| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
B Kinase 
and Phosphorylation of RelA/p651
* Department of Pediatrics, University of Rochester School of Medicine, Rochester, NY 14642; and
Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B 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-
-induced NF-B activation and ICAM-1 expression. The effect of RhoA/ROCK inhibition on thrombin-induced NF-B activation was secondary to inhibition of IB kinase activation and subsequent IB degradation and nuclear uptake and the DNA binding of NF-B. Inhibition of the RhoA/ROCK pathway also prevented phosphorylation of Ser536 within the transactivation domain 1 of NF-B p65/RelA, a critical event conferring transcriptional competency to the bound NF-B. Thus, the RhoA/ROCK pathway signals thrombin-induced ICAM-1 expression through the activation of IB kinase, which promotes NF-B binding to ICAM-1 promoter and phosphorylation of RelA/p65, thus mediating the transcriptional activation of bound NF-B. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2 integrins (CD11/CD18) on the PMN surface (1). The interaction of ICAM-1 with 2 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-B 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-B is a ubiquitously expressed family of transcription factors controlling the expression of numerous genes involved in immunity and inflammation (11). The prototypical NF-B 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 IB, the prototype of a family of inhibitory proteins termed IB proteins (12). Activation of NF-B requires the degradation of IB achieved through serine phosphorylation (Ser32 and Ser36) of IB by a macromolecular IB kinase (IKK) complex (13, 14). Phosphorylation targets IB for polyubiquitination by the E3-SCF-TrCP ubiquitin ligase and subsequently its degradation by the 26S proteasome (15). The released NF-B rapidly translocates to the nucleus to activate the transcription of target genes such as ICAM-1. Another important mechanism regulating NF-B 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-B in the nucleus (16, 17, 18, 19, 20). However, unlike IB 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-B (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 IKK, which, in turn, mediates NF-B binding to ICAM-1 promoter and phosphorylation of RelA/p65, thereby conferring transcriptional competency to the bound NF-B.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human thrombin was purchased from Enzyme Research Laboratories (South Bend, IN). Polyclonal Abs to IKK, IB, p65/RelA, and -actin; an mAb to ICAM-1; and GST-IB fusion protein were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Abs that detect RelA/p65 when phosphorylated at Ser536 or that detect IB when phosphorylated at Ser32 and Ser36 were obtained from Cell Signaling Technology (Beverly, MA). The polyvinylidene difluoride membrane was obtained from Millipore (Bedford, MA); Y27632 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).
Cell culture
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 -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 IKK 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-IB, 20 µM ATP, and 10–15 µCi of [
-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-IB was detected by autoradiography.
Northern analysis
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 [-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 2x SSC with 0.1% SDS, followed by two washes for 15 min each time at 60°C in 0.1x 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 0.1x SSC /0.1% SDS.
Reporter gene constructs, endothelial cell transfection, and luciferase (LUC) assay
The plasmid pNF-B-LUC containing five copies of consensus NF-B 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 IKK 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 IB 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).
EMSA
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-B 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 (0.25x Tris-borate-EDTA). The oligonucleotide used for the gel-shift analysis was NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3') or ICAM-1 NF-B (5'-AGCTTGGAAATTCCGGAGC-TG-3'). The Ig-B oligonucleotide contains the consensus NF-B binding site sequence (underlined) present in pNF-B-LUC. The ICAM-1 NF-B oligonucleotide represents a 21-bp sequence of ICAM-1 promoter encompassing the NF-B binding site located 183 bp upstream of transcription initiation site (32). The sequence motifs within the oligonucleotides are underlined.
Statistical analysis
Results are expressed as the mean ± SE. Data were analyzed by Student’s t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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. 1A). 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. 1B). Because Rho is implicated in a number of TNF- responses (35, 36), we evaluated the function of Rho in the mechanism of TNF--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- (Fig. 1).
|
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- (Fig. 2) in contrast to the effect of Rho inhibition on thrombin-induced ICAM-1 expression (Fig. 1).
|
B activity
Because of the essential role of NF-B in mediating thrombin-induced ICAM-1 transcription, we addressed whether the RhoA/ROCK pathway signals ICAM-1 expression by regulating NF-B activity. We determined the effects of inhibition of RhoA on NF-B-dependent reporter activity. Cotransfection of cells with C3 exoenzyme inhibited thrombin-induced NF-B activity (Fig. 3A). Also, the expression of the dominant negative mutant of RhoA (RhoAmut) inhibited NF-B activity induced by thrombin (Fig. 3C). However, inhibition of RhoA by C3 exoenzyme or RhoAmut had no significant effect on TNF--induced NF-B activity (Fig. 3, B and D).
|
B activity. We found that inhibition of ROCK by exposing the cells to Y-27632 impaired thrombin-induced NF-B activity (Fig. 3A). In parallel experiments, inhibition of ROCK showed no significant effect on TNF--induced NF-B activity (Fig. 3B).
RhoA/ROCK signals thrombin-induced NF-B activation through IKK
We addressed the possibility that RhoA activation is sufficient to induce NF-B activity. We observed that the expression of constitutively active RhoA mutant (RhoAcat) induced NF-B activity in the absence of thrombin challenge (Fig. 4A). In another experiment, cotransfection of RhoAcat increased ICAM-1 promoter-dependent reporter activity (Fig. 4B), consistent with its ability in inducing NF-B activation (Fig. 4A). We next determined the function of RhoA in mediating thrombin-induced IB degradation, a requirement for NF-B activation (39, 40). Because IB degradation is contingent on its phosphorylation by IKK (41, 42, 43), we evaluated the function of IKK in RhoA-mediated NF-B activation. Coexpression of IKKmut prevented RhoAcat-induced NF-B activity (Fig. 4A), indicating that RhoA mediates thrombin-induced NF-B activity through the activation of IKK. To determine the role of ROCK in signaling RhoA activation of IKK, we assessed the effects of ROCK inhibition on thrombin-induced IKK activity using an in vitro kinase assay in which GST-IB was used as an exogenous substrate. We observed that IKK immunoprecipitated from thrombin-stimulated cells showed increased phosphorylation of GST-IB compared with IKK from control cells (Fig. 4C), indicative of activation of IKK. Inhibition of ROCK by Y-27632 prevented thrombin-induced IKK activation (Fig. 4C).
|
activated by RhoA/ROCK catalyzes IB 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 IB phosphorylation, and consequently its degradation (Fig. 4D and Fig. 5, A and B). Because IB degradation results in nuclear localization and DNA binding of NF-B, 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-B (Fig. 5, C and D). In contrast, RhoA/ROCK inhibition had no effect on TNF--induced IB degradation and DNA binding activity of nuclear NF-B (Fig. 5, B–D).
|
Because phosphorylation of Ser536 in the transactivation domain 1 of RelA/p65 regulates transcriptional activity of NF-B in the nucleus (20, 44, 45), we addressed the possibility that RhoA/ROCK contributes to NF-B 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).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B-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-B binding to the ICAM-1 promoter by phosphorylation and degradation of IB as well as enhancing the transactivation capacity of the bound NF-B through Ser536 phosphorylation of RelA/p65. Interestingly, RhoA/ROCK mediated NF-B activation and ICAM-1 expression in response to thrombin, but not TNF-, the prototypic inducer of NF-B activation and ICAM-1 expression. These data show that the RhoA/ROCK pathway activates NF-B-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-B 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 Gq/PKC
-dependent mechanism (5, 10). A recent study has also reported the role of PKC in mediating thrombin-induced RhoA activation in endothelial cells (46). These and other studies (47, 48) suggest that a linkage between the Gq/PKC and RhoA/ROCK pathways signals ICAM-1 expression in response to thrombin challenge of endothelial cells. That the Gq/PKC 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 PKC inhibition on ICAM-1 expression. We also observed that ICAM-1 protein expression was inhibited to a greater extent than NF-B 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- challenge of endothelial cells. These results showed that inhibition of the RhoA/ROCK pathway failed to prevent TNF--induced ICAM-1 expression in these cells. Studies have shown that RhoA/ROCK is required for TNF--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--induced ICAM-1 expression cannot be ascribed to the absence of RhoA/ROCK activation by TNF- in endothelial cells. The reasons for the different effects of RhoA/ROCK inhibition on thrombin- vs TNF--induced ICAM-1 expression are not clear. A possible explanation is that there may be distinct mechanisms of thrombin and TNF- 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- induces ICAM-1 by a PKC
-dependent mechanism, thrombin, as described above, mediates this response via a PKC-dependent pathway (5, 50). Although PKC has been implicated in thrombin-induced RhoA activation (46), the role of PKC in signaling TNF--induced RhoA activation in endothelial cells is not known. It is possible that PKC-dependent activation of RhoA/ROCK by thrombin targets it to an intermediate protein, which, in turn, links it to the NF-B signaling pathway, whereas RhoA/ROCK activated by TNF- 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-B-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-B-dependent reporter activity induced by thrombin. However, inhibition of the RhoA/ROCK pathway using these approaches failed to inhibit the TNF--induced, NF-B-dependent reporter activity. We also used the constitutively active RhoA mutant (RhoAcat) to examine whether activation of RhoA is sufficient to induce NF-B activity in endothelial cells. The expression of RhoAcat induced NF-B activity as well as ICAM-1 promoter activation in the absence of thrombin challenge. In other experiments the expression of kinase-defective IKK mutant (IKKmut) prevented the RhoAcat-induced NF-B activity. These data indicate that the RhoA/ROCK pathway acts upstream of IKK in signaling thrombin-induced NF-B activation in endothelial cells. Consistent with the crucial involvement of IKK, we also showed that RhoA/ROCK-mediated IB phosphorylation and degradation resulted in the migration of NF-B to the nucleus, where its binding to the promoter activated ICAM-1 gene transcription. Cammarano and Minden (24) have reported that RhoA activates NF-B in NIH-3T3 cells in the absence of IKK stimulation. It is not clear what determines IKK-dependent vs -independent activation of NF-B by RhoA in endothelial compared with NIH-3T3 cells. It is also not known whether ROCK is involved in RhoA activation of NF-B in NIH-3T3 cells. Recently, Kato et al. (51) showed that IKK-independent activation of NF-B by UV radiation is mediated by CK2 (formerly casein kinase II) through C-terminal phosphorylation of IB. 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-B 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 IB degradation, and that it plays an important role in conferring transcriptional competency to DNA-bound NF-B (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 IKK is implicated in phosphorylating RelA/p65 at Ser536 (20, 44, 45), it is possible that the RhoA/ROCK-mediated activation of IKK catalyzes Ser536 phosphorylation of RelA/p65 and thus renders bound NF-B transcriptionally competent. This is in addition to the other function of Rho/ROCK discussed above to induce IB phosphorylation and promote NF-B 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 IKK 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-B activation and ICAM-1 expression in endothelial cells. The role of RhoA/ROCK in the thrombin response is distinct because TNF--mediated NF-B 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.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
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: arshad_rahman{at}urmc.rochester.edu
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; EBM2, endothelial basal medium 2; IKK, IB kinase; LUC, luciferase; ROCK, Rho-associated kinase.
Received for publication February 19, 2004. Accepted for publication October 1, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B site of the promoter mediates endothelial ICAM-expression and neutrophil adhesion. J. Immunol. 162:5466. regulates thrombin-induced ICAM-1 gene expression in endothelial cells via activation of p38 mitogen-activated protein kinase. Mol. Cell. Biol. 21:5554.q and G regulate PAR-1-induced NF-B activation and ICAM-1 transcription in endothelial cells. Circ. Res. 91:398.B puzzle. Cell 109:S81.
B and I-B proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[Medline]
B?. Trends Cell Biol. 10:129.[Medline]
B activity. Annu. Rev. Immunol. 18:621.[Medline]
B determines its association with CBP/p300 or HDAC-1. Cell. 9:625.
B p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1). EMBO J. 22:1313.[Medline]
PKC in NF-B transcriptional activation. EMBO J. 22:3910.[Medline]
-induced phosphorylation of RelA/p65 on Ser529 is controlled by casein kinase II. J. Biol. Chem. 275:32592.B kinases phosphorylate NF-B p65 subunit on serine 536 in the transactivation domain. J. Biol. Chem. 274:30353.B by IB kinase (IKK)-dependent and IKK-independent pathways. J. Biol. Chem. 276:25876.B and C/EBP transcription factors. J. Biol. Chem. 274:8506. signals Rho-guanine nucleotide dissociation inhibitor phosphorylation and Rho activation and regulates the endothelial cell barrier function. J. Biol. Chem. 276:22614.-induced reorganization of the actin cytoskeleton and cell-cell junctions by Rho, Rac, and Cdc42 in human endothelial cells. J. Cell Physiol. 176:150.[Medline]
B proteolysis by site-specific, signal-induced phosphorylation. Science 267:1485.B on serines 32 and 36 controls IB proteolysis and NF-B activation in response to diverse stimuli. EMBO J. 14:2876.[Medline]
B kinase complex (IKK) contains two kinase subunits, IKK and IKK, necessary for IB phosphorylation and NF-B activation. Cell 91:243.[Medline]
B kinase activity through IKK subunit phosphorylation. Science 284:309.B kinase 2 gene. Science 284:321.B activation in lymphotoxin receptor signaling depends on the phosphorylation of p65 at serine 536. J. Biol. Chem. 278:919. plays an essential role in the phosphorylation of RelA/p65 on serine 536 induced by lipopolysaccharide. J. Immunol. 170:5630.q and Gq-coupled receptors. J. Biol. Chem. 277:27130.-mediated bovine pulmonary endothelial cell apoptosis. Biochem. Biophys. Res. Commun. 306:244.[Medline]
-induced ICAM-1 gene transcription in endothelial cells. Am. J. Physiol. 279:C906.
B kinase responsible for NF-B activation during the UV response. Mol. Cell 12:829.[Medline]
B through utilization of the IB kinase and activation of the mitogen-activated protein kinase p38. J. Biol. Chem. 276:18934.This article has been cited by other articles:
|
|
 |
|
  F. Fazal, K. M. Bijli, M. Minhajuddin, T. Rein, J. N. Finkelstein, and A. Rahman Essential Role of Cofilin-1 in Regulating Thrombin-induced RelA/p65 Nuclear Translocation and Intercellular Adhesion Molecule 1 (ICAM-1) Expression in Endothelial Cells J. Biol. Chem., July 31, 2009; 284(31): 21047 - 21056. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Xue, P. B. Thippegowda, G. Hu, K. Bachmaier, J. W. Christman, A. B. Malik, and C. Tiruppathi NF-{kappa}B regulates thrombin-induced ICAM-1 gene expression in cooperation with NFAT by binding to the intronic NF-{kappa}B site in the ICAM-1 gene Physiol Genomics, June 10, 2009; 38(1): 42 - 53. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. I. Borissoff, H. M.H. Spronk, S. Heeneman, and H. ten Cate Is thrombin a key player in the 'coagulation-atherogenesis' maze? Cardiovasc Res, June 1, 2009; 82(3): 392 - 403. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Meyer-Schwesinger, S. Dehde, C. von Ruffer, S. Gatzemeier, P. Klug, U. O. Wenzel, R. A. K. Stahl, F. Thaiss, and T. N. Meyer Rho kinase inhibition attenuates LPS-induced renal failure in mice in part by attenuation of NF-{kappa}B p65 signaling Am J Physiol Renal Physiol, May 1, 2009; 296(5): F1088 - F1099. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Minhajuddin, K. M. Bijli, F. Fazal, A. Sassano, K. I. Nakayama, N. Hay, L. C. Platanias, and A. Rahman Protein Kinase C-{delta} and Phosphatidylinositol 3-Kinase/Akt Activate Mammalian Target of Rapamycin to Modulate NF-{kappa}B Activation and Intercellular Adhesion Molecule-1 (ICAM-1) Expression in Endothelial Cells J. Biol. Chem., February 13, 2009; 284(7): 4052 - 4061. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Bair, P. B. Thippegowda, M. Freichel, N. Cheng, R. D. Ye, S. M. Vogel, Y. Yu, V. Flockerzi, A. B. Malik, and C. Tiruppathi Ca2+ Entry via TRPC Channels Is Necessary for Thrombin-induced NF-{kappa}B Activation in Endothelial Cells through AMP-activated Protein Kinase and Protein Kinase C{delta} J. Biol. Chem., January 2, 2009; 284(1): 563 - 574. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Qiao, O. Holian, B.-S. Lee, F. Huang, J. Zhang, and H. Lum Phosphorylation of GTP dissociation inhibitor by PKA negatively regulates RhoA Am J Physiol Cell Physiol, November 1, 2008; 295(5): C1161 - C1168. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Bijli, F. Fazal, M. Minhajuddin, and A. Rahman Activation of Syk by Protein Kinase C-{delta} Regulates Thrombin-induced Intercellular Adhesion Molecule-1 Expression in Endothelial Cells via Tyrosine Phosphorylation of RelA/p65 J. Biol. Chem., May 23, 2008; 283(21): 14674 - 14684. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. M. Wagner and J. Jenkins Effects of airway distension on leukocyte recruitment in the mouse tracheal microvasculature J Appl Physiol, April 1, 2007; 102(4): 1528 - 1534. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Su, M. Hodnett, N. Wu, A. Atakilit, C. Kosinski, M. Godzich, X. Z. Huang, J. K. Kim, J. A. Frank, M. A. Matthay, et al. Integrin {alpha}vbeta5 Regulates Lung Vascular Permeability and Pulmonary Endothelial Barrier Function Am. J. Respir. Cell Mol. Biol., March 1, 2007; 36(3): 377 - 386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Fazal, M. Minhajuddin, K. M. Bijli, J. L. McGrath, and A. Rahman Evidence for Actin Cytoskeleton-dependent and -independent Pathways for RelA/p65 Nuclear Translocation in Endothelial Cells J. Biol. Chem., February 9, 2007; 282(6): 3940 - 3950. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Bijli, M. Minhajuddin, F. Fazal, M. A. O'Reilly, L. C. Platanias, and A. Rahman c-Src interacts with and phosphorylates RelA/p65 to promote thrombin-induced ICAM-1 expression in endothelial cells Am J Physiol Lung Cell Mol Physiol, February 1, 2007; 292(2): L396 - L404. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. Fessler, P. G. Arndt, I. Just, J. A. Nick, K. C. Malcolm, and G. Scott Worthen Dual role for RhoA in suppression and induction of cytokines in the human neutrophil Blood, February 1, 2007; 109(3): 1248 - 1256. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Cui, B. Tieu, A. Recinos, R. G. Tilton, and A. R. Brasier RhoA Mediates Angiotensin II-Induced Phospho-Ser536 Nuclear Factor {kappa}B/RelA Subunit Exchange on the Interleukin-6 Promoter in VSMCs Circ. Res., September 29, 2006; 99(7): 723 - 730. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Sapet, S. Simoncini, B. Loriod, D. Puthier, J. Sampol, C. Nguyen, F. Dignat-George, and F. Anfosso Thrombin-induced endothelial microparticle generation: identification of a novel pathway involving ROCK-II activation by caspase-2 Blood, September 15, 2006; 108(6): 1868 - 1876. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Wojciak-Stothard, L. Y. F. Tsang, E. Paleolog, S. M. Hall, and S. G. Haworth Rac1 and RhoA as regulators of endothelial phenotype and barrier function in hypoxia-induced neonatal pulmonary hypertension Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1173 - L1182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Woods and F. Beier RhoA/ROCK Signaling Regulates Chondrogenesis in a Context-dependent Manner J. Biol. Chem., May 12, 2006; 281(19): 13134 - 13140. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Schmeck, S. Huber, K. Moog, J. Zahlten, A. C. Hocke, B. Opitz, S. Hammerschmidt, T. J. Mitchell, M. Kracht, S. Rosseau, et al. Pneumococci induced TLR- and Rac1-dependent NF-{kappa}B-recruitment to the IL-8 promoter in lung epithelial cells Am J Physiol Lung Cell Mol Physiol, April 1, 2006; 290(4): L730 - L737. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. H. Brown, D. P. Del Re, and M. A. Sussman The Rac and Rho Hall of Fame: A Decade of Hypertrophic Signaling Hits Circ. Res., March 31, 2006; 98(6): 730 - 742. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Cernuda-Morollon and A. J. Ridley Rho GTPases and Leukocyte Adhesion Receptor Expression and Function in Endothelial Cells Circ. Res., March 31, 2006; 98(6): 757 - 767. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Amin, C. S. Haas, K. Zhu, P. J. Mansfield, M. J. Kim, N. P. Lackowski, and A. E. Koch Migration inhibitory factor up-regulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 via Src, PI3 kinase, and NF{kappa}B Blood, March 15, 2006; 107(6): 2252 - 2261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-Y. Wei, K.-C. Huang, Y.-H. Chou, P.-F. Hsieh, K.-H. Lin, and W.-W. Lin The Role of Rho-Associated Kinase in Differential Regulation by Statins of Interleukin-1beta- and Lipopolysaccharide-Mediated Nuclear Factor {kappa}B Activation and Inducible Nitric-Oxide Synthase Gene Expression in Vascular Smooth Muscle Cells Mol. Pharmacol., March 1, 2006; 69(3): 960 - 967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Loirand, P. Guerin, and P. Pacaud Rho Kinases in Cardiovascular Physiology and Pathophysiology Circ. Res., February 17, 2006; 98(3): 322 - 334. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Cuvelier, S. Paul, N. Shariat, P. Colarusso, and K. D. Patel Eosinophil adhesion under flow conditions activates mechanosensitive signaling pathways in human endothelial cells J. Exp. Med., September 19, 2005; 202(6): 865 - 876. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
