PKC inhibitor) could inhibit PKC phosphorylation, but had no effect on p38 and JNK phosphorylation. Thus, our hypothesis, the signaling transduct pathway of MAPK, may be as follows: PTK-PI3K-Akt-JNK/p38 or PTK-PI3KPKC-ERK (As shown in Fig. 10). PI3K may have a negative regulation on the activation of downstream protein. It is well known that Ras is a small G protein, which can independently regulate many different pathways [31]. Raf is a serine/threonine (Ser/Thr) protein kinase (also known as MAPKK). When Ras binds to the N-terminal domain of Raf, Raf is activated and then activates MAPKK. MAPKK activates MAPK by phosporylating the threonine and serine residues [25]. Therefore, Ras ? MAPK pathway is a classic MAPK signaling pathway [32], which plays an important role in the control of a wide variety of cellular processes, including growth, survival and differentiation [33–35]. Our next study is to find out the relationship and interaction between PI3K ? Akt/PKC ? MAPK and Ras ? MAPK,JAK-STAT in AMs of CB rats.
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Conclusion The current study demonstrated an increased expressions of activated PTK, PI3K, PKC, ERK, JNK, p38 MAPK proteins in AMs of CB rats, suggesting that activation of MAPK signaling pathway is a crucial step in the pathogenesis of CB. These findings support the relevance of MAPK as a potential molecular target for the development of effective therapeutic strategies for CB. Acknowledgments This work was supported by the National Science Foundations of China (No. 30572355), Provincial Science Foundation of Anhui (No. 090413134), Outstanding Young Teachers Foundation in Higher Education Institution (No. 2009SQRZ049ZD).
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