We investigated the involvement of the RhoA/Rho-associated kinase (ROCK) pathway in regulating ICAM-1 expression in endothelial cells by the procoagulant, thrombin. Exposure of HUVECs to C3 exoenzyme, a selective inhibitor of Rho, markedly reduced thrombin-induced ICAM-1 expression. Inhibition of ROCK, the downstream effector of Rho, also prevented thrombin-induced ICAM-1 expression. Blockade of thrombin-induced ICAM-1 expression was secondary to inhibition of NF-κB activity, the key regulator of ICAM-1 expression in endothelial cells. In parallel studies we observed that inhibition of the RhoA/ROCK pathway by the same pharmacological and genetic approaches failed to inhibit TNF-α-induced NF-κB activation and ICAM-1 expression. The effect of RhoA/ROCK inhibition on thrombin-induced NF-κB activation was secondary to inhibition of IκB kinase activation and subsequent IκBα degradation and nuclear uptake and the DNA binding of NF-κB. Inhibition of the RhoA/ROCK pathway also prevented phosphorylation of Ser536 within the transactivation domain 1 of NF-κB p65/RelA, a critical event conferring transcriptional competency to the bound NF-κB. Thus, the RhoA/ROCK pathway signals thrombin-induced ICAM-1 expression through the activation of IκB kinase, which promotes NF-κB binding to ICAM-1 promoter and phosphorylation of RelA/p65, thus mediating the transcriptional activation of bound NF-κB.
The recruitment of polymorphonuclear leukocytes (PMN)3 from blood to the site of infection is a highly ordered process. A key step is the stable adhesion of PMN to the endothelium involving the expression of ICAM-1 (CD54) on the endothelial cell surface and activation of its counter-receptor β2 integrins (CD11/CD18) on the PMN surface (1). The interaction of ICAM-1 with β2 integrins enables PMN to adhere firmly and stably to the vascular endothelium and migrate across the endothelial barrier (2, 3). Although ICAM-1 is constitutively expressed in low levels in endothelial cells, its expression can be induced by proinflammatory mediators such as the procoagulant thrombin (4, 5, 6), which is released during intravascular coagulation initiated by tissue injury or sepsis (7, 8, 9). We have shown that the transcription factor NF-κB p65 (RelA) is the key regulator of thrombin-induced ICAM-1 gene transcription in endothelial cells and that this response is mediated through activation of the GTP-binding protein (G protein)-coupled receptor, protease-activated receptor-1 (4, 10).
NF-κB is a ubiquitously expressed family of transcription factors controlling the expression of numerous genes involved in immunity and inflammation (11). The prototypical NF-κB complex, a heterodimer of 50-kDa (p50) and 65-kDa (RelA) subunits, is sequestered in the cytoplasm in an inactive form by its association with IκBα, the prototype of a family of inhibitory proteins termed IκB proteins (12). Activation of NF-κB requires the degradation of IκBα achieved through serine phosphorylation (Ser32 and Ser36) of IκBα by a macromolecular IκB kinase (IKK) complex (13, 14). Phosphorylation targets IκBα for polyubiquitination by the E3-SCFβ-TrCP ubiquitin ligase and subsequently its degradation by the 26S proteasome (15). The released NF-κB rapidly translocates to the nucleus to activate the transcription of target genes such as ICAM-1. Another important mechanism regulating NF-κB activity is through modulation of its transcriptional function by phosphorylation of RelA/p65 (16, 17, 18, 19, 20). Studies have shown that phosphorylation of RelA/p65 at serine 276, 311, 529, or 536 increases the transcriptional capacity of NF-κB in the nucleus (16, 17, 18, 19, 20). However, unlike IκBα phosphorylation, the RelA/p65 phosphorylation site and the kinase involved vary in a stimulus- and cell type-specific manner (16, 17, 18, 19, 20).
The Rho family of monomeric GTPases, Rho, Rac, and Cdc42, serve as molecular switches by cycling between the inactive GDP-bound state and the active GTP-bound state (21). The active conformation facilitates the interaction of Rho GTPases with their downstream targets to activate intracellular signaling. Cycling between the two conformations is regulated by guanine nucleotide exchange factors, which promote the release of GDP and allow GTP to bind, and GTPase-activating proteins, which stimulate the hydrolysis of GTP (21, 22). In addition to the role of Rho GTPases in the regulation of cytoskeletal dynamics, actin stress fiber formation, and myosin L chain phosphorylation, RhoA can affect gene expression through activation of transcription factors such as serum response factor and NF-κB (23, 24, 25). Rho-associated kinase (p160ROCK/Rho kinase), a downstream effector of RhoA, is implicated in a variety of RhoA-mediated responses (26, 27). We have demonstrated that thrombin activation of RhoA is critical in regulating thrombin-induced endothelial barrier function (28). In the present study we addressed the possible role of the RhoA/Rho-associated kinase (ROCK) pathway in signaling ICAM-1 expression in endothelial cells. Our data demonstrate that the RhoA/ROCK pathway signals thrombin-induced ICAM-1 expression in endothelial cells by the activation of IKKβ, which, in turn, mediates NF-κB binding to ICAM-1 promoter and phosphorylation of RelA/p65, thereby conferring transcriptional competency to the bound NF-κB.
Materials and Methods
Human thrombin was purchased from Enzyme Research Laboratories (South Bend, IN). Polyclonal Abs to IKKβ, IκBα, p65/RelA, and β-actin; an mAb to ICAM-1; and GST-IκBα fusion protein were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Abs that detect RelA/p65 when phosphorylated at Ser536 or that detect IκBα when phosphorylated at Ser32 and Ser36Y27632 was purchased from Calbiochem-Novabiochem (La Jolla, CA); the protein assay kit and nitrocellulose membrane were obtained from Bio-Rad (Hercules, CA); the plasmid Maxi Kit was purchased from Qiagen (Valencia, CA). All other materials were obtained from Fisher Scientific (Pittsburgh, PA) or VWR Scientific Products (Gaithersburg, MD).
HUVECs (Cambrex, La Jolla, CA) were cultured as previously described (10) in gelatin-coated flasks using endothelial basal medium 2 (EBM2) with Bullet Kit additives (Cambrex). Confluent cells were incubated in EBM2-containing heat-inactivated 0.5% FBS for 2 h or in 1% FBS for 12 h before thrombin challenge. All experiments were performed with cells between the third and eighth passages, except for the transfection experiments, in which cells were between the third and fifth passages.
Cell lysis and immunoblotting
After thrombin challenge of HUVEC transfected with C3 transferase or pretreated with Y27632, cells were lysed in radioimmune precipitation buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.25 mM EDTA (pH 8.0), 1% deoxycholic acid, 1% Triton X-100, 5 mM NaF, and 1 mM sodium orthovanadate supplemented with complete protease inhibitors (Sigma-Aldrich, St. Louis, MO). Cell lysates were analyzed by SDS-PAGE and transferred onto nitrocellulose (Bio-Rad) or polyvinylidene difluoride membranes, and the residual binding sites on the filters were blocked by incubation with 5% (w/v) nonfat dry milk in TBST (10 mM Tris (pH 8.0), 150 mM NaCl, and 0.05% Tween 20) for 1 h at room temperature or overnight at 4°C. The membranes were subsequently incubated with the indicated Abs and developed using an ECL method as previously described (5).
In vitro IKK assay
Cells were starved by 12-h incubation in 1% FBS containing EBM2. The cells were subsequently challenged with thrombin (2.5–5 U/ml) for 1 h in the absence or the presence of Y-27632 (10 μM), which was added 1 h before thrombin treatment. The cells were then lysed with kinase lysis buffer (20 mM Tris-HCl (pH 7.5), 125 mM NaCl, 100 μM sodium orthovanadate, 25 mM β-glycerophosphate, 50 mM sodium fluoride, 1 mM EDTA, 1 mM magnesium chloride, 1% Triton X-100, 1 mM PMSF, and protease inhibitor mixture (Sigma-Aldrich)). Cell lysates were immunoprecipitated with an Ab against IKKβ using protein A/G-agarose (Santa Cruz Biotechnology) as previously described (5). The immunocomplexes were washed three times with kinase lysis buffer and twice with kinase assay buffer (20 mM HEPES (pH 7.5), 20 mM MgCl2, 0.5 mM EGTA, and 20 mM DTT) and were resuspended in 30 μl of kinase assay buffer containing 1 μg of GST-IκBα, 20 μM ATP, and 10–15 μCi of [γ-32P]ATP. The reaction was incubated for 30 min at 30°C and was terminated by the addition of SDS sample buffer. Proteins were analyzed by SDS-PAGE, and the phosphorylated form of GST-IκBα was detected by autoradiography.
HUVEC transfected with C3 transferase or pretreated with Y27632 were challenged with thrombin, and total RNA was isolated using an RNeasy kit (Qiagen) according to the manufacturer’s recommendations. Quantification and purity of RNA were assessed by A260/A280 absorption, and an aliquot of RNA (20 μg) from samples with a ratio >1.6 was fractionated using a 1% agarose formaldehyde gel. The RNA was transferred to Duralose-UV nitrocellulose membrane (Stratagene, La Jolla, CA) and covalently linked by UV irradiation using a Stratalinker UV cross-linker (Stratagene). Human ICAM-1 (0.96-kb SalI to PstI fragment) (29) and rat GAPDH (1.1-kb PstI fragment) were labeled with [α-32P]dCTP using the random primer kit (Stratagene), and hybridization was conducted as previously described (5). Briefly, the blots were prehybridized for 30 min at 68°C in QuikHyb solution (Stratagene), then hybridized for 2 h at 68°C with random-primed, 32P-labeled probes. After hybridization, the blots were washed twice for 30 min each time at room temperature in 2× SSC with 0.1% SDS, followed by two washes for 15 min each time at 60°C in 0.1× SSC with 0.1% SDS. Autoradiography was performed with an intensifying screen at −70°C for 12–24 h. The nitrocellulose membrane was soaked for stripping the probe with boiled water or with 0.1× SSC /0.1% SDS.
Reporter gene constructs, endothelial cell transfection, and luciferase (LUC) assay
The plasmid pNF-κB-LUC containing five copies of consensus NF-κB sequences linked to a minimal E1B promoter-LUC gene was purchased from Stratagene. The ICAM-1 LUC reporter plasmid containing approximately 1393 bp of ICAM-1 5′-flanking DNA linked to the firefly LUC gene has been described previously (30). The constructs encoding the dominant negative (N19RhoA) and constitutively active (V14RhoA) mutants of RhoA were gifts from Dr. A. Hall (University College London, London, U.K.). The construct encoding the kinase-defective mutant of IKKβ was described previously (5). Transfections were performed using the DEAE-dextran method (30, 31) with slight modifications (5). Briefly, 5 μg of DNA was mixed with 50 μg/ml DEAE-dextran in serum-free EBM2, and the mixture was added to cells that were 70–80% confluent. In some experiments HUVEC were also transfected with C3 transferase. We used 0.125 μg of pTKRLUC plasmid (Promega, Madison, WI) containing Renilla LUC gene driven by the constitutively active thymidine kinase promoter to normalize the transfection efficiencies. After 1 h, cells were incubated for 4 min with 10% DMSO in serum-free EBM2. The cells were then washed twice with EBM2/10% FBS and grown to confluence. We achieved a transfection efficiency of 16 ± 3% (mean ± SD; n = 3) in these cells.
In some experiments we used Superfect (Qiagen) to transfect the cells as previously described (5). Briefly, reporter DNA (1 μg) was mixed with 5 μl of Superfect in 100 μl of serum-free EBM2 (Cambrex). We used 0.1 μg of pTKRLUC to normalize the transfection efficiencies. After a 5- to 10-min incubation at room temperature, 0.6 ml of EBM2/10% FBS was added, and the mixture was applied to the cells that had been washed once with PBS. Three hours later, the medium was changed to EBM2/10% FBS, and the cells were grown to confluence. This protocol resulted in a transfection efficiency of 20 ± 2% (mean ± SD; n = 3). Cell extracts were prepared and assayed for firefly and Renilla LUC activities using a Biotech Dual Luciferase Reporter Assay System (Promega). The data were expressed as the ratio of firefly to Renilla luciferase activity.
Cytoplasmic and nuclear extract preparation
After thrombin challenge of HUVEC transfected with C3 transferase or pretreated with Y27632, cells were washed twice with ice-cold TBS and resuspended in 400 μl of buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF). After 15 min, Nonidet P-40 was added to a final concentration of 0.6%. Samples were centrifuged to collect the supernatants containing cytosolic proteins for determining IκBα degradation by Western blot analysis. The pelleted nuclei were resuspended in 50 μl of buffer B (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF). After 30 min at 4°C, lysates were centrifuged, and supernatants containing the nuclear proteins were transferred to new vials. The protein concentration of the extract was measured using a protein determination kit (Bio-Rad).
EMSAs were performed as previously described (5). Briefly, 10 μg of nuclear extract was incubated with 1 μg of poly(dI-dC) in a binding buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM DTT, and 10% glycerol (20 μl final volume)) for 15 min at room temperature. Then end-labeled, double-stranded oligonucleotides containing an NF-κB site (30,000 cpm each) were added, and the reaction mixtures were incubated for 15 min at room temperature. The DNA-protein complexes were resolved in 5% native PAGE in low ionic strength buffer (0.25× Tris-borate-EDTA). The oligonucleotide used for the gel-shift analysis was NF-κB (5′-AGTTGAGGGGACTTTCCCAGGC-3′) or ICAM-1 NF-κB (5′-AGCTTGGAAATTCCGGAGC-TG-3′). The Ig-κB oligonucleotide contains the consensus NF-κB binding site sequence (underlined) present in pNF-κB-LUC. The ICAM-1 NF-κB oligonucleotide represents a 21-bp sequence of ICAM-1 promoter encompassing the NF-κB binding site located 183 bp upstream of transcription initiation site (32). The sequence motifs within the oligonucleotides are underlined.
Results are expressed as the mean ± SE. Data were analyzed by Student’s t test.
Inhibition of Rho impairs thrombin-induced ICAM-1 expression in endothelial cells
We addressed the role of Rho in signaling thrombin-induced ICAM-1 mRNA expression in endothelial cells. Northern blot analysis showed that transfection of HUVEC with Clostridium botulinum C3 exoenzyme, an inhibitor of Rho (33, 34), markedly reduced ICAM-1 mRNA expression (Fig. 1⇓A). We also determined the effect of inhibition of Rho on thrombin-induced ICAM-1 protein expression. Western blot analysis showed that thrombin challenge of HUVEC resulted in increased ICAM-1 protein expression and that this response was inhibited with C3 exoenzyme (Fig. 1⇓B). Because Rho is implicated in a number of TNF-α responses (35, 36), we evaluated the function of Rho in the mechanism of TNF-α-induced ICAM-1 expression in endothelial cells. We found that inhibition of Rho by C3 exoenzyme failed to prevent ICAM-1 expression induced by TNF-α (Fig. 1⇓).
Rho signals thrombin-induced ICAM-1 expression through ROCK
We investigated the possibility that ROCK, a downstream effector of Rho (26, 27, 37), participates in Rho signaling of thrombin-induced ICAM-1 expression. We found that pretreatment of cells with Y-27632, an inhibitor of ROCK (38), prevented mRNA as well as protein expression of ICAM-1 in response to thrombin challenge of endothelial cells (Fig. 2⇓). In related experiments, inhibition of ROCK failed to prevent ICAM-1 expression by TNF-α (Fig. 2⇓) in contrast to the effect of Rho inhibition on thrombin-induced ICAM-1 expression (Fig. 1⇑).
Inhibition of RhoA/ROCK prevents thrombin-induced NF-κB activity
Because of the essential role of NF-κB in mediating thrombin-induced ICAM-1 transcription, we addressed whether the RhoA/ROCK pathway signals ICAM-1 expression by regulating NF-κB activity. We determined the effects of inhibition of RhoA on NF-κB-dependent reporter activity. Cotransfection of cells with C3 exoenzyme inhibited thrombin-induced NF-κB activity (Fig. 3⇓A). Also, the expression of the dominant negative mutant of RhoA (RhoAmut) inhibited NF-κB activity induced by thrombin (Fig. 3⇓C). However, inhibition of RhoA by C3 exoenzyme or RhoAmut had no significant effect on TNF-α-induced NF-κB activity (Fig. 3⇓, B and D).
We next evaluated whether inhibition of ROCK mimicked the effects of inhibition of RhoA on NF-κB activity. We found that inhibition of ROCK by exposing the cells to Y-27632 impaired thrombin-induced NF-κB activity (Fig. 3⇑A). In parallel experiments, inhibition of ROCK showed no significant effect on TNF-α-induced NF-κB activity (Fig. 3⇑B).
RhoA/ROCK signals thrombin-induced NF-κB activation through IKKβ
We addressed the possibility that RhoA activation is sufficient to induce NF-κB activity. We observed that the expression of constitutively active RhoA mutant (RhoAcat) induced NF-κB activity in the absence of thrombin challenge (Fig. 4⇓A). In another experiment, cotransfection of RhoAcat increased ICAM-1 promoter-dependent reporter activity (Fig. 4⇓B), consistent with its ability in inducing NF-κB activation (Fig. 4⇓A). We next determined the function of RhoA in mediating thrombin-induced IκBα degradation, a requirement for NF-κB activation (39, 40). Because IκBα degradation is contingent on its phosphorylation by IKKβ (41, 42, 43), we evaluated the function of IKKβ in RhoA-mediated NF-κB activation. Coexpression of IKKβmut prevented RhoAcat-induced NF-κB activity (Fig. 4⇓A), indicating that RhoA mediates thrombin-induced NF-κB activity through the activation of IKKβ. To determine the role of ROCK in signaling RhoA activation of IKKβ, we assessed the effects of ROCK inhibition on thrombin-induced IKKβ activity using an in vitro kinase assay in which GST-IκBα was used as an exogenous substrate. We observed that IKKβ immunoprecipitated from thrombin-stimulated cells showed increased phosphorylation of GST-IκBα compared with IKKβ from control cells (Fig. 4⇓C), indicative of activation of IKKβ. Inhibition of ROCK by Y-27632 prevented thrombin-induced IKKβ activation (Fig. 4⇓C).
The above data led us to investigate whether IKKβ activated by RhoA/ROCK catalyzes IκBα phosphorylation (Ser32 and Ser36), and its degradation after thrombin challenge of endothelial cells. The results showed that inhibition of the RhoA/ROCK pathway interfered with the ability of thrombin to induce IκBα phosphorylation, and consequently its degradation (Fig. 4⇑D and Fig. 5⇓, A and B). Because IκBα degradation results in nuclear localization and DNA binding of NF-κB, we determined whether inhibition of RhoA/ROCK would lead to inhibition of these events. We found that inhibition of the RhoA/ROCK pathway abrogated thrombin-induced nuclear translocation and the DNA binding function of nuclear NF-κB (Fig. 5⇓, C and D). In contrast, RhoA/ROCK inhibition had no effect on TNF-α-induced IκBα degradation and DNA binding activity of nuclear NF-κB (Fig. 5⇓, B–D).
Inhibition of ROCK prevents thrombin-induced RelA/p65 phosphorylation
Because phosphorylation of Ser536 in the transactivation domain 1 of RelA/p65 regulates transcriptional activity of NF-κB in the nucleus (20, 44, 45), we addressed the possibility that RhoA/ROCK contributes to NF-κB activity by controlling thrombin-induced phosphorylation of RelA/p65. Western blot analysis showed that thrombin challenge of HUVEC resulted in Ser536 phosphorylation of RelA/p65, and inhibition of RhoA/ROCK after pretreatment of cells with Y-27632 prevented this response (Fig. 6⇓).
We have previously shown that thrombin promotes endothelial adhesiveness toward PMN by a mechanism involving the expression of ICAM-1 via a NF-κB-dependent pathway (4). In this study we provide evidence that activation of the RhoA/ROCK pathway plays an essential role in signaling the thrombin-induced ICAM-1 expression in endothelial cells. Our data demonstrate that the RhoA/ROCK pathway mediates ICAM-1 expression by promoting NF-κB binding to the ICAM-1 promoter by phosphorylation and degradation of IκBα as well as enhancing the transactivation capacity of the bound NF-κB through Ser536 phosphorylation of RelA/p65. Interestingly, RhoA/ROCK mediated NF-κB activation and ICAM-1 expression in response to thrombin, but not TNF-α, the prototypic inducer of NF-κB activation and ICAM-1 expression. These data show that the RhoA/ROCK pathway activates NF-κB-dependent ICAM-1 expression in endothelial cells in an agonist-specific manner, possibly secondary to activation of the G protein-coupled receptor, protease-activated receptor-1, that is ligated by thrombin in endothelial cells (4, 10).
We used pharmacological and genetic approaches to address the role of the RhoA/ROCK pathway in mediating NF-κB activation and ICAM-1 expression after thrombin challenge. We used Clostridium botulinum C3 exoenzyme, specifically inactivates Rho by ADP-ribosylating Asn41 in its effector domain (33, 34), to investigate whether inhibition of RhoA influences ICAM-1 expression. Inhibiting Rho activity markedly reduced thrombin-induced ICAM-1 expression. These data led us to investigate whether RhoA signals thrombin-induced ICAM-1 expression through the activation of the RhoA kinase ROCK, the downstream effector of RhoA (26, 27, 37). Pretreatment of cells with Y-27632, the pyridine-derived smooth muscle relaxant that selectively inhibits ROCK (38), reproduced the effect of RhoA inhibition on ICAM-1 expression induced by thrombin. We have previously shown that thrombin induces ICAM-1 expression via a Gαq/PKCδ-dependent mechanism (5, 10). A recent study has also reported the role of PKCδ in mediating thrombin-induced RhoA activation in endothelial cells (46). These and other studies (47, 48) suggest that a linkage between the Gαq/PKCδ and RhoA/ROCK pathways signals ICAM-1 expression in response to thrombin challenge of endothelial cells. That the Gαq/PKCδ and RhoA/ROCK signaling pathway is important in mediating the thrombin response finds further support from our observation that inhibition of ROCK mimicked the effects PKCδ inhibition on ICAM-1 expression. We also observed that ICAM-1 protein expression was inhibited to a greater extent than NF-κB activity or ICAM-1 mRNA, raising the possibility that ROCK may regulate ICAM-1 expression at post-transcriptional and translational levels.
We evaluated in parallel studies the effects of RhoA/ROCK inhibition on ICAM-1 expression after TNF-α challenge of endothelial cells. These results showed that inhibition of the RhoA/ROCK pathway failed to prevent TNF-α-induced ICAM-1 expression in these cells. Studies have shown that RhoA/ROCK is required for TNF-α-induced reorganization of the actin cytoskeleton and endothelial cell apoptosis (35, 36, 49). Thus, the lack of involvement of the RhoA/ROCK pathway in mediating TNF-α-induced ICAM-1 expression cannot be ascribed to the absence of RhoA/ROCK activation by TNF-α in endothelial cells. The reasons for the different effects of RhoA/ROCK inhibition on thrombin- vs TNF-α-induced ICAM-1 expression are not clear. A possible explanation is that there may be distinct mechanisms of thrombin and TNF-α activation of RhoA/ROCK as well as a specialized set of downstream effectors activated by each agonist. An indication of this difference is that although TNF-α induces ICAM-1 by a PKCζ-dependent mechanism, thrombin, as described above, mediates this response via a PKCδ-dependent pathway (5, 50). Although PKCδ has been implicated in thrombin-induced RhoA activation (46), the role of PKCζ in signaling TNF-α-induced RhoA activation in endothelial cells is not known. It is possible that PKCδ-dependent activation of RhoA/ROCK by thrombin targets it to an intermediate protein, which, in turn, links it to the NF-κB signaling pathway, whereas RhoA/ROCK activated by TNF-α interacts with other downstream targets mediating responses such as endothelial cell apoptosis (49).
In the present study we addressed the mechanism by which the RhoA/ROCK pathway regulates ICAM-1 expression in response to thrombin challenge of endothelial cells. We observed that thrombin-induced NF-κB-dependent reporter activity was significantly reduced in cells pretreated with either C3 exoenzyme or Y-27632. The expression of dominant negative RhoA mutant also prevented NF-κB-dependent reporter activity induced by thrombin. However, inhibition of the RhoA/ROCK pathway using these approaches failed to inhibit the TNF-α-induced, NF-κB-dependent reporter activity. We also used the constitutively active RhoA mutant (RhoAcat) to examine whether activation of RhoA is sufficient to induce NF-κB activity in endothelial cells. The expression of RhoAcat induced NF-κB activity as well as ICAM-1 promoter activation in the absence of thrombin challenge. In other experiments the expression of kinase-defective IKKβ mutant (IKKβmut) prevented the RhoAcat-induced NF-κB activity. These data indicate that the RhoA/ROCK pathway acts upstream of IKKβ in signaling thrombin-induced NF-κB activation in endothelial cells. Consistent with the crucial involvement of IKKβ, we also showed that RhoA/ROCK-mediated IκBα phosphorylation and degradation resulted in the migration of NF-κB to the nucleus, where its binding to the promoter activated ICAM-1 gene transcription. Cammarano and Minden (24) have reported that RhoA activates NF-κB in NIH-3T3 cells in the absence of IKK stimulation. It is not clear what determines IKK-dependent vs -independent activation of NF-κB by RhoA in endothelial compared with NIH-3T3 cells. It is also not known whether ROCK is involved in RhoA activation of NF-κB in NIH-3T3 cells. Recently, Kato et al. (51) showed that IKK-independent activation of NF-κB by UV radiation is mediated by CK2 (formerly casein kinase II) through C-terminal phosphorylation of IκBα. Thus, it is possible that RhoA interacts with different downstream targets depending upon the cell type and stimulus, which, in turn, can dictate whether NF-κB activation occurs by an IKK-dependent or -independent mechanism.
Recent studies have established that signal-induced phosphorylation of RelA/p65 is an additional regulatory pathway activated in parallel with IκBα degradation, and that it plays an important role in conferring transcriptional competency to DNA-bound NF-κB (16, 17, 18, 19, 20). Our results show that thrombin induced the phosphorylation of p65/RelA at Ser536 in transactivation domain 1 mediated by the RhoA/ROCK-dependent pathway. Because IKKβ is implicated in phosphorylating RelA/p65 at Ser536 (20, 44, 45), it is possible that the RhoA/ROCK-mediated activation of IKKβ catalyzes Ser536 phosphorylation of RelA/p65 and thus renders bound NF-κB transcriptionally competent. This is in addition to the other function of Rho/ROCK discussed above to induce IκBα phosphorylation and promote NF-κΒ binding to DNA. However, given that the same serine residue of RelA/p65 can be phosphorylated by more than one kinase (16, 17, 44, 45), the role of other kinases in this response cannot be excluded. We have shown that p38 MAPK also contributes to thrombin-induced ICAM-1 expression by inducing the transactivation capacity of RelA/p65 bound to ICAM-1 promoter (5). Others have shown that p38 MAPK signals downstream of IKK to induce transcriptional activation of RelA/p65 (52). It remains to be determined whether RhoA/ROCK-dependent Ser536 phosphorylation of RelA/p65 by thrombin is directly mediated by IKKβ or it requires the participation of p38 MAPK.
In summary, the present study has identified the important role of the RhoA/ROCK pathway in regulating thrombin-induced NF-κB activation and ICAM-1 expression in endothelial cells. The role of RhoA/ROCK in the thrombin response is distinct because TNF-α-mediated NF-κB activation and ICAM-1 occur independently of this mechanism. Thus, the specific targeting of the RhoA/ROCK pathway may be a useful strategy for preventing the thrombin-activated inflammatory response associated with intravascular coagulation.
We are grateful to Alan Hall for kindly providing the RhoA constructs used, and to Anser Azim for useful discussions.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 This work was supported by National Heart Lung and Blood Institute Grants HL67424, HL46350, and HL64573.
↵2 Address correspondence and reprint requests to Dr. Arshad Rahman, Department of Pediatrics, Box 850, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, NY 14642. E-mail address:
↵3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; EBM2, endothelial basal medium 2; IKK, IκB kinase; LUC, luciferase; ROCK, Rho-associated kinase.
- Received February 19, 2004.
- Accepted October 1, 2004.
- Copyright © 2004 by The American Association of Immunologists