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Originally published online as doi:10.1189/jlb.0107009 on June 22, 2007

Published online before print June 22, 2007
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(Journal of Leukocyte Biology. 2007;82:619-629.)
© 2007 by Society for Leukocyte Biology

Molecular mechanisms of thrombin-induced interleukin-8 (IL-8/CXCL8) expression in THP-1-derived and primary human macrophages

Lei Zheng and Manuela Martins-Green1

Department of Cell Biology and Neuroscience, University of California Riverside, Riverside, California, USA

1 Correspondence: Department of Cell Biology and Neuroscience, University of California Riverside, Riverside, CA 92521, USA. E-mail: manuela.martins{at}ucr.edu


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ABSTRACT
 
Under normal conditions, macrophages provide essential innate immune surveillance in tissues. These cells also play key functions during wound healing and in pathological conditions. When macrophages are exposed to thrombin, an enzyme released from leaky blood vessels, they are stimulated to produce inflammatory cytokines, which are critical for wound healing and can also facilitate tumor growth and invasion. Using antibody cytokine arrays, we identified IL-8/CXCL8, a chemokine that plays important functions in inflammation and angiogenesis and consequently in healing and tumor development, as one of the cytokines that is highly stimulated in macrophages by thrombin. Here, we investigated the signal transduction mechanism by which thrombin stimulates IL-8/CXCL8 expression in THP-1-derived and primary human macrophages. We show that JNK is a crucial mediator of the thrombin signaling pathways in macrophages, and the activation of JNK is dependent on stimulation of the Rho small GTPase. The thrombin-induced Rho/JNK cascade is a novel signaling cascade for IL-8/CXCL8 transcription activation. Understanding the molecular mechanism by which thrombin controls the expression of inflammatory cytokines in macrophages can lead to therapeutic interventions, which can provide better management of healing, inflammation, and tumorigenesis.

Key Words: inflammation • chemokine • Rho • c-jun N-terminal kinase • signal transduction • stress response


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INTRODUCTION
 
Thrombin is a multifunctional serine protease, primarily known for its functions in homeostasis and thrombosis. However, this enzyme also plays other important roles in stress response during inflammation [1 , 2 ], wound healing [3 ], and tumor development [4 ]. Immediately after wounding, the precursor form of thrombin, prothrombin, is activated rapidly to promote clot formation and stop bleeding. Activated thrombin has also been shown to remain at the wound site during the inflammatory phase of healing and to function as a potent activator of stress response genes through distinct signaling pathways in a variety of cell types. For example, thrombin stimulates the release of presynthesized, proinflammatory chemokines and other stress response molecules from endothelial cells via a p38 MAPK-dependent pathway [5 ]. In fibroblasts, an ERK/ets-like protein 1 (Elk-1) signaling pathway has been characterized to mediate thrombin-induced, stress-response chemokines [6 7 8 ]. However, it is still not clear what signaling events can be initiated by thrombin in macrophages, which are critical for proper healing.

Macrophages are important cells in healing because of their immunological functions as phagocytes and APC, as well as their potent ability to produce inflammatory cytokines and growth factors [1 2 3 ]. Normally, tissue-resident macrophages undergo slow turnover and provide continuous innate immune surveillance to the tissues. In the early stages of healing, local macrophages are exposed to thrombin and produce inflammatory chemokines, such IL-8/CXCL8, henceforth referred to as IL-8, subsequently recruiting neutrophils, which in turn, produce the chemokine MCP-1 [3 ], recruiting monocytes. Upon arrival at the wound site, these monocytes differentiate into macrophages, and their functions persist until the end of the acute inflammation phase when they undergo cell death during tissue remodeling. Macrophages are also identified as the major effector cells involved in numerous inflammatory diseases and cancers. For example, during the early phase of atherogenesis, proinflammatory cytokines are expressed, and leukocyte recruitment occurs. Macrophages are also known to play central roles in the development of atherosclerotic plaques, as they are the major producers of proinflammatory cytokines and mitogenic factors within the plaque [3 ]. In addition, macrophages are identified within several types of solid tumors [9 , 10 ]. These tumor-associated macrophages (TAMs) have gained recognition for their roles in promoting tumor growth and metastases through the synthesis of growth factors and chemokines. Whereas these TAMs are subjected to the complex tumor microenvironment, the highly permeable vasculature within the tumor stroma allows extravasation of thrombin into the surrounding tissue [11 , 12 ]. This consistent thrombin exposure results in constant stimulation of the macrophages to produce inflammatory proteins such as chemokine proteins, which in turn, exacerbate the inflammatory environment of solid tumors.

IL-8 was identified initially as a chemotactic protein for leukocytes, but more recently, it has been found that this chemokine functions in wound healing [13 , 14 ], angiogenesis [15 ], T cell homing [16 , 17 ], and various aspects of tumorigenesis [18 ]. Tissue injury and tumor growth result in recruitment of macrophages, which are challenged by the local environment to produce various cytokines, including IL-8, influencing the local physiology. As a result of the physiological relevance of thrombin stimulation on macrophages in numerous traumatic and pathological situations and the importance of IL-8 in the same processes, it is critical to understand the molecular mechanisms by which thrombin regulates the expression of this chemokine in these cells so that therapeutic interventions can be developed to provide better clinical wound management, as well as therapies for various inflammatory diseases and cancers. In the work presented here, we delineate the signal transduction mechanisms of thrombin-induced stimulation of IL-8 gene expression in THP-1-derived macrophages and primary human macrophages.


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MATERIALS AND METHODS
 
Reagents
Bovine thrombin (Sigma Chemical Co., St. Louis, MO, USA), a proven functional substitute for human thrombin [19 , 20 ], was reconstituted in deionized water, filtered, and used at 8 U/ml unless noted otherwise. All the chemical inhibitors (Calbiochem, San Diego, CA, USA) were reconstituted in DMSO and used at the concentrations indicated in the figures. PMA (Calbiochem) was dissolved in DMSO and used at a final concentration of 100 nM to induce differentiation of THP-1 monocytes into macrophages. IL-8 and ß-actin mAb (Sigma Chemical Co.); phosphor (p)-ERK, p-JNK, total-ERK, and total-JNK antibodies (Cell Signaling Technology, Beverly, MA, USA); p-I{kappa}B kinase (p-IKK){alpha}, I{kappa}B, RelA, c-Jun, and c-Fos antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and Rho antibody (Pierce, Rockford, IL, USA) were used for immunoblots. Dura reagent (Amersham Biosciences, Piscataway, NJ, USA) was used as substrate for all the immunoblotting.

Cell culture
Human monocytic THP-1 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI-1640 medium with 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate, and 50 mM ß-ME, supplemented with 5% FBS in a 5% CO2 atmosphere at 37°C. PMA was used to induce THP-1 monocytes to differentiate into macrophages. This was accomplished by washing and resuspending the cells in fresh RPMI 1640 + 10% FBS containing 100 nM PMA for 72 h [21 ]. At this point, the cells were washed with serum-free media and cultured overnight in fresh serum-free RPMI-1640 medium and then processed for the various assays. CD14+ primary human monocytes (Cambrex, East Rutherford, NJ, USA) were cultured in the same media supplemented with 100 ng/ml M-CSF (PeproTech, Rocky Hill, NJ, USA) for 5–6 days with replacement of half of the media every 2–3 days to induce the full differentiation into macrophages. The cells (5x105/well) were stimulated with thrombin (10 U/ml) in 300 µl fresh serum-free RPMI 1640 for the conditions indicated in Results (and see Fig. 8 legend). After the stimulation, the supernatants or the cell extracts were collected for assays. For the experiments involving chemical inhibitors, they were used to pretreat the cells for 30 min followed by thrombin stimulation for 6 h or for 1.5 h, depending on the experiment. MTT assays were performed on cells following inhibitor treatment to ensure that the concentrations of the inhibitors were not compromising the functionality of the cells. For the experiments shown, the MTT readings were less than 5% lower than those of the control. We did not use inhibitors at concentrations that caused more than 5% reduction in the MTT readings when compared with untreated cells or if floating cells were observed under phase microscopy.


Figure 8
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  		Figure 8. Probing the thrombin-induced signaling pathway in primary human macrophages. (A) Various signal transduction inhibitors or agonists (SP600125, 0.3 µM; DEX, 100 nM; TRAP-6, 200 µM; U0126, 1 µM; C3, 10 ug/ml) were used to probe the thrombin-stimulated signal transduction pathway in primary human macrophages. (B) RelA nuclear translocation was determined in primary macrophages with immunostaining. The nuclei were stained with DAPI and pseudo-colored in red. The RelA is immunolabeled in green. The top panel is an immunolabeling of thrombin-treated human macrophage with only the secondary antibody (no anti-RelA primary antibody used) serving as the control for the specificity of the immunolabeling. The results represent one of two independent experiments.

Cytokine arrays
THP-1-derived macrophages were prepared as described above and treated with thrombin (10 U/ml) for 6 and 18 h, respectively. Parallel, negative-control trials were subjected to the same condition, except for the thrombin treatment. Cell culture supernatants were collected and used in the Q-Plex human cytokine array (Quansys Biosciences, Logan, UT, USA) to quantify 12 different human cytokines by bioluminescence-based ELISA. The plates were read with a charged-coupled device (CCD) camera, and each cytokine was evaluated and quantified by densitometry with the imaging analysis software by Quansys Biosciences.

Immunoblots
To quantitatively determine the amount of IL-8 secreted by macrophages, an equal number of primary or THP-1 monocytes were seeded and differentiated into macrophages. An equal volume of fresh, serum-free media (300 µl) containing thrombin (8–10 U/ml) was used to treat an equal number of macrophages (0.5x106). All wells were incubated under the same conditions for each of the experiments. The cell culture supernatant containing the secreted IL-8 was collected after 6 h, and equal volumes (30 µl) were loaded onto 20% polyacrylamide-glycerol gels and electrophoresed at 20 V for ~12 h. Transfer was performed at pH 10.4 using a semidry transfer apparatus (The W.E.P. Co., Las Vegas, NV, USA). To determine the phosphorylation of ERK and JNK, we used immunoblot analysis with specific antibodies. Briefly, cells were treated with varying concentrations of the inhibitors for 30 min before stimulation with thrombin. Cells were then lysed in 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 100 mM NaF, 100 mM sodium orthovanadate, and 1 mM EGTA, followed by centrifugation for 5 min at 12,000 g at 4°C. Protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of total protein were subjected to electrophoresis on 10% SDS-PAGE. The membranes were stripped and reprobed with antibdoes specific for the total ERK and JNK or ß-actin to ensure equal loading.

RT-PCR
Total RNA was isolated using TRIzol reagents (Invitrogen, Carlsbad, CA, USA). IL-8 and ß-actin mRNAs were quantified by RT-PCR with the Access One-Step RT-PCR system (Promega, Madison, WI, USA) following the manufacturer’s instruction. Briefly, 1 µg total RNA was used as template for the RT-PCR reaction containing 5 pmol primer pairs for IL-8 and ß-actin. We used the following primers: IL-8 sense, 5'-CACACTGCGCCAACACAGAA-3', IL-8 antisense, 5'-TGTGGATCCTGGCTAGCAGA-3'; ß-actin sense, 5'-AGAAGAGCTACGAGCTGCCT-3', ß-actin antisense, 5'-CACACGGAGTACTTGCGCTC-3'. The RT-PCR reactions followed a cycling condition of one initial 45 min RT reaction incubation at 48°C and then the conventional PCR with 30 cycles of denaturation at 92°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 1 min. The PCR products were electrophoresed on 1.5% agarose gels and visualized with ethidium bromide staining. Bands were photographed by a CCD camera with National Institutes of Health Image software.

Rho activation assay
Rho activity assay was performed as described [22 ]. THP-1-derived macrophages were stimulated with thrombin for the indicated time interval and then lysed in 50 mM Tris, pH 7.5, 300 mM NaCl, 10% glycerol, 1% Nonidet P-40 (NP-40), 0.1% Triton X-100, 5 mM MgCl2, 0.1 mM PMSF, 10 mg/ml leupeptine, and 10 mg/ml aprotinin. Cleared lysates were incubated with bacterially produced GST-Ras-binding domain (RBD; Rhotekin) [23 ] bound to glutathione-agarose beads for 50 min at 4°C. The beads were washed three times with lysis buffer, and then bound proteins were eluted in SDS-sample buffer and analyzed by immunoblot with Rho mAb.

Tet-On retroviral expression system
Open reading frames (ORFs) of C3 exoenzyme/enhanced GFP (eGFP) and V14Rho/eGFP fusions were cloned into the HRSp Tet-On retroviral vector from the RetroTet Art system (Helen Blau, Stanford University, Stanford, CA, USA) to replace the puromycin ORF. Retroviruses were prepared with a HEK293T package cell line with vesicular stomatitis virus-G pseudotyping. To establish different strains of cells, the wild-type THP-1 was infected first with tet-repressor RTHg(–) retrovirus, followed by infection of tet-activator RTAb(+) retrovirus. Then, the THP-1 monocytes were divided and infected separately with HRS-C3/eGFP or HRS-V14/eGFP. The C3/eGFP or V14/eGFP expression in infected THP-1 monocytes or these cells infected and differentiated into macrophages could be induced with doxycycline (DOX; 10 µM) within 36 h, as demonstrated by confocal microscopy.

Antisense oligonucleotides
All the oligonucleotides used in this assay were synthesized and HPLC-purified by Sigma-Genosys (The Woodlands, TX, USA). The specific oligos were as follows: c-fos antisense, 5'-TGCGTTGAAGCCCGAGAA-3', c-fos sense, 5'-TTCTCGGGCTTCAACGCA-3'; c-jun antisense, 5'-CGTTTCCATCTTTGCAGT-3', c-jun sense, 5'-ACTGCAAAGATGGAAACG-3'. All the oligonucleotides have two terminal phosphorodiester bonds replaced with phosphorothioate bonds during synthesis to extend their half-life inside of cells. Purified oligos were reconstituted in water, and 1 µg oligo was transfected into 5 x 105 THP-1-derived macrophages with metafectene (Biontex, Martinsried, Germany) following the manufacturer’s protocol. Oligos were removed after 2 h of incubation, and cells were incubated in serum-free RPMI medium for another 24 h before thrombin treatment. Thrombin (3 U/ml) was used to treat the transfected, THP-1-derived macrophages for 12 h, and IL-8 secretion was determined by immunoblotting as described earlier.

Immunolabeling and confocal microscopy
To prepare cells for immunostaining, cells cultured in chamber slides (Nunc, Denmark) were fixed in 4% paraformaldehyde for 20 min, rinsed with PBS, and then incubated in PBS containing 0.1 M glycine for 10 min and blocked with 10% nonimmune serum of the species in which the secondary antibodies were generated. Primary antibodies in 1% BSA/PBS were applied to the sample for 1 h at room temperature, washed, and incubated in secondary antibody for 1 h at room temperature; this was followed by staining of the nuclei using 4',6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co.) at room temperature for 10–20 min. After extensive washes, the cells were mounted in Vectashield. Immunofluorescence was imaged using a Leica SP2 laser-scanning confocal microscope.

EMSA
Cells were treated as described earlier, and nuclear extracts were prepared as described previously [6 ]. Extracted nuclear protein (1 µg) was used for each binding reaction with 2 ng [{gamma}-32P]ATP-labeled probe containing the consensus AP-1 (5'-CGCTTGATGAGTCAGCCGGAA-3')- or NF-{kappa}B (5'-AGTTGAGGGGACTTTCCCAGGC-3')-binding duplex oligonucleotides in EMSA-binding buffer [250 mM NaCl, 5 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 2.5 mM EDTA, 2.5 mM DTT, 0.25 mg/ml poly(dI-dC)•poly(dI-dC), and 20% glycerol]. Unlabeled probe at concentrations indicated in the figures was used for the competition assay. Samples were assayed in 6% nondenaturing PAGE.

Chromatin immunoprecipitation (ChIP)
The ChIP experiments were carried out following methods published previously [7 ]. Briefly, ~1 x 107 THP-1-derived macrophages were treated with thrombin for the indicated time intervals, washed twice with PBS, and cross-linked with 1% formaldehyde at room temperature for 15 min. Cells were then rinsed with ice-cold PBS twice, scrapped into collection buffer (100 mM Tris-HCl, pH 9.4, 10 mM DTT), incubated for 15 min at 30°C, and centrifuged for 5 min at 2000 g. Precipitated cells were washed with 1 ml ice-cold PBS, buffer I (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 6.5), and buffer II (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 6.5). Then, the cells were resuspended in 0.3 ml lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, with protease inhibitor cocktail) and sonicated three times, 15 s each (Fisher Scientific, Pittsburgh, PA, USA, Model 100), followed by centrifugation for 10 min. Supernatants were collected and diluted in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1). Immunoprecipitation was performed overnight at 4°C with specific antibodies. After immunoprecipitation, 40 µl protein A/G-sepharose was added and continued for another 1 h of incubation. Precipitates were washed for 10 min each in wash buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), wash buffer II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), and wash buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1). Precipitates were then washed three times with Tris/EDTA buffer and extracted three times with 1% SDS, 0.1 M NaHCO3. Eluates were pooled and heated to 65°C overnight to reverse the formaldehyde cross-linking. Then, pH of the eluate was adjusted, and the DNA was purified with the QIAquick Spin kit (Qiagen, Valencia, CA, USA). Primers encompassing the human IL-8 promoter region from –131 to +61 are as follows: sense, 5'-TGTGATGACTCAGGTTTGC-3', antisense, 5'-TGTGCCTTATGGAGTGCTCC-3'. Primers targeting the downstream genomic region (+2361 to +2489) of the IL-8 gene were used as negative control: sense, 5'-ATCTGGCAACCCTAGTCTGC-3', antisense, 5'-GTGCTTCCACATGTCCTCAC-3'. PCR was carried out (94°C, 30 s; 45°C, 30 s; 72°C, 30 s) for 35 cycles, followed by another 10–15 cycles with new PCR master mix if needed.


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RESULTS
 
Thrombin stimulation of multiple inflammatory cytokines in macrophages
To study the effect of thrombin stimulation of macrophages, we examined the inflammation-related cytokine production using a highly sensitive and quantitative, ELISA-based cytokine antibody array, which can quantify 12 inflammation-related human cytokines simultaneously. THP-1-derived macrophages were treated with thrombin (10 U/ml) for 6 and 18 h, the supernatants were collected, and the cytokines were quantified by the Q-Plex human cytokine array (Quansys Biosciences). A total of five proteins was detected in the cell culture supernatant, including four proinflammatory factors (IL-1ß, IL-6, IL-8, TNF-{alpha}) and one anti-inflammatory factor (IL-10). The chemokine IL-8 was highly produced by these THP-1-derived macrophages and reached saturated levels, ~190 µg/ml within 6 h of thrombin exposure (Fig. 1A ) versus 6.5 µg/ml in the nontreated group. As IL-8 plays key roles in wound healing and pathological conditions involving inflammation, we focused our studies on the stimulation of this chemokine by thrombin.


Figure 1
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  		Figure 1. Thrombin stimulates the chemokine IL-8 in human macrophages. (A) ELISA-based human cytokine arrays were used to evaluate production of various cytokines in the cell culture supernatant of thrombin-treated versus nontreated, THP-derived macrophages (M{phi}) at 6 h and 18 h (10 U/ml). Although other proteins were detected in the culture supernatant after thrombin treatment, the most prominent was IL-8. The results represent two independent experiments with triplicates for each trial. No significant differences were observed between experiments. (B) Time- and (C) dose-dependent thrombin stimulation (10 U/ml) of IL-8 production in THP-1-derived macrophages was confirmed by immunoblot analysis. To ensure equal loading for all the IL-8 immunoblots, all experiments were started with the same number of cells (0.5x106), and an equal volume of media was used for treating the cells (300 µl) and for loading in each well (30 µl). (D) RT-PCR analysis of IL-8 mRNA levels in macrophages stimulated by thrombin. ß-actin levels at each time-point were measured as internal controls. (E) Hirudin (a thrombin-specific inhibitor) at 20 U/ml abolishes thrombin-induced stimulation of IL-8 in 6 h. DMSO (used as vehicle) did not stimulate IL-8 production in macrophages. (F) A thrombin-derived peptide agonist thrombin receptor-activating peptide-6 (TRAP-6; 200 µM) stimulates macrophages to produce IL-8, much like thrombin within 6 h. The immunoblots represent one of four independent experiments.

To delineate the molecular mechanisms of IL-8 up-regulation in macrophages upon thrombin stimulation, we first confirmed our cytokine array data by immunoblot analysis (Fig. 1B) . Based on these results, we decided to use 6 h of thrombin treatment for all subsequent experiments. To optimize the concentration of thrombin treatment, we performed experiments with thrombin concentrations varying from 2 U/ml to 16 U/ml (Fig. 1C) and found a strong, dose-dependent effect on IL-8 production. We use concentrations of 8–10 U thrombin/ml for all treatments unless otherwise mentioned, as we obtain sufficiently high levels of IL-8 for our analysis and avoid the effects of excess thrombin. To control for loading of the immunoblots performed to detect IL-8 in the supernatant, we plated equal numbers of cells and kept all treatments and loadings at the same volume (see details in Materials and Methods).

In addition, we used RT-PCR to determine whether thrombin stimulates IL-8 at the mRNA level. The levels of this chemokine were first detected after 1 h of exposure to thrombin and the peak level at 4 h (Fig. 1D) . This dynamic change of IL-8 mRNA level correlates well with that of the IL-8 protein level, which increases significantly between 3 and 6 h after thrombin treatment (Fig. 1B) .

To confirm that the IL-8 stimulation is thrombin-specific, we preincubated the thrombin with five or 20 antithrombin units of purified hirudin (a natural, direct inhibitor of thrombin) per reaction for 30 min and then exposed the cells to this mixture. The effect of thrombin is neutralized completely by excessive hirudin (Fig. 1E) . To determine whether thrombin stimulates IL-8 through its proteolytically activated receptor 1 (PAR-1) [24 ], we treated THP-1-derived macrophages with the PAR-1-specific peptide agonist TRAP-6, which activates PAR-1, much like thrombin does. TRAP-6 (200 µM) stimulates IL-8 production by these macrophages to similar levels as those stimulated by thrombin (Fig. 1F) . These results show that IL-8 stimulation is a result of direct activation by thrombin through its receptor PAR-1.

JNK activation is critical for thrombin stimulation of IL-8 in macrophages
To identify the signaling pathway involved in thrombin stimulation of IL-8, we used chemical inhibitors to probe for key signaling kinases and/or proteases. We used inhibitors for MEK1/2, p38, JNK, Src, epidermal growth factor receptor tyrosine kinase, Her2, PI-3K, protein kinase A, cathepsins, and matrix metalloproteinases and found that the JNK inhibitor SP600125 (0.1 µM) abolished thrombin stimulation (Fig. 2A ) at doses, which do not affect cell metabolism, as determined by the MTT assay, and by microscope observation of floating cells in these cultures, whereas the MEK1/2 inhibitor U0126 (10 µM) inhibited only IL-8 production at high doses (10 µM Fig. 2B ), and p38 kinase inhibitor SB203580 failed to do so, even at high concentrations (Fig. 2C) . To confirm the results obtained with the SP600125 inhibitor on the JNK pathway, we tested a cell-permeable peptide inhibitor of JNK [25 ] in the presence of thrombin stimulation and found that the IL-8 production was inhibited completely by this peptide (Fig. 2D) . Inhibition of all of the other signaling molecules mentioned above did not lead to inhibition of thrombin-induced IL-8 expression.


Figure 2
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  		Figure 2. JNK activation is critical for thrombin stimulation of IL-8. Macrophages were treated with thrombin at the indicated time intervals in the presence of different inhibitors at increasing concentrations: (A) SP600125 for JNK, (B) U0126 for MEK1/2, (C) SB203580 for p38, and (D) TAT-TI-JIP for JNK. Immunoblot analysis was used in a time-dependent manner to determine the activation dynamics of (E) JNK and (F) ERK with antibodies against their phosphorylated forms. The densitometric measurements of the blots have been shown for the JNK and ERK activation experiments. The immunoblots represent one of three independent experiments.

Based on the result of chemical inhibitors, we used immunoblot analysis to further confirm that the JNK pathway is activated by thrombin stimulation through the phosphorylation of JNK. We found that JNK is activated by thrombin in a time-dependent manner, which peaks 30–45 min poststimulation (Fig. 2E) . We also tested the potential activation of ERK1/2 by examining phosphorylation of these kinases upon thrombin treatment and found that their phosphorylation is not increased (Fig. 2F) . Collectively, these experiments provide evidence that JNK activation is critical for thrombin stimulation of IL-8 in THP-1-derived macrophages.

Rho mediates thrombin-induced activation of JNK in macrophages
Recent evidence has emerged that G-protein-coupled receptors may signal through trimeric G-proteins containing the G{alpha}12 and G{alpha}13 subunits to activate the JNK signaling cascade [26 ]. As G{alpha}12 and G{alpha}13 can mediate the interaction of PAR-1 withregulators of the Rho family of small G-proteins [27 ], we investigated the possibility that in THP-1-derived macrophages, thrombin/PAR-1 interactions activate the JNK cascade through a Rho-dependent mechanism. Rho is a small GTPase, which switches between inactive, GDP-bound and active, GTP-bound forms. To determine whether Rho is activated in response to thrombin stimulation, we used a pull-down assay with Rhotekin, the natural substrate of Rho. A Rhotekin RBD was produced by an expression vector, which contained an engineered fusion protein with the GST motif. The GST-RBD fusion protein was conjugated onto agarose beads, and the beads were used to affinity-precipitate the Rho in its GTP-bound, active form from the protein extracts. Thrombin-treated cells were lysed at different time intervals following treatment, and the Rho-GTP in the cell extracts was precipitated and analyzed by immunoblot. To provide a positive control, one sample of cell extracts without thrombin pretreatment was mixed with 10 mM nonhydrolyzable GTP-{gamma}S, which can stably activate most of the Rho in the cell extract. An aliquot of each cell extract before precipitation of activated Rho was saved and also analyzed by immunoblot as a loading control. We found that thrombin stimulation resulted in a time-dependent activation of Rho, which appeared 15 min after thrombin treatment and continued to increase for at least 60 min (Fig. 3A ). To determine whether Rho activation by thrombin is required for this signaling process, we used a tetracycline-inducible retroviral system [28 ] to stably deliver Rho modulator genes into the THP-1-derived macrophages, including a constitutive form of Rho (V14Rho) and an inhibitor of Rho (C3 exoenzyme). V14Rho-eGFP and C3-eGFP fusion ORFs were subcloned into the HRS retroviral vector of the RetroTet Art system (Helen Blau, Stanford University). THP-1 monocytes were infected sequentially with the retrovirus encoding for the repressor RTHg(–) and activator RTAb(+) and then with retrovirus encoding HRS-V14Rho-eGFP or HRS-C3-eGFP. Infected THP-1 monocytes or macrophages differentiated from these infected monocytes were induced with DOX for 2 days, and the expression of V14Rho-eGFP and C3-eGFP was examined by confocal microscopy (Fig. 3B) . Expression of V14Rho or C3 in THP-1-derived macrophages did not compromise the viability of the cells, which were determined by MTT assay and by morphological observations. Counting of the eGFP-positive cells versus nuclei staining by DAPI revealed a positive transgene expression rate of ~92% for V14 and 90% for C3 lines. We then treated the THP-1-derived macrophages expressing various Rho modulators with thrombin and looked for IL-8 expression. Without DOX induction, V14Rho- and C3-expressing macrophages responded to thrombin stimulation like the wild-type cells with a significant level of IL-8 expression (Fig. 3C) . However, after stimulation of transgene expression by DOX, V14 macrophages expressed considerable levels of IL-8, even without thrombin stimulation (Fig. 3C) , suggesting that constitutive activity of Rho is sufficient to stimulate IL-8 expression. Conversely, thrombin-induced IL-8 expression in C3 macrophages was virtually eliminated (Fig. 3C) , suggesting that Rho activity is required for thrombin-induced IL-8 expression. To determine whether blocking Rho activity can lead to inhibition of JNK activation by thrombin, we used the wild-type, V14, and C3 lines of macrophages, induced them with DOX, then stimulated the cells with thrombin for 45 min, and analyzed for JNK phosphorylation. Unlike the wild-type macrophage, the V14 macrophages displayed constitutive JNK phosphorylation, even without thrombin stimulation (Fig. 3D) . Conversely, thrombin-induced JNK phosphorylation is virtually blocked in C3-expressing macrophages (Fig. 3D) , which confirms our findings that Rho activity is crucial for thrombin-induced JNK activation and supports our hypothesis that Rho is upstream of JNK. Taken together, these results indicate that the Rho/JNK cascade is crucial for thrombin-induced IL-8 expression in THP-1-derived macrophages.


Figure 3
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  		Figure 3. Rho/JNK cascade is important for thrombin-induced IL-8 expression. (A) Affinity precipitation assays were performed using the Rhotekin RBD to determine whether Rho can be activated by thrombin treatment. Precipitated, GTP-bound, activated Rho was analyzed with immunoblot analysis. GTP-{gamma}S-pretreated samples were included in the affinity precipitation as a positive control. Aliquots of total protein before precipitation were used as loading controls. (B) THP-1 monocytes were stably infected with retrovirus expressing tet-controlled V14Rho-eGFP or C3-eGFP genes, and then, DOX (10 µM) was used to induce the differentiated macrophages for ~2 days. The DOX-induced expression of V14Rho-eGFP and C3-eGFP was examined with confocal microscopy. (C) Overexpression of Rho modulators was able to interfere with thrombin stimulation of IL-8 in wild-type (WT) or infected, differentiated macrophages, with or without DOX induction. The IL-8 expression was determined using immunoblot analysis. For loading control, see Figure 1 or Materials and Methods. (D) Macrophages preinfected with retrovirus were induced with DOX for 2 days, and the level of JNK activation was measured by immunoblot analysis to determine the active JNK level. The results represent one of three independent experiments.

The AP-1 transcription factor contributes to thrombin-stimulated IL-8 expression in macrophages
Active JNK is able to phosphorylate a number of substrate proteins, including the prototypic c-Jun at Ser-63 and Ser-73, leading to an increase in activation of the AP-1 transcription factor [29 ]. To determine whether thrombin stimulation of IL-8 through JNK is dependent on AP-1 activation, we prepared nuclear extracts from the cells treated with thrombin for 2 h, probed them with a radioactively labeled AP-1 consensus oligonucleotide (Promega) shown to be specific for this transcription factor, and analyzed the reaction by EMSA. Two hours of thrombin exposure led to a dramatic increase of AP-1 binding to the labeled oligo, whereas 30-fold excessive, unlabeled, competing oligo successfully out-competed the binding of AP-1 to the labeled oligo, showing the specificity of the DNA/protein-binding (Fig. 4A ). When the cells were preincubated with the JNK inhibitor SP600125, the increase of AP-1 binding to DNA was diminished significantly, suggesting that the AP-1 activation is partially JNK-dependent (Fig. 4A) .


Figure 4
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  		Figure 4. AP-1 transcription factor contributes to thrombin-stimulated IL-8 expression. (A) Macrophages were stimulated with thrombin for 2 h, with or without JNK inhibitor SP600125 (0.3 µM). Nuclear extracts containing 2 µg protein were incubated for 1 h with 32P-labeled oligonucleotides corresponding to the AP-1 sequences derived from the IL-8 promoter. Specificity of the AP-1 transcription factor-binding to the IL-8 promoter was done by incubating the nuclear extracts with unlabeled oligonucleotides (30–100x). The nucleotide–protein complex was resolved by PAGE. The AP-1/DNA shift bands are indicated. (B) Antisense (AS) or sense (S) oligos targeting c-Jun or c-Fos were transfected into macrophages, followed by thrombin stimulation, and the supernatants were analyzed for IL-8 by immunoblotting. The results represent one of three independent experiments.

To confirm these results, we used another approach to reduce the AP-1 protein and determine whether this can impede thrombin stimulation to IL-8. We used specifically designed antisense oligonucleotides to knock down AP-1 at the mRNA level [30 , 31 ], which has been shown previously to reduce the biological effects mediated by c-fos and c-jun [30 , 32 ]. The antisense oligos are targeted to the regions surrounding the translation initiation sites on the c-Fos and c-Jun mRNAs. The sense oligos for the same region to both antisense oligos were designed as negative controls. These oligos were synthesized with terminal phosphothioated bonds to increase their stability within cells. THP-1-derived macrophages were transfected with these oligos, allowed to recover in serum-free media for 18 h, and then treated with thrombin or vehicle. The supernatants were collected after 12 h, and IL-8 levels were determined by immunoblot analysis (Fig. 4B) . These results showed that knocking down c-Jun or c-Fos proteins (or both) by antisense oligos only inhibits thrombin stimulation of IL-8 partially. However, the comparison of the cells transfected with the antisense oligos with those transfected with sense oligos demonstrated that the cells with antisense had a lower level of response to thrombin than those with sense oligos (Fig. 4B) . Taken together, our data suggest that AP-1 is in part responsible for the thrombin-induced IL-8 production, but other factors/pathways may also be crucial for this signal transduction.

The NF-{kappa}B pathway is crucial for thrombin stimulation of IL-8 in macrophages
To identify other factors/pathways crucial for thrombin stimulation, we probed the NF-{kappa}B pathway with dexamethasone (DEX), which is a potent, anti-inflammatory glucocorticoid, repressing the activation of many inflammatory genes by activating the glucocorticoid receptor, which in turn, inhibits the binding of a critical inflammatory regulator, NF-{kappa}B, to its cognate cis-element on DNA [33 ]. We found that DEX abolished thrombin stimulation completely (Fig. 5A ). However, DEX is also known to block AP-1 and JNK activity [30 ]; therefore, we tested a NF-{kappa}B activation-specific inhibitor Quinazoline [34 ] and found that Quinazoline also inhibits thrombin-induced IL-8 production (Fig. 5B) . The canonical and noncanonical pathways of NF-{kappa}B activation involve the phosphorylation and activation of a trimeric regulatory complex composed of IKKs, which includes catalytical subunits IKK{alpha} and IKKß and a regulatory subunit IKK{gamma} [35 ]. The activated IKK complex will, in turn, phosphorylate the NF-{kappa}B inhibitory subunit I{kappa}B, which normally binds to the heterodimer of NF-{kappa}B from the cytoplasm to the nucleus in unstimulated cells. When I{kappa}B is phosphorylated, it is targeted into the proteosome-mediated protein degradation pathway, and the degradation of I{kappa}B is followed by liberation of heterodimer RelA/p50 in the cytosol, which then translocates to the nucleus and functions as a transcription activator [36 , 37 ].


Figure 5
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  		Figure 5. The NF-{kappa}B pathway is crucial for thrombin stimulation of IL-8. (A) DEX and (B) NF-{kappa}B activation inhibitor Quinazoline were preincubated for 30 min with macrophages followed by thrombin treatment. IL-8 production was determined with immunoblot analysis. Evaluation of NF-{kappa}B involvement was accomplished by stimulation of macrophages with thrombin for the indicated time intervals followed by protein extraction. p-IKK{alpha} (C) and total I{kappa}B protein levels (D) were determined by immunoblot analysis. (E) Macrophages were stimulated with thrombin for 1 h, with or without JNK inhibitor SP600125 (0.3 µM) and then fixed with paraformaldehyde for immunolabeling to localize the RelA subunit of NF-{kappa}B using confocal microscopy. (F) Macrophages were stimulated with thrombin for 2 h in the presence of the same inhibitor. Nuclear extracts containing 2 µg protein were incubated for 1 h with 32P-labeled oligonucleotides, corresponding to the NF-{kappa}B sequences, derived from the IL-8 promoter. Unlabeled oligonucleotides were included as cold competitors. RelA antibody (2 µg) was added for the supershift assay. The results represent one of three independent experiments.

With immunoblot analysis, we first showed that IKK{alpha}, which is phosphorylated in a canonical and noncanonical NF-{kappa}B pathway, is activated in a time-dependent manner in response to thrombin treatment (Fig. 5C) , and I{kappa}B protein was degraded over time (Fig. 5D) , which collectively suggests that thrombin stimulates the activation of the trimeric IKK complex, controlling the activation of NF-{kappa}B. We demonstrated further, by immunostaining of thrombin-stimulated, THP-1-derived macrophages (Fig. 5E) , that the RelA subunit localizes in the cytoplasm of these cells without stimulation. However, 2 h of thrombin stimulation resulted in significant nucleus translocation of the RelA subunit, and this process is JNK activity-dependent, as the JNK inhibitor SP600125 significantly inhibited translocation of RelA to the nucleus. To provide further evidence of NF-{kappa}B activation by thrombin, we also performed EMSA assays. The nuclear extracts were prepared from the cells treated with thrombin for 2 h and were probed with the radioactively labeled NF-{kappa}B consensus oligos (Promega). We found that thrombin stimulation increases the binding of the active NF-{kappa}B heterodimer (RelA/p50) significantly (Fig. 5F) . These results also show that the increase of NF-{kappa}B-DNA binding is a result of JNK, as the inhibitor SP600125 blocks such increase. Moreover, binding of NF-{kappa}B to DNA could be successfully out-competed by a 30-fold of cold oligos, and preincubation of the complex with an antibody to RelA resulted in a supershift (Fig. 5F , Lane 9). These data indicate that NF-{kappa}B activation is a key pathway for IL-8 expression stimulated by thrombin in THP-1-derived macrophages.

Thrombin activates NF-{kappa}B through the Rho/JNK cascade in THP-1-derived macrophages
As we have identified the JNK and Rho cascade to be the key signal transduction pathway and NF-{kappa}B as the key transcription regulator for thrombin stimulation of IL-8, we examined whether thrombin can activate NF-{kappa}B through the Rho/JNK cascade. First, we used the RetroTet Art system to examine whether changes in Rho activity could affect the activity of the NF-{kappa}B pathway. We found that the V14Rho-expressing macrophages have a constitutive, higher level of active IKK{alpha} compared with wild-type and C3-expressing macrophages (Fig. 6A ). Simultaneously, we also detected a relatively lower level of the NF-{kappa}B inhibitory protein I{kappa}B without thrombin treatment in V14Rho-expressing macrophages versus wild-type and C3-expressing macrophages (Fig. 6B) . Moreover, we found that the constitutive activity of V14Rho, which stimulates the production of IL-8 without thrombin treatment (Fig. 3C) , is JNK and NF-{kappa}B activity-dependent (Fig. 6C) . These results indicate that thrombin signals through the Rho/JNK cascade to activate the NF-{kappa}B transcription factor and transcription of the IL-8 gene.


Figure 6
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  		Figure 6. The NF-{kappa}B pathway is Rho/JNK-dependent. The expression of Rho modulators C3-eGFP and V14-eGFP in infected macrophages was induced by DOX for 2 days. Then the IKK{alpha} (A) and I{kappa}B (B) levels in the cells, after 1.5 h of thrombin treatment, were determined by immunoblotting. (C) The macrophages expressing various Rho modulators were also stimulated with thrombin for 6 h in the presence of different inhibitors (SP600125 0.3 µM or DEX 100 nM) as indicated, and IL-8 production was quantified to determine whether NF-{kappa}B activation was dependent on the Rho/JNK activation. The results represent one of four independent experiments.

Thrombin promotes the recruitment of AP-1 and NF-{kappa}B transcription factors to the IL-8 gene promoter
We performed ChIP assays to determine whether AP-1 and NF-{kappa}B transcription factors have physical interaction with the regulatory region of the IL-8 gene. With the Transcription Element Search Software (TESS; University of Pennsylvania, Philadelphia, PA, USA) program, we identified that the regulatory regions of the IL-8 gene contains AP-1 and NF-{kappa}B consensus-binding sites (Fig. 7A ). PCR primers flanking these cis-elements were designed for the ChIP assay. The THP-1-derived macrophages were treated with thrombin, and then, the DNA–protein physical interactions were stabilized by formaldehyde cross-linking at specific time-points after thrombin treatment. Antibodies against the two transcription factors were used to immunoprecipitate the DNA/transcription factor complex, followed by reversal of the cross-linking and purification of the coprecipitated DNA, which was then quantified by PCR with primers designed to flank the cis-elements in the IL-8 promoter region. The ChIP assays showed that c-Fos and c-Jun start to interact with the IL-8 promoter region ~60 min following thrombin treatment and peak at approximately 90 min and then gradually decline (Fig. 7B) . The active NF-{kappa}B heterodimer RelA/p50 also begins to have elevated physical interaction with the IL-8 promoter after 60 min of thrombin stimulation, and the interaction stays elevated for as long as 120 min (Fig. 7B) . We did not detect any interaction of these transcription factors with the downstream genomic region of the IL-8 gene (Fig. 7B) . The dynamic interaction between these transcription factors and the IL-8 promoter region confirms that AP-1 and NF-{kappa}B are involved in thrombin-induced IL-8 expression, and these interactions occur in a differential manner in terms of time and intensity.


Figure 7
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  		Figure 7. Thrombin promotes the recruitment of AP-1 and NF-{kappa}B transcription factors to the IL-8 gene promoter. (A) The potential transcription factor-binding elements present in the promoter of the IL-8 gene were predicted by the TESS program and labeled relative to the transcription initiation site as +1. (B) Macrophages were treated with thrombin over time as indicated, and the chromatin with associated transcription factors was cross-linked with formaldehyde. Various antibodies were used to precipitate different proteins, and then, DNA was released from the complex and purified to serve as a template for PCR with the primer pair indicated by the arrows (A). The ChIP assay resulted in an approximate 200-bp band after PCR, which indicates the association of each specific protein to the IL-8 promoter region during that specific time interval. The results represent one of two independent experiments. EBP, Element-binding protein; SREBP, sterol response EBP.

Probing the thrombin-induced signaling pathway in primary human macrophages
To determine whether the signaling pathway stimulated by thrombin and leading to IL-8 expression in THP-1-derived macrophages also occurs in primary macrophages, we performed key experiments in CD14+ human peripheral monocytes. These cells were induced by M-CSF for 5–6 days to fully differentiate into macrophages and then pretreated with agonists or inhibitors to the key signaling molecules, identified previously in THP-1-derived macrophages, including JNK, Rho, and NF-{kappa}B, followed by thrombin stimulation. IL-8 was seen in the supernatant of the cultures within 6 h of thrombin stimulation (Fig. 8A ). All of the inhibitors or agonists produced expected results in primary macrophages. The RelA subunit nuclear translocation in primary human macrophages was also shown with immunolabeling (Fig. 8B) . In addition to observing dramatic translocation of the RelA subunit to the nucleus in primary cells when treated by thrombin, there was also discernable shrinkage of the nuclei, which was less obvious in THP-1-derived macrophages.


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DISCUSSION
 
In this study, we investigated the molecular mechanisms by which thrombin stimulates the expression of proinflammatory cytokine IL-8 in THP-1 cells and primary human macrophages. We discovered that thrombin stimulates IL-8 expression in THP-1-derived macrophages in a time- and dose-dependent manner via its receptor PAR-1. This stimulation requires sequential activation of Rho/JNK, and activation of the Rho/JNK cascade results in activation of two transcription factors, NF-{kappa}B and AP-1, both of which contribute to IL-8 gene transcription activation (Fig. 9 ). These conclusions support the data we obtained from primary human macrophages (Fig. 8) , although with primary human microphages, the TRAP-6 produced a weaker response, and the C3 treatment only resulted in partial inhibition to IL-8 expression. The relatively lower level of response to TRAP-6 stimulation potentially results from the different level of PAR-1 expression on THP-1 and primary cells. For the experiments with C3, retroviral delivery of C3 into THP-1 cells can result in expression of this protein in a high level compared with the passive diffusion uptake in the C3 protein-treated primary macrophages, and this can possibly account for the partial inhibition of C3 in primary macrophages. To the best our knowledge, this is the first report of thrombin-induced Rho/JNK activation leading to chemokine gene transcription activation. Our findings contribute to the understanding of the molecular mechanisms of inflammatory responses in macrophages, especially to the link between the Rho family of small G-proteins and downstream transcription factors, which may help in developing therapeutic approaches to modulate such molecular/cellular processes in pathological, inflammatory conditions.


Figure 9
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  		Figure 9. Summary of the molecular mechanism of thrombin-induced IL-8 expression in macrophages. Thrombin interacts with its cell surface receptor PAR-1, turning on a signaling cascade involving Rho, activating JNK, which in turn, phosphorylates and activates the AP-1 and NF-{kappa}B transcription factors, involved in the direct activation of IL-8 gene expression.

Our previous research identified an ERK/Elk-1 pathway, which mediates thrombin-induced chicken IL-8 expression in fibroblasts [6 , 7 ], which is followed by activation of the Elk-1 transcription factor. In contrast, we found that in macrophages, the ERK pathway is constitutively active and does not participate in thrombin-induced signaling (Fig. 2F) . Instead, we identified JNK, another MAPK, as the central mediator of the signaling. JNK is known traditionally as the kinase that phosphorylates the c-Jun subunit of the AP-1 transcription factor, but the JNK pathway was also found to cross-activate the NF-{kappa}B transcription factor, which is a key transcription regulator of inflammatory genes. The IL-8 promoter region has several cis-elements including NF-{kappa}B, AP-1, C/EBP, and SREBP. Using ChIP assays, we showed that AP-1 and NF-{kappa}B interact with the IL-8 promoter, but NF-{kappa}B has a much longer, sustained interaction than AP-1. In addition, the RT-PCR results showed that the IL-8 mRNA is elevated significantly 1–6 h after thrombin stimulation. This correlates well with the interaction of NF-{kappa}B with the IL-8 promoter, which starts to increase between 30 and 60 min and stays on as long as 2 h after treatment. The interaction of AP-1 with the IL-8 promoter is of lower magnitude as well as shorter duration compared with that of the NF-{kappa}B. Moreover, we did not observe significant nuclear concentration of c-Jun or c-Fos with immunolabeling (data not shown). Nonetheless, using antisense oligos to knock down AP-1 subunits did achieve a partial blocking of IL-8 expression, suggesting the minor contribution of AP-1 to the thrombin-induced IL-8 expression, which relies primarily on NF-{kappa}B at the transcription level.

Under normal surveillance conditions, macrophages are not subjected to stress; hence, IL-8 expression is suppressed. In contrast, under abnormal conditions in which macrophages are exposed to a stress-inducing agent, such as thrombin, IL-8 is up-regulated rapidly and can initiate an inflammatory response quickly. The thrombin receptor, PAR-1, has been shown to signal through many different signaling pathways [24 ], and it has been suggested but not yet shown [26 ] that PAR-1 signaling through G{alpha}12/G{alpha}13 may activate JNK, followed by activation of NF-{kappa}B, leading to the activation of multiple inflammatory genes. To our knowledge, this is the first demonstration that PAR-1 leads to JNK activation through the small GTPase Rho. It is also interesting to mention that we observed that F-actin dynamics are changed dramatically in macrophages after thrombin stimulation (data not shown). The changes of actin dynamics involve a quick disappearance of F-actin in cells (0–15 min) and later, reappearance of F-actin at 45 min–1 h poststimulation. The actin reorganization is a complex cellular event and under the regulation of many molecules. Rho/Rac/Cdc42 small GTPases are well-known to regulate actin treadmilling, which further controls specific aspects of cell adhesion and migration. Studies of these molecules have shown that the signaling pathway involving these small GTPases sometimes also dictates the expression of stress-response genes [38 , 39 ]; however, the exact mechanisms are unclear. Our unpublished results suggest that following thrombin stimulation, Rho is activated in a similar time-frame when the F-actin starts to reappear, and Rho activation is required for IL-8 expression in THP-1-derived macrophages and primary human macrophages.

In summary, our study delineates the signaling pathways by which thrombin stimulates the expression of the macrophage-derived, proinflammatory cytokine IL-8. This activation goes through PAR-1 and is regulated by a Rho/JNK pathway coupled to the activation of the NF-{kappa}B and the AP-1 transcription factors. We show for the first time that the JNK pathway, the strongest IL-8 transcription activation pathway, goes through PAR-1 and the Rho family of small G-proteins. Understanding the mechanism of the signal transduction and transcription activation pathways by which thrombin stimulates IL-8 expression in macrophages could lead to modulation of the functions of these leukocytes in inflammatory settings such as during abnormal healing, chronic inflammation, atherogenesis, and tumorigenesis.


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ACKNOWLEDGEMENTS
 
We thank Drs. F. Sladek and J. Shyy of University of California Riverside (UCR; Riverside, CA, USA) for helpful comments about the manuscript. We also thank Dr. Helen Blau (Stanford University) for the RetroTet Art gene expression system and Dr. K. DeFea of UCR for the GST-RBD fusion protein.

Received January 4, 2007; revised May 14, 2007; accepted May 14, 2007.


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M. L. Petreaca, M. Yao, Y. Liu, K. DeFea, and M. Martins-Green
Transactivation of Vascular Endothelial Growth Factor Receptor-2 by Interleukin-8 (IL-8/CXCL8) Is Required for IL-8/CXCL8-induced Endothelial Permeability
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