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Am J Physiol Heart Circ Physiol 288: H2946-H2954, 2005. First published January 28, 2005; doi:10.1152/ajpheart.00919.2004
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Biomechanical stress induces IL-6 expression in smooth muscle cells via Ras/Rac1-p38 MAPK-NF-{kappa}B signaling pathways

Anna Zampetaki, Zhongyi Zhang, Yanhua Hu, and Qingbo Xu

Department of Cardiac and Vascular Sciences, St. George's Hospital Medical School, London, United Kingdom

Submitted 2 September 2004 ; accepted in final form 24 January 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
IL-6, a proinflammatory cytokine, has been implicated in the development of vascular diseases. We previously demonstrated that mechanical stress can initiate signaling pathways leading to smooth muscle cell (SMC) proliferation and apoptosis, but little is known concerning cyclic stress-induced inflammatory response. To explore the role of stretch in the upregulation of cytokine expression in SMCs we performed RNase protection assay for a panel of cytokines and found that mechanical stress resulted in a time-dependent induction of IL-6 mRNA but not other cytokines, e.g., IL-1{alpha}, IL-1{beta}, IL-6, IL-10, IL-12p35, IL-12p40, IL-18, IFN-{gamma}, and macrophage migration inhibitory factor (MIF). This induction also correlated with elevated IL-6 protein levels in the supernatant. Pretreatment of the cells with NF-{kappa}B inhibitors inhibited NF-{kappa}B activity and resulted in marked inhibition (50%) of IL-6 protein. Moreover, SMC lines stably expressing dominant-negative Ras (RasN17) or Rac (RacN17) exhibited a remarkable decrease in p38 MAPK activity and IL-6 mRNA induction by mechanical stress. Furthermore, a significant inhibition of 30 and 40% in IL-6 protein was observed in SMCs pretreated with inhibitors of p38 MAPK and ERK1/2, respectively, but not JNK. Interestingly, SMCs isolated from PKC-{delta}-deficient mice exhibited higher levels of IL-6 compared with wild-type cells. Finally, high levels of IL-6 expression were observed in atherosclerotic lesions of vein bypass grafts, which are related to altered biomechanical stress. Our findings demonstrate that biomechanical stress-induced IL-6 expression occurs via a mechanism that involves Ras/Rac/p38 MAPK/NF-{kappa}B/NF-IL6 signaling pathways, which is downregulated by PKC-{delta}, and suggest that modulation of this event contributes to the pathogenesis of atherosclerosis.

mechanical stress; interleukin-6; signal transduction; atherosclerosis

BLOOD VESSELS are dynamically subjected to biomechanical forces in the form of stretch and shear stress resulting from blood pressure and blood flow, respectively. Accumulating evidence indicates that mechanical forces play a key role in the pathogenesis of atherosclerosis, as atherosclerotic lesions develop mainly in the areas where vessels experience elevated stretch stress and low shear stress (8, 44, 23). Furthermore, veins do not develop atherosclerosis. However, when veins are grafted from their low-pressure environment into the arterial circulation, where they are subject to a 10-fold increase in biomechanical forces, they develop arteriosclerosis, which constitutes the main reason for late vein graft failure (30). Thus mechanical stress could be an important factor for the development of arteriosclerosis.

On the other hand, atherosclerosis is believed to be an inflammatory disease that includes adhesion molecule expression, leukocyte recruitment, and production of cytokines such as IL-6 (10, 31, 43). IL-6 is thought to play a major role in the pathogenesis of atherosclerosis (32, 34). Besides their potential role in local vascular disease progression, the circulating concentrations of these cytokines serve as markers for adverse prognosis (2).

Smooth muscle cells (SMCs) are one of the major constituents of blood vessels and are also central to the formation of neointimal and atherosclerotic lesions (35). SMCs are capable of sensing changes in hemodynamic conditions. Previously, we demonstrated that mechanical stretch results in phosphorylation and activation of PDGF receptor (12) and initiates intracellular signaling pathways, typically used by growth factors (18, 19) related to SMC proliferation. It has also been found that mechanical stress leads to activation of {beta}1-integrin receptor/PKC-{delta}/p38 MAPK signal pathways that mediate SMC apoptosis (26, 41, 42). However, little is known about mechanical stretch-initiated signaling leading to an inflammatory response in SMCs, and no data exist concerning strain stress-induced IL-6 expression. In the present study, we explored the possibility of IL-6 production in cultured SMCs stimulated by mechanical stress and in arteriosclerotic lesions in mouse models. We demonstrate that biomechanical stress induces IL-6 protein and mRNA in SMCs via a mechanism that involves Ras/Rac/p38 MAPK/NF-{kappa}B/NF-IL6 signaling pathways and is negatively regulated by PKC-{delta}.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and cyclic stress. SMCs were isolated by enzymatic digestion of mouse aortas as described elsewhere (13, 17) and were cultured in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 15% FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml). In experiments in which SMCs from PKC-{delta}–/– mice were used, the genotype was confirmed by PCR. For cyclic stress experiments, SMCs were plated on silicone elastomer-bottomed and collagen-coated plates (Flexcell, McKeesport, PA). Cells achieving 90% confluence were subjected to mechanical stress with the computer-controlled Cyclic Stress Unit (Flexcell 4000). The Cyclic Stress Unit consisted of a controlled vacuum unit and a base plate to hold the culture plates. Vacuum (15–20 kPa) was repeatedly applied to the elastomer-bottomed plates via the base plates. Cyclic deformation (60 cycles/min) and 7, 15, or 20% elongation of elastomer-bottomed plates were used (18, 41). This new model of the apparatus generates a homogeneous cyclic stress on the membrane.

Stable transfection. Plasmids expressing dominant-negative Ras and rac1 were provided by G. Baier (Institute for Medical Biology and Human Genetics, University of Innsbruck). Rat SMCs were transfected stably with N17 rac (pEF-rac1 N17) plasmids, using a Superfect Kit (Qiagen) according to the manufacturer's instructions. Transfected cells were selected for neomycin resistance with G418 antibiotic. RasN17, RacN17 SMCs were identified by Western blot analysis with HA-Ras and myc-tagged Rac1 antibodies.

Cell treatment and protein extraction. After strain, SMCs were washed twice with cold (4°C) phosphate-buffered saline (pH 7.4), harvested on ice in RIPA buffer [10 mM Tris (pH 8.0), 10 mM EDTA, 140 mM NaCl, 1% Triton, 1% Na deoxycholate, 0.1% SDS, and 25 mM {beta}-glycerol-PO4, supplemented with 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 100 µM phenylmethylsulfonyl fluoride], and then centrifuged at 13,000 rpm (Eppendorf centrifuge; Osterode, Germany) at 4°C for 15 min. The supernatant was harvested and used for Western blot analysis.

Western blot analysis. Briefly, 30 µg of proteins was separated by electrophoresis through a 10% SDS-polyacrylamide gel, transferred onto nitrocellulose membranes, and blocked for 1 h with blocking buffer [1x Tris-buffered saline, 0.1% Tween 20 (TBST) with 5% (wt/vol) nonfat dry milk]. After the membrane was washed with TBST, an overnight incubation at 4°C was performed. The blots were probed with antibodies against I{kappa}B{alpha}, {alpha}-actin, phospho p65 (Cell Signaling Technology, Medford, MA), phospho p38, phospho ERK1/2, phospho JNK1/2, phospho c-jun, HA-Ras, or myc-tagged rac1 (a gift from G. Baier). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), unless otherwise stated. After overnight incubation, the blots were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody. Specific antibody-antigen complexes were detected by using the ECL Western Blot Detection Kit (Amersham Pharmacia Biotech UK).

Kinase assay. For kinase assays, 0.5 mg of proteins were incubated with 10 µl of antibodies against mammalian p38 MAPK for 2 h at 4°C with rotation. Subsequently, 40 µl of protein G agarose suspension (Santa Cruz Biotechnology) was added, and rotation was continued for 1 h at 4°C. Immunocomplexes were precipitated by centrifuge and washed twice with buffer A (in mM: 500 LiCl and 100 Tris) and buffer B (1 mM DTT with 0.1% Triton X-100, pH 7.6), and C (in mM: 20 MOPS, 2 EGTA, 10 MgCl2, and 1 DTT with 0.1% Triton X-100, pH 7.2), respectively. Immunocomplexes were incubated with myelin basic protein (MBP, 6 µg; Upstate Biotechnology, Lake Placid, NY) and [{gamma}-32P]ATP (5 µCi) for 20 min. To stop the reaction, 15 µl of 4x Laemmli buffer was added, and the mixture was boiled for 5 min. Proteins in the kinase reaction were resolved by SDS-polyacrylamide gel electrophoresis (15% gel) and subjected to autoradiography.

RNase protection assay. Total RNA was extracted with the Qiagen kit according to the manufacturer's instructions. To estimate the expression of cytokines, an RNase protection assay (RPA) (RiboQuant; Pharmingen, San Diego, CA) was performed according to the manufacturer's recommendations. Briefly, radiolabeled antisense cytokine murine RNA was synthesized with the "In vitro transcription kit" Mck2b multiprobe template set (Pharmingen) and [{alpha}-32P]UTP (Amersham Biosciences). The labeled probe was hybridized overnight in excess to the same amounts of RNA isolated from different SMCs. Free probe and other single-stranded RNA were subsequently digested with ribonuclease A and T1. The remaining "RNase-protected" fragments were purified and resolved on a 5% sequencing gel and autoradiographed. For quantification, the IL-6 signals for each sample of the blot were normalized to the housekeeping gene L32.

RT-PCR. The procedure used for RT-PCR was similar to that described elsewhere (1). In brief, 2 µg of RNA was converted to cDNA with the Promega Reverse Transcription System (Promega, Madison, WI). cDNA products were amplified by PCR using the following gene specific primers: IL-6 (5'-TTG CCT TCT TGG GAC TGA TGC T-3' and 5'-GTA TCT CTC TGA AGG ACT CTG G-3'); GAPDH (5'-CGG AGT CAA CGG ATT TGG TCG TAT-3' and 5'-AGC CTT CTC CAT GGT GGT GAA GAC-3'). PCR conditions were as follows: 94°C for 2 min and then 35 cycles at 94°C for 30 s, 58°C for 1 min, and 72°C for 1 min, followed by 72°C for 10 min. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining.

Real-time PCR experiments were performed with the Brilliant SYBRgreen QPCR core reagent kit (Stratagene), using the Mx4000 (Stratagene) real-time thermocycler according to the manufacturer's recommendations. Amplification was performed with 40 cycles and an annealing temperature of 58°C. Copy numbers were calculated by the instrument's software from standard curves. The specificity of the amplification reaction was determined by a melting curve analysis. For quantification, IL-6 mRNA expression was normalized to the expressed housekeeping gene GAPDH.

Electrophoretic mobility shift assays. Nuclear extracts were prepared by the method of Dignam et al. (7). In experiments in which NF-{kappa}B inhibitors were used, the cells were pretreated for 1 h with the respective inhibitor and then stressed in the presence of inhibitors. A double-stranded oligonucleotide corresponding to either NF-{kappa}B (AGT TGA GGG GAC TTT CCC AGG C), or NF-IL6 binding site (TGC AGA TTG CGC AAT CTG CA) was used as a probe. Probes were 5' end-labeled with [32P]ATP (3,000 Ci/mmol), using polynucleotide kinase according to standard procedures (33). Binding reactions were performed in a final volume of 15 µl by preincubating 5 µg of nuclear extracts in 4 mM Tris, pH 7.8, 1 mM EDTA, 4% glycerol, 4 mM DTT, and 2 µg of poly(dI-dC) at room temperature for 20 min. For supershift and competition studies, nuclear extracts were incubated in the presence of nonlabeled RelA, mutated RelA, p65, or p50 antibodies (Santa Cruz Biotechnology).

ELISA. Cytokine concentrations in the media of cultured SMCs was determined with a murine IL-6 ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Samples were run in triplicate for each condition and normalized to total cellular proteins.

Mouse sample preparation. All animal experiments were performed according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals. The vein graft procedure was similar to that described previously (13, 17). Briefly, 3-mo-old C57BL/6J mice were anesthetized, and the vena cava was harvested. In the recipient, the right common carotid artery was cut across the middle and the vein segment was grafted between the two ends of the carotid artery with a cuff technique. The grafts were harvested at 8 wk postoperatively (5 mice/group).

Immunohistochemical and immunofluorescent staining. The procedure used in the present study was similar to that described previously (28). Briefly, serial 5-µm-thick frozen sections were overlaid with rabbit anti-mouse IL-6 antibodies (Santa Cruz Biotechnology). Sections were incubated with swine anti-rabbit Ig conjugated with phosphatase (Dakopatts) and developed with a substrate solution (Sigma). The procedure used for immunofluorescent staining was similar to that described previously (28). Frozen sections were labeled with a mouse monoclonal antibody against {alpha}-actin conjugated with Cy3 (Sigma), or a rat monoclonal antibody against mouse MAC-1 (CD11b/18) leukocytes (Pharmingen), and a rabbit antibody against IL-6. The sections were visualized with swine anti-rat Ig- tetramethylrhodamine isothiocyanate or anti-rabbit Ig conjugated with FITC (Dakopatts).

Statistics. Statistical analyses were performed by one-way ANOVA, and P < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cyclic strain stress induces IL-6 in SMCs. To explore the possibility that mechanical stress upregulates the expression of inflammatory mediators, including IL-1{alpha}, IL-1{beta}, IL-1Ra, IL-6, IL-10, IL-12p35, IL-12p40, IL-18, IFN-{gamma}, and macrophage migration inhibitory factor (MIF), we used a global approach—RPA—to detect their mRNA in cultured SMCs. Theonly cytokine that showed an elevated level was IL-6, which was observed after 2 h of stimulation and peaked at 3 h. IL-Ra and MIF mRNAs were detectable in both treated and untreated SMCs (Fig. 1A), although no significant difference was observed. Ribosomal L32 RNA was used as a loading control. Additionally the expression of RANTES, macrophage inflammatory protein (MIP)-1{beta}, MIP-1{alpha}, MIP-2, inducible protein-10, monocyte chemoattractant protein-1, T cell activation gene 3 (TCA-3), and eotaxin was examined in SMCs that were stretched for 1 or 3 h, but no significant change was observed (data not shown). The increase in IL-6 mRNA in mechanically stressed SMCs was also confirmed by RT-PCR (Fig. 1B). To determine whether mechanical stress also induces IL-6 protein, supernatant was harvested at different time points and analyzed by ELISA. As shown in Fig. 1C, increased levels of IL-6 protein were detected in the media as early as 3 h and peaked after 8 h of stimulation.



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  		Fig. 1. Mechanical stress induces IL-6 in smooth muscle cells (SMCs). A: total RNA was isolated from cells after mechanical stress (15% elongation) for the indicated times. Samples were analyzed by RNase protection assay (RPA). L32, a ribosomal protein mRNA, was used as a loading control. B: induction of IL-6 mRNA in mechanically stressed SMCs was confirmed by RT-PCR. Data were normalized to GAPDH mRNA expression. C: cells were stressed (15% elongation) or left untreated for the indicated times, and IL-6 protein was measured in the supernatant by ELISA. Values are means (SD). *Significant difference from untreated controls, P < 0.05. MIF, macrophage migration inhibitory factor; GIF, interferon gamma-inducing factor.

 
To determine whether the IL-6 induction in stretched SMCs depends on the strength of the mechanical force applied, a tensile strength-response analysis was performed with different degrees of elongation. As shown in Fig. 2, when SMCs were stretched to elongation of 7, 15, or 20% of the original size for 2 h, there was a corresponding increase in the IL-6 mRNA level as detected by RPA (Fig. 2A) or RT-PCR (Fig. 2B). This tendency was also observed for IL-6 protein levels in the supernatant after 8 h of stress (Fig. 2C).



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  		Fig. 2. Intensity-dependent IL-6 production in stretched SMCs. Cells were exposed to the indicated elongation, and IL-6 mRNA induction in cells after 2 h of mechanical stress at the indicated elongation was assessed by RPA (A) or RT-PCR (B). C: cells were exposed to the indicated elongation, and IL-6 protein in the supernatant was measured after 8 h by ELISA. Values are means (SD). *Significant difference from untreated controls, P < 0.05.

 
NF-{kappa}B is a key regulator of IL-6 induction in mechanically stressed SMCs. It was previously shown that transcription of the IL-6 gene is partly regulated by the transcription factor NF-{kappa}B (25). The most abundant form of this factor is a heterodimer of p50 and p65 subunits. In resting cells, NF-{kappa}B exists in the cytosol as an inactive complex bound to its inhibitor I{kappa}B{alpha}. Phosphorylation and subsequent degradation of I{kappa}B{alpha} occurs on stimulation of the cells, and the released NF-{kappa}B translocates to the nucleus, binds to DNA, and activates gene transcription (9).

To determine whether I{kappa}B{alpha} degradation occurs in response to mechanical stress, SMCs were exposed to cyclic stress for various time points and total protein extracts were analyzed by Western blot analysis. A significant reduction in the levels of I{kappa}B{alpha} was observed after 15 and 30 min of mechanical stress (Fig. 3A). The detected levels increased again after 1 h of stimulation.



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  		Fig. 3. IL-6 induction is regulated by NF-{kappa}B. A: SMCs were stressed for the indicated times at 15% elongation, and Western blot analysis for I{kappa}B{alpha} was performed. Results were normalized to {alpha}-actin. B: DNA binding of NF-{kappa}B in cyclic stressed cells as detected by EMSA. C: increased phosphorylation at Ser536 on the p65 subunit of NF-{kappa}B in stretched SMCs. Results were normalized to {alpha}-actin. D: effect of pretreatment with various concentrations of pyrrolidinedithiocarbamate (PDTC) on NF-{kappa}B DNA binding activity in cyclic stressed SMCs. E: effect of pretreatment with 100 µM PDTC or 18 µM SN50 on NF-{kappa}B DNA binding activity in stressed SMCs. F: cells were pretreated with NF-{kappa}B inhibitors and then stressed for 8 h. IL-6 protein levels were measured by ELISA in the supernatant. Values are means (SD). *Significant difference from strain stressed, P < 0.05.

 
To examine whether I{kappa}B{alpha} degradation leads to nuclear translocation and increased DNA binding of NF-{kappa}B, an electrophoretic mobility shift assay was performed with an oligonucleotide corresponding to NF-{kappa}B DNA binding site as a probe. Nuclear extracts from untreated and mechanically stretched SMCs were prepared, mixed with the labeled probe, and electrophoresed in a nondenaturing polyacrylamide gel. As shown in Fig. 3B, strong NF-{kappa}B binding was observed after 60 min of mechanical stress. This binding persisted for 120 min. Supershift analysis using antibodies against p65 and p50 subunits and competition experiments with either nonlabeled oligonucleotides corresponding to NF-{kappa}B DNA binding site or mutated oligonucleotides confirmed that the detected complexes were NF-{kappa}B specific.

Regulation of NF-{kappa}B-mediated transcription may occur at multiple levels after cell stimulation. Posttranscriptional modification of the p65 subunit that affects its transactivation potential has been proposed to constitute such an additional layer of NF-{kappa}B regulation (9, 38, 40, 45). Phosphorylation of the p65 subunit on Ser536 was detected in mechanically stressed SMCs (Fig. 3C), providing further evidence that in these cells NF-{kappa}B is activated.

To define the role of NF-{kappa}B in the induction of IL-6 in mechanically-stressed SMCs, we used two pharmacological inhibitors of NF-{kappa}B: pyrrolidinedithiocarbamate (PDTC), an antioxidant that has been shown to inhibit NF-{kappa}B, and SN50, a cell-permeant peptide that interrupts translocation of NF-{kappa}B. As shown in Fig. 3D, pretreatment with 100 µM PDTC completely eliminated NF-{kappa}B DNA binding induced by mechanical stress, whereas lower concentrations only showed a partial inhibition. Pretreatment with 18 µM SN50 could also efficiently inhibit NF-{kappa}B DNA binding (Fig. 3E). Interestingly, in the presence of these inhibitors a significant attenuation of IL-6 induction as measured in the supernatant by ELISA (Fig. 3F) was observed, indicating that NF-{kappa}B plays a crucial role in the expression of this cytokine in mechanically stretched SMCs.

MAPK signaling pathways mediate induction of IL-6 by mechanical stress in SMCs. Cross talk between MAPK signaling and NF-{kappa}B-dependent transcription was identified in previous studies (3, 38). MAPKs are activated by a cascade of intracellular phosphorylation events and transduce signals to the nucleus (4, 11). A strong and rapid activation of p38 MAPK, ERK, and JNK, as determined by phosphorylation levels, occurs after mechanical stress in SMCs (Fig. 4A). To evaluate the contribution of these signaling pathways to the induction of IL-6 by cyclic stressed SMCs, specific kinase inhibitors were applied. These compounds were used at concentrations that inhibited the respective kinases (Fig. 4B). Pretreatment of SMCs with these reagents significantly affected IL-6 protein production after mechanical stress (Fig. 4C). Both p38 and ERK1/2 pathway inhibitors (SB-202190 and PD-90859, respectively) reduced these levels by 30 and 40%, respectively. In contrast, pretreatment with JNK1/2 inhibitor (SP-600125) resulted in significant increase of IL-6 protein in the supernatant media.



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  		Fig. 4. MAPK pathways are involved in IL-6 expression. A: cells were stressed for the indicated times at 15% elongation, and Western blot analysis for phospho (p)-p38 MAPK, phospho-ERK1/2, and phospho-JNK1/2 was performed. Results were normalized to {alpha}-actin. B: pretreatment of cells with SB-202190 (500 nM), PD-98059 (50 µM), or SP-600125 (18 µM) inhibits p38 MAPK and ERK1/2 and JNK1/2 activities in response to strain stress. MBP, myelin basic protein. C: effect of kinase inhibitors on IL-6 protein production in mechanically stressed SMCs, as measured by ELISA in the supernatant after 8 h. Values are means (SD). *Significant difference from untreated controls, P < 0.05.

 
Rac1 and Ras signaling pathways mediate induction of IL-6 by strain in SMCs. The above results suggest that IL-6 induction in mechanically stressed SMCs is regulated by the NF-{kappa}B pathway and MAPK signaling. Ras and Rac small GTPases have been shown to interact with these pathways (16, 36). To investigate any possible role of these signal transducers in the induction of IL-6 in cyclic stress, SMCs were stably transfected with either dominant-negative Rac (Rac1N17) or Ras (RasN17) or a vector plasmid as a control (Fig. 5A). Western blot analysis of these cells showed that the presence of dominant-negative Ras or Rac1 inhibits the activation of p38 MAPK and ERK1/2 by mechanical stress but does not affect the activation of JNK1/2 (Fig. 5B). Interestingly, in these mutants the levels of IL-6 mRNA both at baseline and after cyclic stress were dramatically lower than the control (Fig. 5, C and D). To further define the mechanism of this inhibition, nuclear extracts were isolated from transfected cells that had been stressed and an electrophoretic mobility shift assay was performed. Enhanced NF-{kappa}B DNA binding that was observed in the cells transfected with the control vector was diminished in Rac1N17-transfected cells (Fig. 5E), suggesting that NF-{kappa}B activation is impaired in these mutants.



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  		Fig. 5. Involvement of Ras and Rac1 in IL-6 expression. A: SMCs were stably transfected with constructs expressing dominant-negative Rac1, Ras, or vector plasmid. SMCs expressing Rac N17 and Ras N17 were identified with anti-Myc-tag or anti-HA-Ras antibodies by Western blot analysis. B: effect of RacN17 and Ras N17 on phosphorylation of 3 members of the MAPKs in response to mechanical stress as determined by Western blot analysis. C: IL-6 mRNA levels were assessed by RT-PCR in stably transfected cells after 2 h of stress (15% elongation). Data were normalized to GAPDH mRNA expression. D: quantitative real-time PCR of IL-6 mRNA levels in stably transfected cells after 2 h of stress (15% elongation). Data were normalized to GAPDH mRNA expression. Values are means (SD). *Significant difference from stressed vector samples, P < 0.05. E: NF-{kappa}B DNA binding activity in stably transfected cells exposed to mechanical stress was determined by EMSA. Neo, neomycin-resistant vector.

 
PKC-{delta} negatively regulates IL-6 expression in SMCs. NF-IL6 is a transcriptional regulatory element known to affect IL-6 expression (25). PKC-{delta} has been shown to regulate IL-6 by controlling the DNA binding of this factor (29). To identify a possible role of this kinase in the induction of IL-6 in SMCs, cells were isolated by enzymatic digestion of aortas of PKC-{delta}–/– mice and exposed to altered mechanical forces. PKC-{delta}–/– cells displayed a remarkable increase in IL-6 protein expression compared with the wild type (Fig. 6A), and higher levels of IL-6 mRNA were also detected by RPA (Fig. 6B) and confirmed by RT-PCR (Fig. 6C). This induction seems to depend on the degree of elongation applied, as stressing cells with higher elongations, and thus exposing them to enhanced mechanical forces, led to a further increase in the IL-6 protein (Fig. 7A) and mRNA (Fig. 7B) levels. Interestingly, in nuclear extracts from stressed SMCs, an increase in the DNA binding of NF-IL6 was observed (Fig. 7C). This enhanced binding was even stronger in PKC-{delta}–/– SMCs.



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  		Fig. 6. Elevated IL-6 production in PKC{delta}–/– SMCs. A: cells were stretched (15% elongation) or left untreated for the indicated times, and IL-6 protein was measured in the supernatant by ELISA. Values are means (SD). *Significant difference from untreated controls, P < 0.05. B and C: IL-6 mRNA induction in mechanically stressed (15% elongation) SMCs at the indicated times, as assessed by RPA (B) or RT-PCR (C).

 


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  		Fig. 7. IL-6 production and NF-IL6 activation in PKC-{delta}–/– SMCs. A: cells were exposed to the indicated elongation, and IL-6 protein was measured after 8 h in the supernatant by ELISA. Values are means (SD). *Significant difference from untreated controls, P < 0.05. B: IL-6 mRNA induction in cells after 2 h of mechanical stress at the indicated elongation, as assessed by RPA. C: NF-IL6 DNA binding activity as detected by EMSA.

 
IL-6 expression in neointimal lesions. Because biomechanical stress plays a key role in the development of neointimal lesions in vein grafts, it would be interesting to determine whether neointimal SMCs express IL-6. Immunohistochemical staining revealed significantly increased IL-6 production in neointimal lesions 8 wk after grafting (Fig. 8B), whereas no staining was seen in freshly harvested veins or in vein segments (Fig. 8A). Furthermore, some cells in the adventitia of vein grafts also positively stained for IL-6 (Fig. 8C).



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  		Fig. 8. Immunohistochemical staining for IL-6 and cell markers. Normal veins (A) and 8-wk vein grafts (B and C) from mice were sectioned and labeled with antibodies against mouse IL-6. Sections were developed with fast red and counterstained with hematoxylin (blue). Arrows indicate examples of positive-stained cells (red). D–F: for immunofluorescence double staining, cryostat sections from 8-wk vein grafts of mice were labeled with antibodies against IL-6 (D). The reaction was visualized by a swine anti-rabbit Ig-FITC-conjugated Ig. Sections were incubated with a monoclonal antibody against {alpha}-actin conjugated with Cy3 (E) visualized by anti-rat Ig-tetramethylrhodamine isothiocyanate. F is a merged image of D and E. Arrows indicate the surface of the intima.

 
To clarify cell types expressing high levels of IL-6 in the animal model, double staining for IL-6, SMC, and macrophage markers was performed for neointimal lesions of vein grafts. In neointimal lesions of vein grafts, {alpha}-actin-positive SMCs were mostly stained for IL-6 (Fig. 8, D–F), whereas this was rarely seen for Mac-1+ cells (data not shown). As macrophages are less abundant in neointimal lesions of vein grafts, SMCs are likely to be the main source of IL-6 protein in response to biomechanical stress.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Leukocyte infiltration, enhanced gene expression of adhesion molecules, and proinflammatory cytokine production in early and late atherosclerotic lesions indicate that inflammation mediates all stages of the disease (22, 24, 31). Cyclic stress, the elevated mechanical forces sensed by SMCs because of altered hemodynamic blood flow, also contributes to the pathogenesis of atherosclerosis (27, 44). In the present study, we demonstrate for the first time that, in response to cyclic stress, SMCs produce IL-6 protein and release it into culture medium. The elevated protein levels also correlate with increased IL-6 mRNA levels, indicating upregulation of the IL-6 gene in mechanically stressed SMCs. These findings provide a link between mechanical forces and the inflammatory response in the vessel wall and thus highlight the molecular mechanisms of biomechanical stress that contribute to the initiation of atherosclerosis.

Aiming to identify signaling pathways involved in this induction, we studied the activation of NF-{kappa}B, a known transcriptional regulator of IL-6, in response to other stimuli.(25) Previous reports indicate the involvement of NF-{kappa}B in IL-6 induction in mechanically stressed endothelial and intestinal epithelial cells (14, 15). In the present study, rapid degradation of the NF-{kappa}B inhibitor I{kappa}B{alpha} and increased NF-{kappa}B DNA binding were identified in cyclic stressed SMCs, suggesting that NF-{kappa}B is activated under these experimental conditions. Moreover, a posttranscriptional modification of p65 subunit (phosphorylation of Ser536) was detected in these SMCs, indicating the existence of additional mechanisms that enhance the ability of NF-{kappa}B to activate transcription in stretched SMCs. Further evidence supporting the involvement of NF-{kappa}B activation is the finding that PDTC and SN50, both pharmacological inhibitors of NF-{kappa}B, can inhibit IL-6 protein production. Thus mechanical stress-induced NF-{kappa}B DNA binding is responsible for IL-6 gene expression in SMCs.

On the other hand, the finding that inhibition of the NF-{kappa}B pathway does not completely abrogate IL-6 induction in stretched SMCs suggests that other mediators are also involved. MAPK signaling pathways have been implicated previously in the expression of cytokines in response to other stimuli (3, 38). We found that cyclic stress activates all three major MAPK pathways. However, the three cascades seem to have distinct roles in IL-6 induction and differentially regulate the expression of this cytokine. p38 MAPK and ERK1/2 pathways contribute to IL-6 upregulation in stretched SMCs, and their inhibition significantly attenuates IL-6 protein levels. The JNK signaling, though, seems to provide the negative feedback mechanism that downregulates the expression of IL-6 in mechanically stressed SMCs. In experiments in which the pharmacological inhibitor of this pathway was used, significantly higher levels of IL-6 protein were observed, indicating that mediators controlled by this cascade may act as "molecular brakes" that block the sustained induction of IL-6 in stretched SMCs.

What upstream signal transducers are involved in mechanical stress-induced IL-6 production? Ras and Rac1 small GTPases are recognized as key mediators for a diverse array of cellular events. Most importantly, they interact and can affect MAPK signaling (16, 39). In our model, SMCs stably transfected with dominant-negative constructs for Ras and Rac1 show a dramatic decrease in p38 MAPK and ERK1/2 phosphorylation, but JNK1/2 activation is not affected. Interestingly, as was the case when chemical compounds were used, inhibition of p38 and ERK1/2 signaling led to inhibition of IL-6 induction. Mutant cells induced significantly lower levels of cytokine after cyclic stress, suggesting that IL-6 expression in stretched SMCs is Ras/Rac1/p38 MAPK dependent. Besides controlling MAPK signaling, an additional relation between Rac1 and NF-{kappa}B activation was found in stretched SMCs. In cells transfected with the dominant-negative form of Rac1 attenuated NF-{kappa}B DNA binding was observed, implicating that Rac1 may also be an upstream regulator of NF-{kappa}B pathways in mechanically stressed SMCs.

In addition to Ras/Rac/NF-{kappa}B/MAPK signaling, IL-6 induction in mechanically stressed SMCs seems to involve PKC-{delta}. Significantly higher levels of IL-6 protein were detected in the supernatant of PKC-{delta}–/– SMCs. We have also found IL-6 overexpression in SMCs of vein grafts in PKC-{delta}–/– mice (data not shown), which are characterized by exacerbated arteriosclerotic lesions (17). Furthermore, mechanical stress results in PKC-{delta} activation, as identified by analysis of protein translocation and activity in SMCs (20). The mechanism for IL-6 expression that we propose involves NF-IL6, a transcription factor known to regulate IL-6 expression (25). This element can be phosphorylated in vitro by PKC at Ser240, which is located in the DNA binding domain, and this modification results in decreased DNA binding (29). PKC-{delta} could downregulate IL-6 by inhibiting NF-IL6 DNA binding through phosphorylation of this residue. Indeed, markedly enhanced DNA binding of NF-IL6 transcription factor was observed in PKC-{delta}–/– B cells after endotoxin treatment (29). Concomitantly, higher NF-IL6 DNA binding activity occurs in PKC-{delta}–/– SMCs, suggesting that this may be the mechanism of negative regulation in IL-6 gene expression by PKC-{delta}.

Obviously, mechanical stress is a strong stimulus for IL-6 induction in SMCs in vitro. As discussed above, altered biomechanical forces in vivo are crucial for the development of arteriosclerosis. One of the best models for studying elevated cyclic stress in vivo is vein bypass grafting in animals, as the grafted veins are exposed to increased blood pressure, i.e., arterial vs. venous blood pressure (44). Arteriosclerotic lesions in this model develop rapidly in wild-type (46) and apolipoprotein (apo)E–/– (6) mice. The main cell component in lesions of vein grafts is SMCs (46). In the present study, we demonstrate that the elevated IL-6 production appeared predominantly in SMCs of arteriosclerotic lesions in this model. These findings have several implications. First, our data support the role of biomechanical forces contributing to IL-6 induction in SMCs in vivo, because increased IL-6 induction was only seen in vein grafts and not in normal vessels. Second, our in vitro observations of the signaling pathways involved might also be relevant to the in vivo situation, although there is a more complicated environment for SMCs in vein grafts. Finally, biomechanical stress-stimulated IL-6 production in SMCs might be responsible for both local as well as systemic elevations in IL-6 in patients with vascular diseases.

In conclusion, our results demonstrate that biomechanical stress can trigger IL-6 expression in SMCs in vivo and in vitro. The signal transduction pathways involve Ras/Rac1/p38 MAPK/NF-{kappa}B/NF-IL6, which is negatively regulated by PKC-{delta}. IL-6 induced by mechanical stimulation in SMCs can be released into the intercellular space, where it exerts its role in initiation of an inflammatory response. If altered biomechanical stress in the vessel persists, e.g., in the branching areas of arteries, local elevation of IL-6 may initiate an inflammatory response and thus lead to the formation of early lesions.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the British Heart Foundation and the Oak Foundation.


   ACKNOWLEDGMENTS
 
We thank Drs. Ali R. Afzal (for help in RT-PCR experiments) and Evelyn Torsney (for critical reading of the manuscript).


   FOOTNOTES
 

Address for reprint requests and other correspondence: Q. Xu, Dept. of Cardiac and Vascular Sciences, St. George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK (E-mail: q.xu{at}sghms.ac.uk)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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