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Am J Physiol Cell Physiol 295: C432-C439, 2008. First published June 11, 2008; doi:10.1152/ajpcell.00085.2008
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RECEPTORS AND SIGNAL TRANSDUCTION

Mechanical stress induces tumor necrosis factor-{alpha} production through Ca2+ release-dependent TLR2 signaling

Han Geun Kim,1,2,* Joo Yun Kim,1,3,* Min Geun Gim,1 Jung Min Lee,1,2 and Dae Kyun Chung1,2

1Graduate School of Biotechnology and Institute of Life Science and Resources and 2Skin Biotechnology Center, Kyung Hee University, Yongin; and 3Division of Cardiology, Samsung Medical Center and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea

Submitted 14 February 2008 ; accepted in final form 4 June 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We studied centrifugation-mediated mechanical stress-induced tumor necrosis factor-{alpha} (TNF-{alpha}) production in the monocyte-like cell line THP-1. The induction of TNF-{alpha} by mechanical stress was dependent on the centrifugation speed and produced the highest level of TNF-{alpha} after 1 h of stimulation. TNF-{alpha} production returned to normal levels after 24 h of stimulation. Mechanical stress also induced Toll-like receptor-2 (TLR2) mRNA in proportion to the expression of TNF-{alpha}. The inhibition of TLR2 signaling by dominant negative myeloid differentiation factor 88 (MyD88) blocked TNF-{alpha} expression response to mechanical stress. After transient overexpression of TLR2 in HEK-293 cells, mechanical stress induced TNF-{alpha} 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-{alpha} production were inhibited, indicating that centrifugation-mediated mechanical stress induces both TLR2 and TNF-{alpha} production through Ca2+ releases from intracellular Ca2+ stores following TLR2 phosphorylation. In addition, TNF-{alpha} treatment in THP-1 cells induced TLR2 production in response to mechanical stress, whereas the preincubation of anti-TNF-{alpha} antibody scarcely induced the mechanical stress-mediated production of TLR2, indicating that TNF-{alpha} produced by mechanically stimulated THP-1 cells affected TLR2 production. We concluded that TNF-{alpha} production induced by centrifugation-mediated mechanical stress is dependent on MyD88-dependent TLR2 signaling that is associated with Ca2+ release and that TNF-{alpha} production induced by mechanical stress affects TLR2 production.

centrifugation; Toll-like receptor; THP-1 cells

MECHANICAL STRESS INDUCES the production of growth-related proto-oncogene such as c-fos or c-myc in osteoblasts (20). c-fos and c-myc are considered significant genes because they 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 tumor necrosis factor-{alpha} (TNF-{alpha}) 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, leading to the expression of inflammatory cytokines such as TNF-{alpha}, IL-6, fibrinogen, and soluble vascular cell adhesion molecule 1. These molecules have well-described functions in inflammation that have been associated with atherosclerotic progression (19, 22). Furthermore, TNF-{alpha} is a major mediator in the induction of the 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-{kappa}B activation (25). In addition, mechanical stress induces Toll-like receptor (TLR) expression as well as cytokine expression. Liang et al. (8) reported that TLR4 mRNA was upregulated after shear stress. 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 stress-mediated 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, 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 proinflammatory 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-{alpha}, 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-{alpha} production.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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, Seoul, Korea) and maintained in RPMI 1640 and MEM, respectively. All media were supplemented with 10% FBS, antibiotics (100 units of penicillin and 100 µg/ml streptomycin) in a 37°C incubator with 5% CO2.

A 5804R centrifuge (Eppendorf, Germany) with a microtiter plate rotor was used to apply mechanical stress on the THP-1 and HEK-293 cells. The average shear stress on the cell surface during centrifugation was calculated by Dr. Michael R. King (University of Rochester, Rochester, NY; personal communication) according to the following formulation (14): {tau} = 0.75 x µ x U/a, where µ 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 µm), a centrifugation speed of 280 g 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 speeds of 10, 180, and 461 g correspond to average shear stress on the cell surface of 0.074, 1.3, and 3.4 dyn/cm2, respectively.

RNA preparation and real-time PCR. Total RNA was extracted from THP-1 or HEK-293 cells by using the guanidinium thiocyanate-acid phenol-chloroform method. cDNA was synthesized with the Improm-II reverse transcription system (Promega) according to the manufacturer's instructions. To quantify the TNF-{alpha} and TLR2 mRNA, we carried out real-time PCR amplification with the ABI Prism 7000 sequence detection system (Applied BioSystems) and detected the PCR products with SYBR green. The following primers were used: TNF-{alpha} forward, 5'-CTCTTCTGGCTCAAAAAGAGAATT-3'; TNF-{alpha} reverse, 5'-AGGCCCCAGTTTGAATTCTT-3'; TLR2 forward, 5'-ACCTAGGGGAAACATCTCT-3'; and TLR2 reverse, 5'-AGCTCTGTAGATCTGAAGCATC-3'.

Establishment of dominant negative MyD88 and transient transfection. Dominant negative myeloid differentiation factor 88 (dnMyD88) was manufactured using the method described by Wang et al. (27) with minor modifications. dnMyD88 (amino acids 156–296) that contains the Toll/IL-1 receptor domain was amplified from total RNA extracted from THP-1 cells by RT-PCR and cloned into pCMV-Tag 2A (Stratagene). THP-1 and HEK-293 cells were transfected with the expression vector using the WelFec-Q transfection reagent (JBI, Daegu, 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 were as follows: dnMyD88 forward, 5'- GCTAGCATGCCTGAGCGTTTCG-3'; and dnMyD88 reverse, 5'-GGATCCTCAGGGCAGGGACAA-3'.

NF-{kappa}B luciferase reporter assay. THP-1 cells (5 x 105 cells/well) were seeded onto 12-well plates. After 24 h, the cells were transiently cotransfected with the pNF-{kappa}B-Luc and pRL-SV40 vectors. Thirty-six hours after transfection, the cells were stimulated by centrifugation at 180 g for 5 min. The cells were then 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 polyclonal anti-TLR2 (Santa Cruz Biotechnology) or anti-TNF-{alpha} (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 means ± SD of triplicate cultures. A paired t-test was used to determine the significance of the data. P values <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
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-{alpha} expression after mechanical stress.

Mechanical stress induced TNF-{alpha} and TLR2 production from THP-1 cells. To examine the effect of mechanical stress on the cultured cells, we studied centrifugation-mediated TNF-{alpha} and TLR2 production. Both TLR2 and TNF-{alpha} mRNA production peaked at 1 h after mechanical stimulation and then decreased slowly (Fig. 1A, top). The TLR2 and TNF-{alpha} mRNA levels recovered to their normal levels after 24 h. The levels of TLR2 mRNA were confirmed by measuring the levels of TLR2 protein using Western blotting (Fig. 1A, middle). RT-PCR of TNF-{alpha} expression using 32P-labeled dCTP showed clearly that the production of TNF-{alpha} mRNA peaked 1 h after the cells were stimulated by centrifugation at 180 g for 5 min (Fig. 1A, bottom).


Figure 1
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  		Fig. 1. Induction of tumor necrosis factor-{alpha} (TNF-{alpha}) and Toll-like receptor-2 (TLR2) by mechanical stress. THP-1 cells were cultured for the indicated times after centrifugation (CFG) at 180 g for 5 min (A) or THP-1 cells were exposed to the indicated centrifugation forces for 5 min (B). After treatment, cells were incubated for 1 h. The amounts of TNF-{alpha} and TLR2 were measured using real-time PCR (top), Western blotting with TLR2 mouse monoclonal IgG2a (middle), and RT-PCR with 32P-labeled dCTP (bottom). Data are means ± SD. *P < 0.05; **P < 0.01 vs. 0 h or 0 g. Expression of intracellular TNF-{alpha} (C) and surface TLR2 (D) was examined using immunofluorescence staining.

 
To examine the effect of mechanical stress on TNF-{alpha} and TLR2 expression, we exposed THP-1 cells to various ranges of centrifugation speeds. Previous study demonstrated that centrifugation at 3 g altered c-fos and osteocalcin gene expression in MC3T3-E1 osteoblasts (9). Because a centrifugation force of 3 g was not available in our experimental system, we used a force of 10 g as the lowest mechanical stress source. We also used a centrifugal force of 180 g (1,000 rpm) in our routine cell transfer experiments. To define our observation of the change in gene expression, we used a centrifugal force of 461 g (1,800 rpm) in this experiment. The centrifugation speeds of 10, 180, and 461 g correspond to an average shear stress on the cell surface of 0.074, 1.3, and 3.4 dyn/cm2, respectively.

The productions of TNF-{alpha} and TLR2 mRNA were increased in a speed-dependent manner (Fig. 1B, top). In addition, we used Western blotting to examine the expression of TLR2 (Fig. 1B, middle) and 32P-labeled RT-PCR for the detection of TNF-{alpha} (Fig. 1B, bottom). These alternative experimental methods showed results similar to those obtained using real-time PCR.

To confirm the TNF-{alpha} and TLR2 expression induced by mechanical stress, we performed immunofluorescence staining with antibodies for TNF-{alpha} and TLR2. Confocal microscopy showed that both TLR2 (Fig. 1C) and TNF-{alpha} (Fig. 1D) were induced in a speed-dependent manner. These results suggested that mechanical stress upregulated TLR2 expression and that TNF-{alpha} production was proportional to TLR2 expression.

Effect of polymyxin B treatment on mechanical stress-induced TNF-{alpha} production. THP-1 cells are very sensitive to media containing endotoxin. Therefore, we performed certification experiments with polymyxin B to verify that the mechanical stress-mediated gene expression was not due to endotoxin. THP-1 cells showed a significant elevation in TNF-{alpha} production compared with that of untreated cells when challenged with a known concentration of endotoxin. The endotoxin-induced TNF-{alpha} showed a 35 and 90% decrease after a pretreatment with 5 and 50 µg/ml polymyxin B, respectively. However, the TNF-{alpha} production induced by mechanical stress was not diminished in these experiments, demonstrating that the media did not contain endotoxin (Fig. 2).


Figure 2
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  		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 CFG at 180 g for 5 min and then incubated for 1 h in the presence or absence of polymyxin B (5 or 50 µg/ml). TNF-{alpha} production was measured using real-time PCR. Data are means ± SD. *P < 0.05; **P < 0.01 vs. polymyxin B-free sample.

 
TNF-{alpha} expression in dnMyD88 and TLR2 transfectants after exposure to mechanical stress. We investigated the role of TLR2 on mechanical stress-induced TNF-{alpha} production. Figure 3A, top, shows the expression of dnMyD88 truncated protein in the pCMV/dnMyD88 transfectants. After exposure to centrifugation at 180 g for 5 min, the production of TNF-{alpha} mRNA was significantly induced in pCMV-Tag2A-transfected cells. However, it was not induced in the mechanical stress-stimulated pCMV/dnMyD88-transfected cells compared with unstimulated pCMV/dnMyD88-transfected cells (Fig. 3A, bottom).


Figure 3
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  		Fig. 3. TNF-{alpha} production induced by mechanical stress was dependent on TLR2 and myeloid differentiation factor 88 (MyD88). A: expression of dominant negative MyD88 (dnMyD88) was confirmed by Western blotting (top), and pCMV/dnMyD88 or pCMV-Tag2A transfectants were incubated for 1 h after stimulation with or without CFG at 180 g for 5 min. The total RNA was extracted from the cells and then subjected to real-time PCR (bottom). B: expression of TLR2 from HEK-293 cells was examined using Western blotting (top), and TNF-{alpha} mRNA production was measured from transfectants using real-time PCR after mechanical stimulation with or without CFG at 180 g (bottom). Data are means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 vs. unstimulated cells. C: transfectants containing pCMV-Tag2A, pCMV/dnMyD88, and pCMV/TLR2 were stimulated with or without CFG at 180 g for 5 min and then incubated for 1 h. Mechanical stress-mediated TNF-{alpha} production was examined using immunofluorescence staining.

 
To investigate the role of TLR2 alone in mechanical stress-mediated TNF-{alpha} production, we transfected HEK-293 cells with a TLR2-expressing vector (pCMV/TLR2) or pCMV-Tag2A and confirmed TLR2 expression by Western blotting (Fig. 3B, top). Figure 3B, bottom, shows that the production of TNF-{alpha} was significantly induced in the pCMV/TLR2-transfected cells after stimulation by centrifugation at 180 g for 5 min, whereas the induction of TNF-{alpha} 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 Fig. 3C, immunofluorescence staining with the anti-TNF-{alpha} antibody in THP-1 cells showed that mechanical stress-mediated TNF-{alpha} production was not increased in the pCMV/dnMyD88-transfected cells, whereas TNF-{alpha} was increased in the pCMV-Tag2A-transfected cells. Interestingly, pCMV/TLR2 transfectants had a higher level of TNF-{alpha} production. These results suggest that TLR2-MyD88 signaling has an important role in the mechanical stress-mediated production of TNF-{alpha}.

Mechanical stress induced TLR2 tyrosine phosphorylation. Recent studies have reported that in response to bacterial ligands, Src family kinases initiate TLR2-associated signaling, which in turn is followed by recruitment of phosphatidylinositol 3-kinase (PI 3-K) and phospholipase C{gamma} (PLC{gamma}). 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 Fig. 4, tyrosine phosphorylation of TLR2 was induced by 5 min after stimulation with centrifugation at 180 g 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.


Figure 4
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  		Fig. 4. Mechanical stress induced c-Src-dependent TLR2 phosphorylation. A: THP-1 cells were stimulated by CFG at 180 g for 5 min with or without 50 µM PPI and incubated for the indicated times. The cell lysates were blotted with specific antibodies against TLR2 (Tl2.1) and phosphotyrosine (pY; clone 4G10). B: amount of tyrosine phosphorylation of TLR2 was quantified using ImageJ software (NIH, Bethesda, MD). Data are means ± SD of 3 independent experiments and normalized for unphosphorylated TLR2 proteins. Values from cells with PP1 (w/PP1) were significantly lower (P < 0.01) than those from cells without (w/o) PP1.

 
Mechanical stress induced NF-{kappa}B activation in THP-1 cells. In general, TLR2 signaling induces NF-{kappa}B translocation into the nucleus, resulting in cytokine expression. In our present study, we examined whether mechanical stress-mediated TLR2 signaling induces NF-{kappa}B activation. I{kappa}B-{alpha} and I{kappa}B-β degradation from mechanically stimulated THP-1 cells was examined by Western blotting with anti-I{kappa}B-{alpha} and anti-I{kappa}B-β antibodies. Mechanical stress caused a rapid degradation of I{kappa}B-{alpha} within 10 min, whereas I{kappa}B-β was cleaved more weakly after 10 min (Fig. 5A). Moreover, the degradation of both I{kappa}B-{alpha} and I{kappa}B-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 centrifugation at 180 g for 5 min. However, it was completely inhibited in the pCMV/dnMyD88-transfected cells after their stimulation with centrifugation at 180 g for 5 min (Fig. 5B). These results indicate that mechanical stress induced NF-{kappa}B activation and therefore might result in TNF-{alpha} production.


Figure 5
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  		Fig. 5. NF-{kappa}B activity in THP-1 cells is stimulated by mechanical stress. A: THP-1 cells were incubated for the indicated times after CFG at 180 g. The separated cell lysates were immunoblotted with phosphospecific antibodies against I{kappa}B-{alpha} and I{kappa}B-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 cotransfected with the pNF-{kappa}B-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 CFG at 180 g 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 means ± SD of 3 experiments performed in triplicate and normalized for Renilla luciferase activity. **P < 0.01 vs. unstimulated cells.

 
Mechanical stress-induced TNF-{alpha} and TLR2 production were induced by inositol 1,4,5-trisphosphate-sensitive Ca2+ release. A previous study (17) described that vortex-mediated mechanical stress induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-dependent Ca2+ release from intracellular Ca2+ stores. Therefore, we also examined the potential role of Ca2+ in TNF-{alpha} production that has been induced by centrifugation-mediated mechanical stress. Pretreatment of the cells with BAPTA-AM, an intracellular Ca2+ chelator, inhibited centrifugation-mediated TNF-{alpha} production (Fig. 6A), indicating that intracellular Ca2+ is necessary for TNF-{alpha} production. We next pretreated the cells with NiCl2·6H2O, a nonspecific Ca2+ influx inhibitor. NiCl2·6H2O, however, did not affect centrifugation-mediated TNF-{alpha} production (Fig. 6B), indicating that this type of TNF-{alpha} 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 Ins(1,4,5)P3-dependent 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 Ca2+ mediated by Ca2+ influx (12), inhibited centrifugation-mediated TNF-{alpha} production (Fig. 6B). These results suggest that Ins(3,4,5)P3-dependent Ca2+ release from intracellular Ca2+ stores plays a key role in centrifugation-mediated TNF-{alpha} production, which is similar to the finding that was published on vortex-mediated cell adhesion to fibronectin (17).


Figure 6
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  		Fig. 6. TNF-{alpha} and TLR2 production by centrifugation-mediated mechanical stress depend on inositol 1,4,5-trisphosphate-sensitive Ca2+ release from intracellular stores. THP-1 cells were preincubated with 50 µM BAPTA-AM for 1 h (A and C). THP-1 cells were preincubated 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 pretreated with or without either human recombinant TNF-{alpha} or anti-TNF-{alpha} antibody for 1 h (E). After incubation, cells were stimulated with or without CFG at 180 g for 5 min. TNF-{alpha} mRNA (A and B) and TLR2 mRNA production (C–E) was measured using real-time PCR. Data are means ± SD. *P < 0.05.

 
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-{alpha} activity with anti-TNF-{alpha} 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 TLR2 mRNA expression (Fig. 6D). These results suggest that the expression of TLR2 by centrifugation-mediated mechanical stress was regulated by Ins(1,4,5)P3-dependent Ca2+ release from intracellular Ca2+ stores.

We next examined whether TNF-{alpha} produced from mechanically stimulated THP-1 cells affects TLR2 expression. When THP-1 cells were treated with recombinant human TNF-{alpha}, the expression of TLR2 mRNA was higher than that of untreated THP-1 cells. 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-{alpha} antibody did not induce the TLR2 mRNA expression by THP-1 cells in response to mechanical stress (Fig. 6E). These data indicate that TNF-{alpha} secreted from mechanically stimulated cells affect upregulation of TLR2 mRNA expression.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Adhesion and migration molecules such as vascular cell adhesion molecule 1 (VCAM-1), monocyte chemoattractant protein 1 (MCP-1), and intracellular adhesion molecule 1 (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-{alpha} (4). Our experiments provide evidence that centrifugation-mediated mechanical stress can induce TNF-{alpha} production in THP-1 cells, processes that occur during the progression of atherosclerosis. Furthermore, TNF-{alpha} 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-{alpha}, IFN-{gamma}, and platelet-derived growth factor (22). Mechanical stress, such as shear stress, causes a 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 proatherogenic 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 ligand-mediated TLR signaling in the gene expression of cytokines, chemokines, growth factors, and transcription factors, suggesting an interrelation between mechanical stress and TLR. Recently, Liang et al. (8) showed that overexpression of TLR4 could be induced by mechanical stress. In our present study, TLR2 was significantly induced by mechanical stress, whereas other TLRs were only moderately induced. Therefore, we examined whether TLR2 signaling participated in mechanical stress-mediated TNF-{alpha} production. When THP-1 cells were transfected with dnMyD88, the expression of TNF-{alpha} after the exposure to mechanical stress was not induced, whereas TLR2-transfected cells, after their exposure to mechanical stress, had higher TNF-{alpha} production compared with unstimulated TLR2-transfected cells. These results indicate that mechanical stress-mediated TNF-{alpha} expression was induced through MyD88-dependent TLR2 signaling. By using a genetic loss-of-function approach, a recent study (16) 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 (7) has suggested that laminar flow induced SP1 serine phosphorylation and thereby blocked SP1 binding to the TLR2 promoter, which is required for TLR2 expression. 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-{alpha} 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 PI 3-K activates PLC{gamma} and is involved in intracellular Ca2+ release by generating Ins(1,4,5)P3 (1). PLC{gamma} activity is regulated by plasma membrane localization and tyrosine phosphorylation. Furthermore, PLC{gamma} 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-{alpha} and TLR2 production through Ins(1,4,5)P3-dependent Ca2+ release from intracellular Ca2+ stores. These results were confirmed by the pretreatment of cells with Ca2+ inhibitors. Moreover, the TNF-{alpha} produced from the mechanically stimulated-THP-1 cells affected TLR2 mRNA production. In addition, TLR2 signal transduction by mechanical stress induced TNF-{alpha} production through a MyD88-dependent pathway that included NF-{kappa}B activation. These results suggest that mechanical stress on THP-1 cells leads to mutual action between TLR2 and TNF-{alpha}, which may occur via intracellular Ca2+ release and a MyD88-dependent NF-{kappa}B signal pathway.

In conclusion, we showed that centrifugation-mediated mechanical stress upregulated TNF-{alpha} and TLR2 production in THP-1 cells and that c-Src-dependent TLR2 phosphorylation followed by MyD88-dependent NF-{kappa}B activation and Ca2+ release-dependent TNF-{alpha} production were involved in this mechanism. These findings might elucidate another aspect of the signaling of mechanical stress-mediated atherosclerosis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Regional Innovation Center Grant RIC 07-06-04 (Skin Biotechnology Center), funded by the Ministry of Knowledge Economy, Republic of Korea.


   ACKNOWLEDGMENTS
 
We thank Michael R. King for calculation of the amount of shear stress on the cell surface during centrifugation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. K. Chung, Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee Univ., Yongin 449-701, Korea (e-mail: dkchung{at}khu.ac.kr)

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.

* H. G. Kim and J. Y. Kim contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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