Articles in PresS. Am J Physiol Cell Physiol (June 11, 2008). doi:10.1152/ajpcell.00085.2008
Mechanical stress induces tumor necrosis factor-alpha production through Ca2+ releasedependent TLR2 signaling
Han Geun Kim1, 2 §, Joo Yun Kim1, 3 §, Min Geun Gim1, Jung Min Lee1, 2, and Dae Kyun Chung1, 2
1
Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung
Hee University, Yongin 449-701, Korea. 2
3
Skin Biotechnology Center, Kyung Hee University, Yongin 449-701, Korea. Division of Cardiology, Samsung Medical Center & Samsung Biomedical Research
Institute, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea.
Running head: TLR2 signaling via Ca2+ release in mechanical stress
Address for reprint requests and other correspondence: D. K. Chung, Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 449-701, Korea. Tel.: +82-31-201-2465; Fax: +82-31-202-8333. (e-mail address:
[email protected])
§
H.G.K and J.Y.K contributed equally to this work.
Copyright © 2008 by the American Physiological Society.
ABSTRACT
We studied centrifugation-mediated mechanical stress induced Tumor necrosis factor (TNF)-α production in the monocyte-like cell line THP-1. The induction of TNF-α by mechanical stress was dependent on the centrifugation speed and produced the highest level of TNF-α after 1 h of stimulation. TNF-α production returned to normal levels after 24 h of stimulation. Mechanical stress also induced Toll-like Receptor (TLR) 2 mRNA in proportion to the expression of TNF-α. The inhibition of TLR2 signaling by dominant negative myeloid differentiation factor 88 (MyD88) blocked TNF-α expression response to mechanical stress. After transient over-expression of TLR2 in HEK293 cells, mechanical stress induced TNF-α mRNA production. Interestingly, mechanical stress activated the c-Src-dependent TLR2 phosphorylation, which is necessary to induce Ca2+ fluxes. When THP-1 cells were pretreated with BAPTA-AM, thapsigargin, and NiCl2·6H2O followed by mechanical stimulation, both TLR2 and TNF-α production were inhibited, indicating that centrifugation-mediated mechanical stress induces both TLR2 and TNF-α production through Ca2+ releases from intracellular Ca2+ stores following TLR2 phosphorylation. In addition, TNF-α treatment in THP-1 cells induced TLR2 production in response to mechanical stress, whereas the
pre-incubation of anti-TNF-α antibody scarcely induced the mechanical stress-mediated production of TLR2, indicating that TNF-α produced by mechanically simtulated-THP1 cells affected TLR2 production. We concluded that TNF-α production induced by centrifugation–mediated mechanical stress is dependent on MyD88-dependent TLR2 signaling that is associated with Ca2+ release and that TNF-α production induced by mechanical stress affects TLR2 production.
Key words: Mechanical stress, Centrifugation, Toll-like receptor, Tumor necrosis factoralpha, Ca2+ release.
1
INTRODUCTION
Mechanical stress induces the production of growth-related proto-oncogene such as cfos and c-myc in osteoblasts (20). c-fos and c-myc are considered significant genes because these genes play an important role in promoter regions of several genes related to the growth and mineralization of bone, including osteocalcin, alkaline phosphatase, and collagen type 1 (9, 10). Vortex- or shear stress-mediated mechanical stress induces cell adhesion of THP-1 cells. In addition, THP-1 cells, stimulated by platelet-derived microparticles produced by high shear stress, induce cytokine expression such as TNF-α and IL-1β. These alterations in monocytes can induce the progression of atherosclerosis on the vessel wall (11, 17, 23). Atherosclerosis is regarded as a chronic inflammatory disease of the vessel wall and is characterized by the accumulation of lipid-laden macrophages and foam cells in the large arteries (15, 21). In general, monocytes respond to endogenous or exogenous ligands through pattern-recognition receptors (PRRs), leading to the expression of inflammatory cytokines such as TNF-α, IL-6, fibrinogen, and soluble vascular cell adhesion molecule 1. These molecules have welldescribed functions in inflammation, which have been associated with atherosclerotic progression (19, 22). Furthermore, TNF-α is a major mediator in the induction of the
2
acute phase response (13) and affects the leukocyte-endothelial cell adhesion (2).
Recently, research has focused on the mechanisms by which cytokine and chemokine expressions, as well as biofunctional changes, are induced by mechanical stress. The early cellular response to mechanical stress is the influx of Ca2+, which leads to increased cytosolic Ca2+, and then induces changes in the intracellular activation of numerous molecules and NO production (11). Although more studies on the mechanical stress-mediated signaling pathway are necessary, mechanical stress may induce cytokine expression through NF-κB activation (25). In addition, mechanical stress induces Tolllike receptor (TLR) expression as well as cytokine expression. Liang F et al reported that TLR4 mRNA was up-regulated after shear stress (8). Previous studies have shown that TLR signaling has an important function in the link between atherosclerosis and defense against both foreign pathogens and endogenously generated inflammatory ligands (6, 16). These results suggest that TLR may be linked to mechanical stressmediated cytokine production and thus may induce inflammation, atherosclerosis, and arthritis.
In blood vessels, monocytes may be affected by blood flow as well as the size, shape,
3
branching, and partial obstructions of the vessel. However, there is little evidence that monocytes induce the inflammatory cytokines in response to the mechanical stress caused by such an environment. The regulatory mechanism of pro-inflammatory cytokine expression by mechanical stress-mediated TLR signaling is also insufficient. In this study, therefore, we examined the expression pattern of cytokines, such as TNF-α, that are induced in THP-1 monocyte-like cells by mechanical stress. We also studied that TLR signaling and Ca2+ release are required for mechanical stress-mediated TNF-α production.
4
MATERIALS AND METHODS
Cell culture and cell stimulation experiments. The human monocyte-like cell line, THP-1, and human embryonic kidney (HEK) 293 cell lines were obtained from Korean Cell Line Bank (KCLB, Korea) and maintained in RPMI 1640 and MEM, respectively. All media were supplemented with 10% FBS, antibiotics (100 U penicillin and 100 μg/ml streptomycin) in a 37℃ incubator with 5% CO2. A 5804R centrifuge (Eppendorf, Germany) with a microtiter plate rotor was used to apply mechanical stress upon the THP-1 and HEK293 cells. The average shear stress on the cell surface during centrifugation was calculated by Dr. Michael R. King (University of Rochester, private communication) according to the following formulation (14). Tau = 0.75 × mu × U/a. Where mu is the buffer viscosity, U is the sedimentation velocity of the cell, and a is the cell radius. For a cell that is approximately the size of a neutrophil (a=4 micons), then a centrifugation speed of 280 xg corresponds to an average shear stress on the cell surface of 2.06 dyn/cm2. In this regime of flow, the shear stress will scale linearly with the centrifugation speed. In this study, the centrifugation speed of 10 xg, 180 xg, and 461 xg corresponds to an average shear stress on the cell surface of 0.074 dyn/cm2, 1.3 dyn/cm2, and 3.4 dyn/cm2, respectively.
5
RNA preparation and Real-time PCR. Total RNA was extracted from THP-1 or HEK293 cells by using the guanidium thiocyanate-acid phenol-chloroform method. cDNA was synthesized with the Improm-IITM reverse transcription system (Promega corporation, USA) according to the manufacturer’s instructions. To quantify the TNF-α and TLR2 mRNA, real-time PCR amplification was carried out with the ABI prism 7000 sequence detection system (Applied BioSystems, USA) and the PCR products were detected with SYBR Green. The following primers were used: TNF-α forward, 5′CTCTTCTGGCTCAAAAAGAGAATT-3′;
TNF-α
reverse,
5′-
AGGCCCCAGTTTGAATTCTT-3′;
TLR2
forward,
5′-
ACCTAGGGGAAACATCTCT-3′;
TLR2
reverse,
5′-
AGCTCTGTAGATCTGAAGCATC-3′.
Establishment of dominant-negative MyD88, and transient transfection. dnMyD88 was manufactured using the method described by Wang et al with minor modifications (27). dnMyD88 (amino acids 156-296) that contains the toll/interleukin-1 receptor domain was amplified from total RNA extracted from THP-1 cells by reverse transcription polymerase chain reaction (RT-PCR) and cloned into pCMV-Tag 2A
6
(Stratagene, USA). THP-1 and HEK293 cells were transfected with the expression vector using the WelFec-QTM transfection reagent (JBI, Korea) according to the manufacturer’s instructions. Thirty-six hours after transfection, the cells were used for further analysis. The PCR primers used for RT-PCR are as follows: dnMyD88 forward, 5′-
GCTAGCATGCCTGAGCGTTTCG-3′;
dnMyD88
reverse,
5′-
GGATCCTCAGGGCAGGGACAA-3′.
NF-κB luciferase reporter assay. 5 X 105 THP-1 cells /well were seeded onto 12-well plates. After 24 h, the cells were transiently co-transfected with the pNF-κB-Luc and the pRL-SV40 vectors. Thirty-six hours after transfection, the cells were stimulated by 180 xg of centrifugation for 5 min. Afterwards, the cells were incubated for another 18 h. Total cell lysates were prepared and the luciferase activity of these lysates were measured using a reporter assay system (Promega). The Renilla luciferase reporter gene (10 ng/well) was used as an internal control.
Immunofluorescence staining. After stimulation, THP-1 cells were fixed with 4% paraformaldehyde. Cells were permeabilized with 0.5% Triton X-100, blocked with phosphate-buffered saline containing 1% bovine serum albumin, and incubated with
7
polyclonal anti-TLR2 (Santa Cruz Biotechnology, USA) or anti-TNF-α (Sigma) antibody in blocking buffer at room temperature. Donkey anti-mouse IgG-FITC (Santa Cruz Biotechnology) in blocking buffer was added as a secondary antibody. Fluorescence was examined using a confocal microscope.
Statistical analysis. All experiments were performed at least three times. The data shown are representative results of the mean ± the standard deviation of triplicate cultures. A paired t-test was used to determine the significance of the data * p < 0.05; ** p < 0.01; *** p < 0.001.
8
RESULTS
TLR2 mRNA was increased in response to mechanical stress. To date, 10 members of the TLR family have been identified in humans (26). Different types of ligands induce distinct types of immune responses based on the activation of immune cell subsets that express corresponding TLR profiles. In our experiment, THP-1 cells that were stimulated by mechanical stress induced TLR2 and TLR4 mRNA. In particular, TLR2 mRNA was significantly induced (data not shown). Therefore, we investigated the effect of TLR2 signaling on TNF-α expression after mechanical stress.
Mechanical stress induced TNF-α and TLR2 production from THP-1 cells. To examine the effect of mechanical stress on the cultured cells, we studied centrifugationmediated TNF-α and TLR2 production. Both TLR2 and TNF-α mRNA production peaked at 1 h after mechanical stimulation and then decreased slowly (Fig. 1A). The TLR2 and TNF-α mRNA levels recovered to their normal levels after 24 h. The levels of TLR2 mRNA were confirmed by measuring the levels of TLR2 protein by western blotting (middle panel). RT-PCR of TNF-α expression using
32
P-labeled dCTP showed
clearly that the production of TNF-α mRNA peaked 1 h after the cells were stimulated
9
by a 180 xg centrifugation for 5 min (lower panel). To examine the effect of mechanical stress on TNF-α and TLR2 expression, THP-1 cells were exposed to various ranges of centrifugation speeds. Previous study demonstrated that 3 xg centrifugation altered c-fos and osteoscalcin gene expression in MC3T3-E1 osteoblasts (9). Because 3 xg centrifugation force was not available in our experimental system, we used 10 xg force as the lowest mechanical stress source. We also used 180 xg of centrifugal force (1,000 rpm) in our routine cell transfer experiments. To define our observation of the change in gene expression, we used 461 xg (1,800 rpm) of centrifugal force in this experiment. The centrifugation speed of 10 xg, 180 xg, and 461 xg corresponds to an average shear stress on the cell surface of 0.074 dyn/cm2, 1.3 dyn/cm2, and 3.4 dyn/cm2, respectively. The productions of TNF-α and TLR2 mRNA were increased in a speed-dependent manner (Fig. 1B). In addition, we used western blotting to examine the expression of TLR2 (middle panel) and 32P-labeled RT-PCR for the detection of TNF-α (lower panel). These alternative experimental methods showed results similar to those obtain by using real-time PCR. To confirm the TNF-α and TLR2 expression induced by mechanical stress, immunofluorescence staining was performed with antibodies for TNF-α and TLR2.
10
Confocal microscopy showed that both TLR2 (Fig. 1C) and TNF-α (Fig. 1D) were induced in a speed-dependent manner. These results suggested that mechanical stress up-regulated TLR2 expression and that TNF-α production was proportional to TLR2 expression.
Effect of polymyxin B treatment on mechanical stress-induced TNF-α production. THP-1 cells are very sensitive to media containing endotoxin. Therefore, we performed certification experiments with polymyxin B to verify that the mechanical stressmediated gene expression was not due to endotoxin. THP-1 cells showed a significant elevation in TNF-α production compared with that of untreated cells when challenged with a known concentration of endotoxin. The endotoxin-induced TNF-α showed a 35% and 90% decrease after a pretreatment with 5 μg/ml and 50 μg/ml of polymyxin B, respectively. However, the TNF-α production induced by mechanical stress was not diminished in these experiments, demonstrating that the media did not contain endotoxin (Fig. 2).
TNF-α expression in dnMyD88 and TLR2 transfectants after exposure to mechanical stress. We investigated the role of TLR2 on mechanical stress-induced TNF-α
11
production. Figure 3A shows the expression of dnMyD88 truncated protein in the pCMV/dnMyD88 transfectants (upper panel). After exposure to 180 xg of centrifugation for 5 min, the production of TNF-α mRNA was significantly induced in pCMV-Tag2A transfected cells. However, it was not induced in the mechanical stressstimulated
pCMV/dnMyD88
transfected
cells
compared
with
unstimulated
pCMV/dnMyD88 transfected cells (Fig. 3A, lower panel). To investigate the role of TLR2 alone in mechanical stress-mediated TNF-α production, HEK 293 cells were transfected with a TLR2 expressing vector (pCMV/TLR2) or pCMV-Tag2A, and TLR2 expression was confirmed by western blotting (Fig. 3B, upper panel). Figure 3B (lower panel) shows that the production of TNF-α was significantly induced in the pCMV/TLR2 transfected cells after stimulation by 180 xg of centrifugation for 5 min, whereas the induction of TNF-α production was modestly induced in the pCMV-Tag2A transfected cells, indicating that TLR2 was involved in the signal transduction events that are triggered by mechanical stress. As shown in Figure 3C, immunofluorescence staining with the anti-TNF-α antibody in THP-1 cells showed that mechanical stress-mediated TNF-α production was not increased in the pCMV/dnMyD88 transfected cells, whereas TNF-α was increased in the pCMV-Tag2A transfected cells. Interestingly, pCMV/TLR2 transfectants had a
12
higher level of TNF-α production. These results suggest that TLR2-MyD88 signaling has an important role in the mechanical stress-mediated production of TNF-α.
Mechanical stress induced TLR2 tyrosine phosphorylation. Recent studies have reported that in response to bacterial ligands, Src family kinases initiate TLR2associated signaling, which in turn, is followed by recruitment of PI3K and phospholipase Cγ (pLCγ). These events affect the release of Ca2+ from intracellular stores, which is necessary for the downstream activation of proinflammatory gene transcription (5). Therefore in our present study, we examined TLR2 tyrosine phosphorylation after mechanical stress. As shown in Figure 4, tyrosine phosphorylation of TLR2 was induced by 5 min after stimulation with 180 xg of centrifugation for 5 min. This phosphorylation event requires c-Src, which was previously shown to be associated with TLR2 phosphorylation (5). Furthermore, when THP-1 cells were treated with the c-Src inhibitor PP1, TLR2 tyrosine phosphorylation by mechanical stress was decreased.
Mechanical stress induced NF-κB activation in THP-1 cells. In general, TLR2 signaling induces NF-κB translocation into the nucleus, resulting in cytokine expression.
13
In our present study, we examined whether mechanical stress-mediated TLR2 signaling induces NF-κB activation. IκB-α and IκB-β degradation from mechanically stimulatedTHP-1 cells were examined by western blotting with anti-IκB-α and anti-IκB-β antibodies. Mechanical stress caused a rapid degradation of IκB-α within 10 min, whereas IκB-β was cleaved more weakly after 10 min (Fig. 5A). Moreover, the degradation of both IκB-α and IκB-β was restored after 40 min. In another study, the relative luciferase activity was clearly increased in pCMV-Tag2A transfected cells after they had been stimulated by 180 xg centrifugation for 5 min. However, it was completely inhibited in the pCMV/dnMyD88 transfected cells after their stimulation with 180 xg centrifugation for 5 min (Fig. 5B). These results indicated that mechanical stress induced NF-κB activation and therefore might result in TNF-α production.
Mechanical stress-induced TNF-α and TLR2 production were induced by IP3sensitive Ca2+ release. A previous study described that vortex-mediated mechanical stress-induced IP3-dependent Ca2+ release from intracellular Ca2+ stores (17). Therefore, we also examined the potential role of Ca2+ in TNF-α production that has been induced by centrifugation-mediated mechanical stress. Pretreatment of the cells with BAPTAAM, an intracellular Ca2+ chelator, inhibited centrifugation-mediated TNF-α production
14
(Fig. 6A), indicating that intracellular Ca2+ is necessary for TNF-α production. We next pretreated the cells with NiCl2·6H2O, a nonspecific Ca2+ influx inhibitor. NiCl2·6H2O, however, did not affect centrifugation-mediated TNF-α production (Fig. 6B), indicating that this type of TNF-α production does not depend on Ca2+ influx from outside the cells. To confirm the mechanism of Ca2+ release from intracellular Ca2+ stores, we pretreated the cells with thapsigargin (THG), an inhibitor of Ca2+-ATPase that inhibits IP3dependent Ca2+ release from intracellular stores (17). Pretreatment of the cells with THG with or without NiCl2·6H2O, which was added to block Ca2+ influx because THG itself induces sustained elevation of intracellular calcium mediated by Ca2+ influx (12), inhibited centrifugation-mediated TNF-α production (Fig. 6B). These results suggest that IP3-dependent Ca2+ release from intracellular Ca2+ stores plays a key role in centrifugation-mediated TNF-α production, which is similar with the finding that was published on vortex-mediated cell adhesion to fibronectin (17). To determine the regulation of TLR2 expression in THP-1 cells stimulated by mechanical stress, we blocked Ca2+ release with Ca2+ inhibitors and inactivated TNF-α activity with anti-TNF-α antibody. When THP-1 cells were pretreated with BATA-AM, the TLR2 mRNA expression was not induced by centrifugation-mediated mechanical stress (Fig. 6C). The treatment of THG with or without NiCl2·6H2O also did not induce
15
TLR2 mRNA expression (Fig. 6D). These results suggest that the expression of TLR2 by centrifugation-mediated mechanical stress was regulated by IP3-dependent Ca2+ release from intracellular Ca2+ stores. We next examined whether TNF-α produced from mechanically stimulated THP-1 cells affects TLR2 expression. When THP-1 cells were treated with recombinant human TNF-α, the expression of TLR2 mRNA was higher than that of un-treated. After mechanical stimulation, the TLR2 expression was more highly induced than that of mechanically unstimulated THP-1 cells. On the other hand, pretreatment with TNF-α antibody did not induce the TLR2 mRNA expression by THP-1 cells in response to mechanical stress (Fig. 6E). These data indicate that TNF-α secreted from mechanically stimulated cells affect up-regulation of TLR2 mRNA expression.
16
DISCUSSION
Adhesion and migration molecules such as VCAM-1, MCP-1, and ICAM-1 regulate the interaction between the endothelium and monocytes, and these interactions result in the atherogenic process. The expression of these molecules on the endothelial surface and in the vessel wall cause increased monocyte adhesion and migration (18). A previous report showed that the expression of adhesion molecules was regulated by TNF-α (4). Our experiments provide evidence that centrifugation-mediated mechanical stress can induce TNF-α production in THP-1 cells, processes that occur during the progression of atherosclerosis. Furthermore, TNF-α production induces the expression of many endothelial genes that contribute to the complex processes involved in atherogenesis (19, 21).
Atherosclerosis is a pathological process that affects blood vessels and leads to the development of cardiovascular disease. In addition, the immune system is involved in atherogenesis as well as the pathogenesis of atherosclerosis. Cells within atherosclerotic plaques secrete cytokines, including IL-1, IL-2, IL-6, IL-8, IL-12, IL-10, TNF-α, IFN-γ, and platelet-derived growth factor (22). Mechanical stress, such as shear stress, causes a
17
frictional force that the flow of blood exerts at the endothelial surface of the vessel wall and results in the progression of atherosclerosis (24). Shear stress is defined as the viscous drag of blood over the endothelium. Both animal and human models have shown that atherosclerotic lesions occur predominantly at specific arterial regions with low or disturbed flow, where endothelial shear stress (ESS) occurs and where the atheroprotective genes are suppressed, whereas the pro-atherogenic genes are upregulated, thereby promoting the atherosclerotic process (3). These results suggest that mechanical stress plays an important role in the atherosclerotic process.
The response of cells to mechanical stress is similar to the response of ligandmediated TLR signaling in the gene expression of cytokines, chemokines, growth factors, and transcription factors, suggesting an interrelation between mechanical stress and TLR. Recently, Liang F et al showed that overexpression of TLR4 could be induced by mechanical stress (8). In our present study, TLR2 was significantly induced by mechanical stress, while other TLRs were only moderately induced. Therefore, we examined whether TLR2 signaling participated in mechanical stress-mediated TNF-α production. When THP-1 cells were transfected with dnMyD88, the expression of TNFα after the exposure to mechanical stress was not induced, while TLR2 transfected cells
18
after their exposure to mechanical stress had higher TNF-α production compared with that of un-stimulated TLR2-transfected cells. These results indicate that mechanical stress-mediated TNF-α expression was induced through MyD88-dependent TLR2 signaling. Using a genetic loss of function approach, a recent study showed that MyD88 has an important role in the development of atherosclerosis in murine models of atherogenesis. This study showed that a deficiency in MyD88 led to a significant reduction in plaque size, lipid content, and expression of proinflammatory genes (16). Another study has suggested that laminar flow induced SP1 serine phosphorylation and thereby blocked SP1 binding to the TLR2 promoter, which is required for TLR2 expression (7). This regulatory mechanism of TLR2 production may contribute to an atheroprotective role in atherosclerotic lesion formation. Therefore, our study, which shows that TLR2 and MyD88 signaling mediated mechanical stress-mediated TNF-α production, may provide further insight into the mechanisms of mechanical stress in atherosclerotic formation.
Signal transduction through TLR2 is initiated by c-Src-dependent phosphorylation of TLR2, which is necessary to induce the association of PI3K and TLR2 signaling. The generation of phosphatidylinositol (3,4,5) trisphosphate (PI(3,4,5)P3) by PI3K activates
19
PLC-γ and is involved in intracellular Ca2+ release by generating inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) (1). PLCγ activity is regulated by plasma membrane localization and tyrosine phosphorylation. Furthermore, PLCγ activity is associated with TLR2 in response to the TLR2 agonist (5). In the present study, we showed that centrifugation-mediated mechanical stress induced TNF-α and TLR2 production through IP3-dependent Ca2+ release from intracellular Ca2+ stores. These results were confirmed by the pretreatment of cells with Ca2+ inhibitors. Moreover, the TNF-α produced from the mechanically stimulated-THP-1 cells affected TLR2 mRNA production. In addition, TLR2 signal transduction by mechanical stress induced TNF-α production through a MyD88-dependent pathway that included NF-κB activation. These results suggest that mechanical stress on THP-1 cells leads to mutual action between TLR2 and TNF-α, which may occur via intracellular Ca2+ release and a MyD88dependent NF-κB signal pathway.
In conclusion, we showed that centrifugation-mediated mechanical stress upregulated TNF-α and TLR2 production in THP-1 cells and that c-Src dependent TLR2 phosphorylation followed by MyD88-dependent NF-κB activation and Ca2+ releasedependent TNF-α production were involved in this mechanism. These findings might
20
elucidate another aspect of the signaling of mechanical stress-mediated atherosclerosis.
ACKNOWLEDGMENTS We thank Michael R. King for calculation of the amount of shear stress on the cell surface during centrifugation.
GRANTS This work was supported by a grant of Regional Innovation Center funded by Ministry of Commerce, Industry and Energy, Republic Korea (RIC07-06-04).
DISCLOSURES No conflicts declared.
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Figure legends
Fig. 1. The induction of TNF-α and TLR2 by mechanical stress. THP-1 cells were cultured for the indicated times after a 180 xg centrifugation for 5 min (A) or THP-1 cells were exposed to the indicated centrifugation forces for 5 min. Following treatment, cells were incubated for 1 h (B). The amount of TNF-α and TLR2 were measured by real-time PCR (upper panel), western blotting with TLR2 mouse monoclonal IgG2a (middle panel), and reverse transcriptase (RT) PCR with
32
P labeled
dCTP (lower panel). Data represent the mean ± standard deviation. *, p ＜ 0.05; **, p ＜ 0.01 versus 0 h or 0 xg (A). The expressions of intracellular TNF-α (C) and surface TLR2 (D) were examined by an immunofluorescence staining.
Fig. 2. Effect of polymyxin B on THP-1 cells stimulated by mechanical stress. THP-1 cells were incubated with LPS (0.1 μg/ml) or exposed to a 180 xg centrifugation for 5 min and then incubated for 1 h in the presence or absence of polymyxin B (5 or 50 μg/ml). TNF-α production was measured by real-time PCR. Data represent the mean ± standard deviation. *, p ＜ 0.05; **, p ＜ 0.01 versus polymyxin B-free sample.
27
Fig. 3. TNF-α production induced by mechanical stress was dependent upon TLR2 and MyD88. (A) Expression of dnMyD88 was confirmed by western blotting (upper panel), and pCMV/dnMyD88 or pCMV-Tag2A transfectants were incubated for 1 h after stimulation with or without 180 xg centrifugation for 5 min. The total RNA was extracted from the cells and then subjected to real-time PCR (lower panel). (B) Expression of TLR2 from HEK293 cells was examined by western blotting (upper panel), and TNF-α mRNA production was measured from transfectants using real-time PCR after mechanical stimulation with or without 180 xg centrifugation (lower panel). Data represent the mean ± standard deviation. *, p ＜ 0.05; **, p ＜ 0.01; ***, p ＜ 0.001 versus un-stimulated cells. (C) Transfectants containing pCMV-Tag2A, pCMV/dnMyD88, and pCMV/TLR2 were stimulated with or without 180 xg centrifugation for 5 min and then incubated for 1 h. Mechanical stress-mediated TNF-α production was examined by immunofluorescence staining.
Fig. 4. Mechanical stress induced c-Src-dependent TLR2 phosphorylation. (A ) THP-1 cells were stimulated by a 180 xg centrifugation for 5 min with or without 50 withoutugation incubated for the indicated times. The cell lysates were blotted with
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specific antibodies against TLR2 (Tl2.1), and phosphotyrosine (clone 4G10). (B) Amount of tyrosine phosphorylation of TLR2 was quantified using an image J software (NIH, Bethesda, USA). Data are expressed as the mean ± standard deviation of three independent experiments and normalized for un-phosphorylated TLR2 proteins. Data from w/ PP1 was significantly lower (p < 0.01) than w/o PP1.
Fig. 5. NF-κB activity in the THP-1 cells is stimulated by mechanical stress. (A) THP-1 cells were incubated for the indicated times after 180 xg centrifugation. The separated cell lysates were immunoblotted with phospho-specific antibodies against IκB-α and IκB-β. To verify the amount of loaded protein, the cell lysates were also probed with anti-β-actin antibody. (B) THP-1 cells were transiently co-transfected with the pNF-κB-luc and pRL-SV40 control vectors and either pCMV-Tag2A or pCMV/dnMyD88. Twenty-four hours after transfection, the cells were stimulated with or without 180 xg centrifugation for 5 min and then incubated for 18 h. Cells were lysed, and the lysates were measured for the activity of firefly and Renilla luciferase. Data are expressed as the mean ± standard deviation of three experiments performed in triplicate and normalized for Renilla luciferase activity.
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Fig. 6. TNF-α and TLR2 production by centrifugation-mediated mechanical stress depend on IP3-sensitive Ca2+ release from intracellular stores. THP-1 cells were pre-incubated with 50 μM BAPTA-AM for 1 h (A and C). THP-1 cells were pre-incubated with 1 mM NiCl2·6H2O for 1 h followed by treatment with or without 1 μM THG for 3 h (B and D). THP-1 cells were pre-treated with or without either human recombinant TNF-α or anti-TNF-α antibody for 1 h (E). After incubation, cells were stimulated with or without 180 xg centrifugation for 5 min. TNF-α mRNA (A and B) and TLR2 mRNA (C, D and E) production were measured by real-time PCR, and the data represent the mean ± standard deviation. *, p ＜ 0.05.
A
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