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(Circulation Research. 2002;91:398.)
© 2002 American Heart Association, Inc.

Molecular Medicine

G_q and Gß Regulate PAR-1 Signaling of Thrombin-Induced NF-B Activation and ICAM-1 Transcription in Endothelial Cells

Arshad Rahman^*, Andrea L. True^*, Khandaker N. Anwar, Richard D. Ye, Tatyana A. Voyno-Yasenetskaya, Asrar B. Malik

From the Department of Pharmacology, College of Medicine, The University of Illinois, Chicago, Ill.

Correspondence to Arshad Rahman, Dept of Pharmacology, The University of Illinois, College of Medicine, 835 South Wolcott Ave (M/C 868), Chicago, IL 60612-7343. E-mail ARahman{at}uic.edu

	Abstract

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As thrombin binding to the G protein–coupled proteinase activated receptor-1 (PAR-1) induces endothelial adhesivity to leukocytes through NF-B activation and intercellular adhesion molecule-1 (ICAM-1) expression, we determined the signaling pathways mediating the response. Studies showed that the heterotrimeric G proteins, G_q, and the Gß dimer were key determinants of the PAR-1 agonist peptide (TFLLRNPNDK)-induced NF-B activation and ICAM-1 expression in endothelial cells. Cotransfection of RGS3T, a regulator of G-protein signaling that inhibits G_q, or -transducin (G_t), a scavenger of the Gß, markedly decreased NF-B activity induced by PAR-1 activation. We determined the downstream signaling targets activated by G_q and Gß that mediate NF-B activation. Expression of the kinase-defective protein kinase C (PKC)- mutant inhibited NF-B activation induced by the constitutively active G_q mutant, but had no effect on NF-B activity induced by Gß₁₂. In related experiments, NF-B as well as ICAM-1 promoter activation induced by Gß₁₂ were inhibited by the expression of the dominant-negative mutant of 85-kDa regulatory subunit of PI 3-kinase; however, the expression of this mutant had no effect on the response induced by activated G_q. Cotransfection of the catalytically inactive Akt mutant inhibited the NF-B activation induced by the constitutively active PI 3-kinase mutant as well as that by the activated forms of G_q and PKC-. These results support a model in which ligation of PAR-1 induces NF-B activation and ICAM-1 transcription by the engagement of parallel Gq/PKC- and Gß/PI3-kinase pathways that converge at Akt.

Key Words: G proteins • protein kinase C- • Akt • nuclear factor-B • intercellular adhesion molecule-1

	Introduction

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The procoagulant thrombin, a serine protease activated during thrombosis, induces the expression of intercellular adhesion molecule-1 (ICAM-1) in endothelial cells.¹ ICAM-1, the ligand for the leukocyte ß₂ integrins (CD11/CD18), promotes the firm adhesion of leukocytes, and facilitates their migration across the endothelial barrier.² ICAM-1 is constitutively present on the endothelial cell surface, but its expression can be induced by the activation of the transcription factor nuclear factor (NF)-B.^1,3 NF-B is typically a heterodimer of 50-kDa (p50) and 65-kDa (RelA) subunits sequestered in the cytoplasm in association with IB proteins that mask the NF-B nuclear localization signal.⁴ Stimulation with proinflammatory mediators such as thrombin results in serine phosphorylation (Ser³² and Ser³⁶) of IB by IBß kinase (IKKß).⁵ Phosphorylation targets IB for ubiquitination and proteasome-mediated degradation.⁶ The released NF-B translocates to the nucleus where it binds to cis-regulatory element present in the ICAM-1 promoter.¹

Thrombin activates protease activated receptor-1 (PAR-1) by the cleavage of its NH₂-terminal domain at Arg-41 and Ser-42, unmasking the tethered ligand (SFLLRN),⁷ which interacts with the extracellular loop 2 of the receptor (amino acids 248 to 268).⁸ PAR-1 activation induces endothelial adhesivity toward human neutrophils (PMNs) and monocytes.^1,9 The coupling of PAR-1 with G_q, G_i, and G12/13 in endothelial cells is required for the activation of multiple cellular responses.¹⁰ In the present study, we addressed the pathways by which PAR-1 signals NF-B activation and ICAM-1 transcription in endothelial cells. The results demonstrate that ligation of PAR-1 mediates NF-B activation and ICAM-1 transcription through the heterotrimeric G protein, G_q, and the released Gß dimer. The downstream signaling involves activation of parallel protein kinase C (PKC)- and PI 3-kinase pathways, which converge at Akt to induce NF-B–dependent ICAM-1 transcription.

	Materials and Methods

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Materials
Human thrombin (activity of 3170 NIH U/mg) was purchased from Enzyme Research Laboratories (South Bend, Ind). PAR-1 agonist peptide (TFLLRNPNDK) and inactive control peptide (FTLLRNPNDK) were synthesized.¹¹ Superfect and plasmid maxi kit were from QIAGEN Inc. DEAE-dextran was purchased from Sigma. The construct pTKRLUC containing Renilla luciferase gene driven by the constitutively active thymidine kinase promoter and Dual Luciferase Assay System were purchased from Promega (Madison, Wis). Plasmids encoding Gß₁ and G₂,¹² subunit of bovine retinal transducin (G_t),¹³ activated forms of G_q, pG_qR138C (G_qRC),¹⁴ and G₁₃, pG₁₃Q226L (G₁₃QL)¹⁵ were prepared as described. ICAM-1-LUC construct containing 1393 bp of 5'-regulatory region of ICAM-1 promoter linked to firefly luciferase reporter gene was provided by Dr C. Stratowa (Ernst Boehringer Institut, Vienna, Austria).¹⁶ Expression vectors of the constitutively active catalytic subunit (p110*) and dominant-negative regulatory subunit (*p85) of PI 3-kinase were prepared as described.^17,18 Constructs of wild-type, constitutively active, and kinase-defective mutants of Akt/PKB and expression vector encoding RGS3T are described.^19–21 PKC-, -, and - mutants lacking the functional catalytic domain due to substitution of lysine 368, 437, or 376 for arginine, respectively, were gifts from Dr J. Soh (Columbia University, New York, NY).²² Construct encoding kinase-defective mutant of IKKß is described.¹⁹ Construct pNF-B-LUC containing 5 copies of consensus NF-B sequences linked to a minimal E1B promoter-firefly luciferase gene was purchased from Stratagene.

Endothelial Cell Culture
Human umbilical vein endothelial cells (HUVECs; Clonetics) were cultured²³ in gelatin-coated flasks using endothelial basal medium 2 (EBM2) with bullet kit additives. Confluent cells were incubated for 2 to 12 hours in heat inactivated 0.5% to 1% FBS containing EBM2 before thrombin or PAR-1 agonist peptide challenge. All experiments were made in cells under the 8th passage, except where otherwise indicated.

Northern Analysis
Total RNA was isolated and Northern blotting was performed using the labeled human ICAM-1 cDNA probe (0.54 kb SalI–PstI fragment).²³

Cell Lysis and Immunoblotting
After treatment, cells were lysed in SDS-sample buffer (10 mmol/L Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.4% dithiothreitol, 1 mmol/L orthovanadate with bromphenol blue). Cell lysates were analyzed for ICAM-1 expression by immunoblotting.²³

Transfection and Luciferase Assay
Transfections were performed using DEAE-dextran method.²³ Briefly, 5 µg DNA was mixed with 50 µg/mL DEAE-dextran in serum-free EBM, and the mixture was added onto 70% to 80% confluent cells. We used 0.125 µg pTKRLUC to normalize the transfection variation from plate to plate. After 1 hour, cells were incubated for 4 minutes with 10% dimethyl sulfoxide (DMSO) in serum-free EBM2. Cells were then washed 2x with EBM2 containing 10% FBS and grown to confluence. This protocol resulted in a transient transfection efficiency of 11±2% (mean±SD; n=3). In some experiments, we used Superfect (Qiagen) to transfect the cells.²³ Briefly, reporter DNA (1 µg) was mixed with 5 µL of Superfect in 100 µL serum-free EBM2. We used 0.1 µg pTKRLUC to normalize the transfection variation from plate to plate. After 5 to 10 minutes incubation at room temperature, 0.6 mL EBM2 containing 10% FBS was added, and the mixture was applied onto cells prewashed once with PBS. The medium was changed 3 hours later to EBM2 containing 10% FBS and cells were grown to confluence. Using this protocol, we achieved a transient transfection efficiency of 21±2% (mean±SD; n=3). Cell extracts were prepared and assayed for reporter gene activity using the Promega Biotech Dual Luciferase Assay System. Firefly (Photinus pyralis) luciferase activity was normalized to sea pansy (Renilla reniformis) luciferase activity and expressed as relative light units (RLU)/µg cell protein or fold increase. Protein content was determined using the Bio-Rad protein determination kit.

PMN Adhesion Assay
PMN adhesion assay was performed as described.²⁴ HUVECs grown in 12-well plates were stimulated with PAR-1 agonist peptide or thrombin for 6 to 8 hours followed by labeling with 3 µmol/L fluorescent (red) Cell Tracker dye for 30 minutes. Freshly isolated PMNs were stained with 5 µmol/L fluorescent (green) Cell Tracker dye, coincubated with endothelial cells for 20 minutes, washed with PBS, and visualized using a fluorescent microscope. The number of adherent PMN/0.8 mm² of endothelial cell was counted and expressed as fold increase relative to untreated control.

Statistical Analysis
Data are presented as mean±SEM. Comparisons between groups were made by ANOVA (Tukey’s post-test). Differences were considered significant at P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PAR-1 Induces ICAM-1 Expression and Endothelial Adhesivity PAR-1 agonist peptide (TFLLRNPNDK) was used to address the role of PAR-1 in activation of ICAM-1 expression and endothelial adhesivity toward PMNs. PAR-1 agonist peptide mimicked the effects of thrombin in inducing these responses; however, the magnitude of these responses was 30% to 40% less even at the maximal concentration of TFLLRNPNDK (Figures 1A through 1C). In the control experiment, the inactive peptide (FTLLRNPNDK) failed to induce ICAM-1 transcription (Figure 1A). To address the role of PAR-1 in signaling NF-B activation, HUVECs were transfected with pNF-B-LUC, in which the firefly luciferase reporter gene is driven by multiple NF-B sequences. Results showed that TFLLRNPNDK, like thrombin, induced NF-B–dependent firefly luciferase activity but to a lesser extent (Figure 1D). We also determined whether NF-B activation leads to increased ICAM-1 promoter activity. Exposure of cells transfected with ICAM-1LUC to thrombin induced ICAM-1 promoter activity (Figure 1D) consistent with its effect on ICAM-1 expression and endothelial adhesivity toward PMNs (Figure 1A through 1C).

View larger version (23K): [in this window] [in a new window]   Figure 1. PAR-1 induces NF-B activation, ICAM-1 expression, and endothelial adhesivity toward PMN. HUVECs were challenged with PAR-1 agonist peptide (TFLLRNPNDK) or thrombin at the indicated concentrations. A, After 3 hours, total RNA was isolated and analyzed by Northern hybridization with a human ICAM-1 cDNA. In some experiments, inactive PAR-1 peptide (FTLLRNPNDK) was used as a control. Blots were stripped and re-probed to determine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression as a measure of RNA loading. B, At 6 to 8 hours, total cell lysates were separated by SDS-PAGE and immunoblotted with an antibody against ICAM-1. Blots were stripped and re-probed with an antibody against actin to indicate equal loading of the gel. C, After 8 hours, endothelial adhesivity toward naïve PMN was determined (see Materials and Methods). Data are mean±SE; n=3 for each condition. *Different from control (P<0.05). D, HUVECs were transfected with NF-BLUC or ICAM-1LUC construct using DEAE-dextran (see Materials and Methods). At 12 to 24 hours, cells were incubated for 2 to 4 hours in 1% FBS/EBM2 before challenge with TFLLRNPNDK or thrombin. At 8 or 12 hours, cells were lysed and firefly luciferase activity normalized to Renilla luciferase activity was determined and expressed as fold increase. Data are mean±SE; n=3 for each condition. *Different from control (P<0.05).

G_q and Gß Transduce PAR-1–Induced NF-B Activation
We used the NF-B–dependent firefly luciferase activity as the readout to address the PAR-1–activated signaling mechanisms mediating ICAM-1 transcription. Because PAR-1 couples to multiple heterotrimeric G proteins,^25,26 we initially addressed the contributions of G_q, G_i, and G₁₃ in mediating NF-B activation. RGS3T, a regulator of G-protein signaling that inhibits G_q,²⁷ was used to address the function of G_q. RGS3T significantly reduced NF-B activity after TFLLRNPNDK or thrombin challenge (Figure 2A). As further evidence of the importance of G_q, the expression of constitutively active G_q mutant (G_qRC) induced NF-B activity in the absence of PAR-1 activation (Figure 2B). In contrast, expression of activated Gi₂ (G_i2QL) or G13 (G₁₃QL) had no effect on NF-B activity (Figure 2B). In control experiments, G₁₃QL induced SRE-LUC (serum response element-luciferase) (Figure 2B) and G_i2QL activated PAR-1-Luc (PAR-1 promoter-luciferase)²⁶ (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 2. A and B, Role of Gq in PAR-1–induced NF-B activation. A, HUVECs were cotransfected with NF-BLUC and RGS3T expression construct using DEAE-dextran method. Cells were challenged with thrombin or TFLLRNPNDK, and luciferase activity was determined as in Figure 1D. Data are mean±SE; n=3 for each condition. *Different from vector control (P<0.05); #Different from thrombin- or TFLLRNPNDK-stimulated controls (P<0.05). B, HUVECs were cotransfected with (1) NF-BLUC and constitutively active mutant of Gq (GqRC), Gi2 (Gi2QL), or G13 (G13QL) or (2) SRELUC and G13QL using Superfect (see Materials and Methods). After 12 to 14 hours, cells were lysed and firefly luciferase activity normalized to Renilla luciferase activity was determined and expressed as RLU/µg protein. Data are mean±SE; n=3 to 6 for each condition. *Different from vector controls (P<0.05). C, Role of Gß dimer in PAR-1–induced NF-B activation. HUVECs were cotransfected with pNF-BLUC and subunit of -transducin (Gt) using DEAE-dextran (see Materials and Methods). Cells were challenged with thrombin or TFLLRNPNDK, and luciferase activity was determined (as in Figure 1D). Data are mean±SE; n=3 to 6 for each condition. *Different from vector controls (P<0.05); #Different from thrombin or TFLLRNPNDK-stimulated controls (P<0.05). D and E, Expression of Gß12 induces NF-B activation. HUVECs were cotransfected with (D) NF-BLUC or (E) ICAM-1LUC and equimolar ratios of Gß12 alone or in combination with Gt using Superfect (see Materials and Methods). After 12 to 14 hours, luciferase activity was determined (as in B). Data are mean±SE; n=3 to 6 for each condition. *Different from vector controls (P<0.01); #Different from Gß12 control (P<0.05).

As the released Gß dimer of heterotrimeric G proteins can activate signaling pathways that may lead to NF-B activation, we studied the effects of -transducin (Gt), which sequesters Gß and antagonizes Gß signaling.¹³ Cotransfection of Gt inhibited the NF-B activity induced by thrombin or TFLLRNPNDK (Figure 2C). In another experiment, cotransfection of equimolar concentrations of constructs encoding Gß₁ and G₂ with pNF-B-LUC induced NF-B activity in the absence of PAR-1 activation (Figure 2D); this response was inhibited by expression of Gt (Figure 2D). Coexpression of Gß₁₂ also induced ICAM-1 promoter activity in a Gt-sensitive manner (Figure 2E). These data indicate that both G_q and Gß can independently transduce NF-B activation after PAR-1 activation.

G_q Induces NF-B Activation Through PKC-
Because PKC- is required for thrombin-induced NF-B activity,²³ we addressed the possibility that PKC- functions downstream of G_q in signaling NF-B activation. Cotransfection of kinase-defective mutant of PKC- (PKC-^mut) abrogated the NF-B activity induced by G_qRC, indicating the requirement of PKC- in the response (Figure 3A). Cotransfection of kinase-defective mutants of PKC- (PKC-^mut) and PKC- (PKC-^mut) also reduced the G_qRC-induced NF-B activity, although to a significantly lesser extent (P<0.05) than the PKC-^mut (Figure 3A).

View larger version (25K): [in this window] [in a new window]   Figure 3. Inhibition of PKC- prevents Gq-mediated NF-B activation. HUVECs were cotransfected with (A) pNF-BLUC and GqRC or (B) pNF-BLUC and Gß12 in combination with the constructs encoding kinase-defective mutant of PKC- (PKC-mut), - (PKC-mut), or - (PKC-mut) using Superfect (see Materials and Methods). After 12 to 14 hours, luciferase activity was determined (as in Figure 2B). *Different from vector controls (P<0.01); #Different from GqRC controls (P<0.05).

We also addressed the possibility that PKC- contributes to NF-B activity induced by Gß. Cotransfection of PKC-^mut failed to inhibit the Gß₁₂-induced NF-B activity (Figure 3B), suggesting that PKC- did not contribute to the Gß₁₂-dependent component of the response.

Gß Induces NF-B Activation Through PI 3-Kinase
Dominant-negative mutant of the regulatory 85-kDa subunit of PI 3-kinase (p85) was used to address the role of PI 3-kinase in the mechanism of NF-B activation. Coexpression of p85 inhibited NF-B activity (Figure 4A) induced by thrombin or TFLLRNPNDK. Inhibition of PI 3-kinase activity by p85 also inhibited ICAM-1 promoter activity (data not shown). We also determined the effects of constitutively active mutant of PI 3-kinase (p110*), created by joining the catalytic (p110) and regulatory (p85) subunits through a hinge peptide.¹⁷ Expression of p110* induced NF-B and ICAM-1 promoter activities in the absence of PAR-1 activation (Figures 4B and 4C). These data show that PI 3-kinase is required for PAR-1–induced NF-B activity and ICAM-1 promoter activation.

View larger version (18K): [in this window] [in a new window]   Figure 4. A, Inhibition of PI-3 kinase inhibits PAR-1–induced NF-B activation. HUVECs were cotransfected with pNF-BLUC and a dominant-negative mutant of the regulatory p85 subunit of PI 3-kinase, *p85, using DEAE-dextran (see Materials and Methods). Cells were challenged with thrombin or TFLLRNPNDK for 6 hours, and luciferase activity was determined (as in Figure 1D). *Different from vector controls (P<0.05); #Different from thrombin- or TFLLRNPNDK-stimulated controls (P<0.05). B and C, Expression of activated PI 3-kinase mutant induces NF-B and ICAM-1 promoter activation. HUVECs were cotransfected with (B) NF-BLUC or (C) ICAM-1LUC and the activated form of catalytic subunit of PI 3-kinase, p110*, using Superfect (see Materials and Methods). Luciferase activity was determined (as in Figure 2B). Data are mean±SE; n=3 to 6 for each condition. #Different from vector controls (P<0.001).

To address whether PI 3-kinase functions downstream of Gß, we determined the effects of p85 on the Gß-induced NF-B activity. Cotransfection of p85 prevented the Gß₁₂–induced activation of NF-B (Figure 5A). We also addressed the possibility that PI 3-kinase is a downstream effector of G_q in mediating NF-B activation. However, this study showed that coexpression of p85 failed to prevent NF-B activity induced by G_qRC (Figure 5B), suggesting that PI 3-kinase does not participate in G_q-activated signaling of NF-B activation. These data demonstrate that PI 3-kinase lies downstream of Gß in signaling NF-B activity.

View larger version (19K): [in this window] [in a new window]   Figure 5. PI 3-kinase signals downstream of Gß in mediating NF-B activation: HUVECs were cotransfected with (A) NF-B-LUC and Gß12 or (B) NF-BLUC and GqRC in combination with *p85 using Superfect (see Materials and Methods). Luciferase activity was determined as described in Figure 2B. Data are mean±SE; n=3 to 6 for each condition. *Different from vector controls (P<0.05); #Different from Gß controls (P<0.005).

G_q/PKC- and Gß/PI 3-Kinase Pathways Converge at Akt
We determined whether the serine/threonine kinase Akt lies downstream of both PKC- and PI 3-kinase in mediating PAR-1–induced NF-B activation. Inhibition of Akt by the kinase-defective Akt mutant (Akt^mut) inhibited the NF-B activity in response to thrombin or TFLLRNPNDK challenge, indicating the involvement of Akt in the response (Figure 6A). Cotransfection of Akt^mut also prevented thrombin-induced ICAM-1 promoter activity (data not shown). In another experiment, we determined the effects of inhibition of Akt on NF-B activity induced by constitutively active mutant of PI 3-kinase (p110*). We found that cotransfection of the Akt^mut prevented the NF-B activity induced by p110* (Figure 6B). We also addressed the other possibility that Akt functions downstream of G_q/PKC- in signaling NF-B activation. Expression of wild-type Akt (Akt^wt) augmented G_qRC-induced NF-B activity, whereas expression of Akt^mut inhibited the response (Figure 7A). These findings show a critical role of Akt in signaling the G_q-transduced response. As PKC- functions downstream of G_q (Figure 3), we next determined the effects of inhibition of Akt on NF-B activity induced by the constitutively active PKC- (PKC-^CAT). Results of this experiment showed that cotransfection of Akt^mut inhibited the PKC-^CAT–induced NF-B activity (Figure 7B). In contrast, expression of PKC-^mut had no effect on NF-B activity induced by the constitutively active Akt (Akt^CAT) (Figure 7C). Taken together, these data show that G_q/PKC- and Gß/PI 3-kinase pathways converge at Akt to activate NF-B.

View larger version (21K): [in this window] [in a new window]   Figure 6. A, Involvement of Akt in PAR-1–induced NF-B activation. HUVECs were cotransfected with NF-BLUC and a construct encoding kinase-defective mutant of Akt (Aktmut) using DEAE-dextran (see Materials and Methods). Cells were challenged with thrombin or TFLLRNPNDK and luciferase activity was determined (as described in Figure 1D). *Different from vector controls (P<0.05); #Different from thrombin- or TFLLRNPNDK-stimulated controls (P<0.005). B, Inhibition of Akt prevents PI 3-kinase–mediated NF-B activation. HUVECs were cotransfected with NF-BLUC in combination with kinase-defective mutant of Akt and activated form of the catalytic subunit of PI 3-kinase, p110* using Superfect (see Materials and Methods). Luciferase activity was determined (as in Figure 2B). *Different from vector controls (P<0.05); #Different from p110* control (P<0.005).

View larger version (21K): [in this window] [in a new window]   Figure 7. Inhibition of Akt prevents Gq-and PKC-–mediated NF-B activation. HUVECs were cotransfected with (A) pNF-BLUC and GqRC or (B) pNF-BLUC and activated form of PKC-, PKC-CAT, in combination with wild-type (Aktwt) or kinase-defective mutant of Akt (Aktmut) using Superfect (see Materials and Methods). Luciferase activity was determined (as in Figure 2B). *Different from (A) GqRC control or (B) vector control (P<0.05); #Different from (A) GqRC control or (B) PKCCAT control (P<0.05). C, Inhibition of PKC- fails to prevent Akt-mediated NF-B activation. HUVECs were cotransfected with pNF-BLUC and constitutively active mutant of Akt, AktCAT in combination with the constructs encoding PKCmut using Superfect (see Materials and Methods). Luciferase activity was determined (as in Figure 2B). *Different from vector controls (P<0.05).

Akt Signals PAR-1–Induced NF-B Activation Through IKKß
As NF-B activation involves the degradation of IB requiring its phosphorylation by IKKß, we determined the involvement of Akt in mediating the PAR-1–induced NF-B activity through the activation of IKKß. HUVECs were cotransfected with pNF-BLUC in combination with the kinase-defective IKKß mutant (IKKß^mut). Results showed that expression of IKKß^mut prevented the PAR-1–induced NF-B activity (Figure 8A), demonstrating the requirement of IKKß in the response. We next addressed the function of IKKß in the Akt-mediated NF-B activation. Cotransfection of Akt^CAT activated NF-B in the absence of PAR-1 activation (Figure 8B). Moreover, cotransfection of IKKß^mut prevented Akt^CAT-induced NF-B activity (Figure 8B), indicating the critical role of Akt in mediating the PAR-1–induced NF-B activity through the activation of IKKß.

View larger version (28K): [in this window] [in a new window]   Figure 8. A and B, Inhibition of IKKß prevents Akt-mediated NF-B activation. HUVECs were cotransfected with pNF-BLUC and kinase-defective mutant of IKKß in the absence (A) or presence (B) of AktCAT. Cells were challenged with thrombin or TFLLRNPNDK (A) or left untreated (B) and luciferase activity was determined (as in Figure 1D). *Different from vector controls (P<0.05); #Different from thrombin- or TFLLRNPNDK-stimulated controls (inset) or AktCAT control. C, Signaling events regulating PAR-1–induced NF-B activation and ICAM-1 transcription in endothelial cells. Activation of Gq and dissociation of Gß complex after thrombin stimulation of PAR-1 results in the parallel activation of the PKC-– and PI 3 kinase–dependent pathways. These pathways converge at Akt to activate IKKß and induce NF-B activation and ICAM-1 transcription in endothelial cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thrombin induces endothelial cell adhesivity and firm adhesion of leukocytes by the expression of ICAM-1.^1,9 ICAM-1 expression involves the activation of the transcription factor NF-B¹; however, the signaling mechanisms responsible for NF-B–dependent ICAM-1 transcription remain unclear. In the present study, we addressed the role of the heterotrimeric G proteins coupled to PAR-1 in transducing NF-B activation in endothelial cells. The data showed that PAR-1 induces NF-B activation and resultant ICAM-1 expression through G_q and the Gß dimer. We observed that G_q and Gß independently signal through PKC- and PI 3-kinase, with the signals converging at Akt to induce NF-B activation (Figure 8C).

We used the selective PAR-1 agonist (TFLLRNPNDK)¹¹ to address the role of PAR-1 in mediating NF-B activation. The present study focused on PAR-1 because it is the predominant thrombin receptor in endothelial cells.^26,28 PAR-1 agonist peptide induced NF-B activation and ICAM-1 expression in endothelial cells, much like thrombin itself. PAR family consists of 4 described receptors of which 3 are activated by thrombin (PAR-1, PAR-3, and PAR-4); PAR-2 is activated by trypsin or mast cell tryptase.²⁹ In addition to PAR-1, endothelial cells express PAR-2 and PAR-3.^30,31 As TFLLRNPNDK is not a ligand for PAR-2 and PAR-3,²⁸ NF-B activation and ICAM-1 expression in the present study can only be ascribed to PAR-1. However, because the magnitude of NF-B activation and ICAM-1 expression induced by TFLLRNPNDK was less than thrombin, we cannot exclude the involvement of other PARs in contributing to the thrombin response.

PAR-1 may trigger signaling in endothelial cells via its coupled heterotrimeric G proteins, G_i, G_q, and G_12/13.^10,25,26 We addressed the role of G_i2 and G₁₃ by expression of constitutively active G_i2 (Gi₂QL) or G₁₃ (G₁₃QL) forms. Both G_i2QL and G₁₃QL failed to activate NF-B, indicating that G_i2 and G₁₃ are not required for this response in endothelial cells. The inability of G_i2QL and G₁₃QL to activate NF-B cannot be the result of a lack of activity of these constructs because in positive control experiments the constructs activated PAR-1-Luc (the PAR-1 promoter-luciferase construct)²⁶ and SRE-LUC (serum response element-luciferase), respectively. Because G₁₃QL activates NF-B in HeLa cells, ³² its lack of effect in endothelial cells suggests that G₁₃QL may function in a cell-specific manner. To address the role of G_q, we used RGS3T, a regulator of G-protein signaling that inhibits G_q.^21,27 These findings showed that inhibition of G_q signaling prevented NF-B activation after PAR-1 stimulation. Moreover, the expression of constitutively active G_q mutant (G_qRC) or Gß₁₂ dimer was sufficient in itself to activate NF-B. Because the released Gß complex can induce G protein–coupled receptor signaling, ^12,33 we investigated the possible role of Gß in the mechanism of PAR-1–induced NF-B activity. The inhibition of Gß signaling by -transducin (Gt), which sequesters the Gß subunits,¹³ prevented the Gß- as well as PAR-1–induced NF-B activation responses. Thus, these studies show that both G_q and Gß are important in signaling the PAR-1–induced NF-B activation.

Because the novel PKC isoform PKC- activated by thrombin is required for NF-B activation and ICAM-1 transcription in endothelial cells,²³ we addressed its contribution in signaling NF-B activation downstream of G_q. The results showed that PKC- is in fact critical in signaling NF-B activation in this sequence. The results also show that PKC- and PKC- contribute to the G_q-activated response but to a lesser extent. Thus, the data are consistent with an important role of G_q (which activates both classical and novel PKC isoforms PKC-, PKC-, and PKC-³⁴) in signaling the PAR-1–mediated NF-B activation. These results also support the notion of possible synergism between PKC isoforms to activate NF-B.

In contrast to G_q, the stimulatory effect of Gß on NF-B activity was the result of PI 3- kinase, which catalyzes the addition of phosphate to the 3-OH position of the inositol ring of phosphatidylinositol lipids.³⁵ Two of the ubiquitously expressed PI 3-kinase catalytic subunits, p110 and p110ß, form a heterodimer complex with the regulatory subunits of p85 family.³⁵ Another PI 3-kinase catalytic subunit, p110, lacks the binding site for p85, but instead associates with the regulatory subunit p101.³⁶ Studies have shown that these PI 3-kinase isoforms are activated by Gß released from ligand-activated G protein–coupled receptors.^19,36,37 The present data show that the expression of p85 (ie, dominant-negative mutant of p85 subunit) inhibited both thrombin- and TFLLRNPNDK- as well as Gß-induced NF-B activation. Similar results were obtained with the PI 3-kinase inhibitors, LY294002 and wortmannin (data not shown). These findings are consistent with Trumel et al³⁸ showing the signaling function PI 3-kinase in PAR-1–induced platelet aggregation. Thus, the present studies point to an important role of the p110/p85 heterodimer in mediating PAR-1–induced NF-B activation.

We observed that Akt functions as the downstream effector of both PKC- and PI 3-kinase in mediating the PAR-1–induced NF-B activation. Inhibition of Akt prevented the NF-B activation, indicating that Akt is required for the induction of this response. In addition, inhibition of Akt prevented NF-B activation induced by either GqRC or PKC-^CAT. Although there is controversy concerning the role of Gq in regulating Akt activation in COS-7 cells,^33,39 our evidence indicates that Akt signals downstream of GqRC/PKC-^CAT in mediating NF-B activation in endothelial cells. As additional evidence of the importance of Akt, we showed that inhibition of Akt prevented NF-B activation induced by the constitutively active PI 3-kinase mutant (p110*). These data demonstrate that the Gq/PKC- and Gß/PI 3-kinase pathways converge at Akt to activate NF-B. The data are consistent with other studies showing that both PI 3-kinase and PKC can independently activate Akt.⁴⁰ We showed that each pathway contributes equally to the PAR-1–induced activation of Akt and the downstream activation of NF-B.

We addressed possible mechanisms by which Akt induces NF-B activation. Expression of the kinase-defective mutant of IKKß (IKKß^mut) prevented NF-B activity induced by thrombin, TFLLRNPNDK, and Akt^CAT. These data are in accord with the transient association of Akt with IKK in vivo that induces IKK activation leading to phosphorylation and degradation of IB, and the subsequent NF-B DNA-binding activity.⁴¹ Another mechanism by which Akt can regulate NF-B activity may involve phosphorylation of RelA/p65 subunit of NF-B.⁴² Although the precise basis of Akt-induced NF-B activation is not clear, the present studies indicate that Akt serves as a node for PAR-1–activated signaling in endothelial cells. Thus, the targeting of Akt may be useful in preventing the thrombin-activated inflammatory responses.

	Acknowledgments

 
This work was supported by NHLBI grants HL67424, HL46350, HL64573, and HL45638.

	Footnotes

 
*Both authors contributed equally to this study.

Received February 8, 2002; revision received July 9, 2002; accepted July 31, 2002.

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