We addressed the regulatory function of mammalian target of rapamycin (mTOR) in the mechanism of thrombin-induced ICAM-1 gene expression in endothelial cells. Pretreatment of HUVECs with rapamycin, an inhibitor of mTOR, augmented thrombin-induced ICAM-1 expression. Inhibition of mTOR by this approach promoted whereas over-expression of mTOR inhibited thrombin-induced transcriptional activity of NF-κB, an essential regulator of ICAM-1 transcription. Analysis of the NF-κB signaling pathway revealed that inhibition of mTOR potentiated IκB kinase activation resulting in a rapid and persistent phosphorylation of IκBα on Ser32 and Ser36, a requirement for IκBα degradation. Consistent with these data, we observed a more efficient and stable nuclear localization of RelA/p65 and, subsequently, the DNA binding activity of NF-κB by thrombin following mTOR inhibition. These data define a novel role of mTOR in down-regulating thrombin-induced ICAM-1 expression in endothelial cells by controlling a delayed and transient activation of NF-κB.
The procoagulant thrombin, a serine protease released during intravascular coagulation initiated by tissue injury or sepsis (1, 2), promotes endothelial adhesivity toward neutrophils (polymorphonuclear leukocytes) by inducing the endothelial cell surface expression of ICAM-1, a counter receptor for leukocyte β2 integrins LFA-1 and Mac-1 (CD11a/CD18 and CD11b/CD18) (3). The engagement of ICAM-1 with β2 integrins ensures a firm and stable adhesion of leukocytes to the vascular endothelium and thus enables them to migrate across endothelial barrier (4, 5). Additionally, ICAM-1 is implicated in the pathogenesis of various inflammatory disorders including allograft rejection (6, 7, 8, 9). We have shown that activation of the transcription factor NF-κB is essential for thrombin-induced ICAM-1 expression in endothelial cells (10, 11) and that this response is mediated through activation of the GTP-binding protein (G-protein)-coupled receptor, protease-activated receptor-1 (12).
NF-κB is a ubiquitously expressed family of transcription factors controlling the expression of numerous genes involved in immunity and inflammation (13, 14). NF-κB, typically 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 (13, 14). Activation of NF-κB is initiated through phosphorylation of IκBα on two specific serine residues (Ser32 and Ser36) by a macromolecular cytoplasmic IκB kinase (IKK) 4 complex composed of the catalytic subunits IKKα and IKKβ and the regulatory subunit NEMO/IKKγ (13, 14). Phosphorylation marks IκBα for polyubiquitination by the E3-SCFβ-TrCP ubiquitin ligase and, subsequently, its degradation by the 26 S proteasome (14, 15). Degradation of IκBα unmasks the nuclear localization signal of NF-κB, thus allowing its translocation to the nucleus where it activates transcription of target genes including ICAM-1.
Mammalian target of rapamycin (mTOR), alternatively termed as FRAP (FK506-binding protein 12 (FKBP12) and rapamycin associated protein) or RAFT (rapamycin and FKBP12 target), is a 289-kDa protein (16, 17, 18) belonging to the family of phosphatidylinositol kinase-like kinases (19). Like its homologues in Saccharomyces cerevisae, Tor1p and Tor2p, mTOR directs a wide range of cellular processes and signaling activities (20, 21). The best studied functions of mTOR include the cell growth and protein synthesis through activation of p70 S6 kinase and phosphorylation of the eukaryotic initiation factor 4E binding protein 1 (4E-BP1) (21, 22). mTOR was discovered during studies into the mechanism of action of rapamycin (16, 17, 18), an immunosuppressive drug used to prevent rejection of organ grafts (20, 23). Rapamycin complexes with high affinity with its cellular receptor FKBP12, and the resulting rapamycin-FKBP12 complex specifically binds to mTOR and inhibits mTOR-dependent downstream signaling (16, 20, 21).
Recent studies have described an important function of IKKβ in promoting cell growth and tumorigenesis (24) and also in linking inflammation to tumorigenesis (25). These findings, coupled with the role of ICAM-1 in promoting and the use of rapamycin in preventing allograft rejection (6, 7, 8, 9, 20, 23), prompted us to investigate the possible involvement of mTOR in regulating thrombin-induced IKK activation and ICAM-1 expression. In the present study, we describe a novel function of mTOR in down-regulating thrombin-induced ICAM-1 expression in endothelial cells. Our data establish that inhibition of mTOR signaling by rapamycin augments thrombin-induced ICAM-1 expression by potentiating IKK activation.
Materials and Methods
Endothelial cell culture
HUVEC cultures were established as described previously (26, 27) by using umbilical cords collected within 48 h of delivery. Cells were cultured as described (12) in gelatin-coated flasks using endothelial basal medium 2 (EBM2) with bullet kit additives (BioWhittaker). HUVEC used in the experiments were between three and six passages.
Total RNA was isolated using RNeasy kit (QIAGEN) and RT was performed using oligo(dT) primers and superscript reverse transcriptase (Invitrogen Life Sciences) following the manufacturer’s recommendations. Human ICAM-1 and GAPDH were amplified using the following primer set: ICAM-1 (forward, 5′-AGCAATGTGCAAGAAGATAGCCAA-3′ and reverse, 5′-GGTCCCCTGCGTGTTCCACC-3′); GAPDH (forward, 5′-TATCGTGGAAGGACTCATGACC-3′ and reverse, 5′-TACATGGC AACTGTGAGGGG-3′). RT product (2 μl) was amplified in a 50-μl volume containing 0.5 μmol of primers and 2.5 units of TaqDNA polymerase. The reaction conditions were as follows: 95°C for 30 s, 67°C for 30 s, and 72°C for 30 s for 25 cycles for ICAM-1 amplification, and 95°C for 30 s, 55°C for 30 s, 72°C for 1 min for 25 cycles for GAPDH amplification as described (28). Normalization of ICAM-1 expression was achieved by comparing the expression of GAPDH for the corresponding sample.
Northern blot analysis
Total RNA was isolated using RNeasy kit and fractionated using a 1% agarose-formaldehyde gel. The RNA was transferred to a Duralose-UV nitrocellulose membrane (Stratagene) and covalently linked by UV irradiation using a Stratalinker UV cross-linker (Stratagene). Human ICAM-1 (0.96-kb SalI to PstI fragment) (29), E-selectin (1.35-kb EcoR I fragment) (30), 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 described (31). Autoradiography was performed with an intensifying screen at −70°C. The nitrocellulose membrane was soaked for stripping the probe with boiled water or 0.1× SSC with 0.1% SDS.
Cell lysis and immunoblotting
Cells were lysed in a phosphorylation lysis buffer (50 mM HEPES, 150 mM NaCl, 200 μM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, 0.5–1% Triton X-100, 1 mM PMSF, and protease inhibitor cocktail obtained from Sigma-Aldrich) or in radioimmune precipitation assay (RIPA) buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% deoxycholic acid, 1% Triton X-100, 0.25 mM EDTA, 5 mM sodium fluoride, and protease inhibitor cocktail obtained from Sigma-Aldrich. Cell lysates were analyzed by SDS-PAGE and transferred onto PVDF membranes, and the residual binding sites on the filters were blocked by incubating with 5% (w/v) nonfat dry milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature. The membranes were subsequently incubated with indicated Abs overnight at 4°C and developed using an ECL method as described (11).
In vitro IKK assay
Cells were lysed in kinase lysis buffer (20 mM Tris-HCl, pH 7.5, 125 mM NaCl, 0.1 mM sodium orthovanadate, 25 mM β-glycerophosphate, 50 mM sodium fluoride, 1 mM EDTA, 1 mM MgCl2, 1% Triton X-100, 1 mM PMSF, and protease inhibitor cocktail obtained from Sigma-Aldrich). Cell lysates were immunoprecipitated with an Ab against IKKα/β as described (11). The immunocomplexes were washed three times with kinase lysis buffer and two times with kinase assay buffer (20 mM HEPES (pH 7.5), 20 mM MgCl2, 2 mM DTT), and then resuspended in 30 μl of kinase assay buffer containing 0.5 μg of GST-IκBα and 20 μM ATP. After 5 min, 10–15 μCi of [γ-32P]ATP was added, and the mixture was incubated at 30°C for 30 min. The reaction was terminated by the addition of SDS sample buffer, and each sample was resolved by SDS-PAGE, transferred to PVDF membrane, and the phosphorylated form of GST-IκBα was detected by autoradiography.
Cytoplasmic and nuclear extract preparation
After treatment, 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 to determine IκBα degradation by Western blotting. 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 Bio-Rad protein determination kit.
EMSA was performed as described (11). 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, 10% glycerol (20 μl final volume)) for 15 min at room temperature. Then end-labeled double-stranded oligonucleotides containing a 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 Ig-κB 5′-AGTTGAGGGGACTTTCCCAGGC-3′. The Ig-κB oligonucleotide contains the consensus NF-κB binding site sequence present in mouse Igκ L chain gene (32). The NF-κB sequence motif within the oligonucleotides is underlined.
Reporter gene constructs, endothelial cell transfection, and luciferase assay
The plasmid pNF-κB-LUC containing five copies of consensus NF-κB sequences linked to a minimal E1B promoter-luciferase gene was purchased from Stratagene. The construct pRK5mTOR encoding the wild-type mTOR (mTOR-WT) was a kind gift from David Sabatini (Massachusetts Institute of Technology, Boston, MA). Transfections were performed using DEAE-dextran method (33) with slight modifications (11). Briefly, 5 μg of DNA was mixed with 50 μg/ml DEAE-dextran in serum-free EBM2, and the mixture was added onto cells that were 60–80% confluent. We used 0.125 μg of pTKRLUC plasmid (Promega) containing Renilla luciferase 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 two times with EBM2–10% FBS and grown to confluency. We achieved transfection efficiency of 16 ± 3 (mean ± SD; n = 3) in these cells. Cell extracts were prepared and assayed for firefly and Renilla luciferase activities using Promega Biotech Dual Luciferase reporter assay system. The data expressed as a ratio of firefly to Renilla luciferase activity.
Thrombin activates mTOR in endothelial cells
We evaluated the ability of thrombin to activate mTOR in endothelial cells by determining the phosphorylation of p70 S6 kinase, a well established downstream target of mTOR (20, 21, 22). Western blot analysis showed that thrombin induced the phosphorylation of p70 S6 kinase (Thr421/Ser424) in endothelial cells (Fig. 1⇓A). We used rapamycin, an inhibitor of mTOR, to address the involvement of mTOR in mediating the phosphorylation of p70 S6 kinase by thrombin. Pretreatment of cells with rapamycin prevented thrombin-induced p70 S6 kinase phosphorylation (Fig. 1⇓A), indicating the activation of mTOR by thrombin. We also assessed the effect of rapamycin on the phosphorylation of p70 S6 kinase in response to phorbol ester, a known activator of mTOR (34). As expected, phorbol ester induced phosphorylation of p70 S6 kinase, and rapamycin prevented this response (Fig. 1⇓B), consistent with the inhibitory effect of rapamycin on mTOR signaling (20, 21, 34).
Inhibition of mTOR potentiates thrombin-induced ICAM-1 expression in endothelial cells
We determined the role of mTOR in regulating thrombin-induced ICAM-1 expression. We found that pretreatment of cells with rapamycin augmented ICAM-1 protein expression following thrombin challenge of endothelial cells (Fig. 2⇓A). We next determined if the effect of rapamycin on thrombin-induced ICAM-1 protein expression is secondary to increased ICAM-1 mRNA expression. RT-PCR and Northern blot analyses showed that thrombin challenge of endothelial cells resulted in increased ICAM-1 mRNA expression and that this response was potentiated in cells pretreated with rapamycin (Fig. 2⇓, B and C). We also assessed the effect of rapamycin on the transcription of E-selectin, another adhesive protein induced similarly by thrombin (35, 36). Inhibition of mTOR potentiated thrombin-induced E-selectin mRNA expression in a dose-dependent manner (Fig. 2⇓C), consistent with the effect of rapamycin on ICAM-1 mRNA expression.
Inhibition of mTOR augments thrombin-induced NF-κB activity by accelerating and stabilizing the nuclear localization and DNA binding function of NF-κB
The essential role of NF-κB in mediating thrombin-induced ICAM-1 gene transcription (6) led us to examine whether rapamycin promotes ICAM-1 expression by enhancing NF-κB activity. We found that inhibition of mTOR by rapamycin markedly enhanced NF-κB-dependent reporter gene activity induced by thrombin (Fig. 3⇓A). In another experiment, we investigated the effect of mTOR inhibition on TNF-α-induced NF-κB activity. We observed that rapamycin also promoted TNF-α-induced NF-κB-dependent reporter gene activity, albeit at a higher concentration (Fig. 3B⇓). In reciprocal experiments, we determined the effect of over-expression of mTOR on thrombin-induced NF-κB activity. Results showed that over-expressing mTOR inhibited thrombin-induced NF-κB activity but had no significant effect on basal NF-κB activity (Fig. 3⇓C). We next determined if the increase in thrombin-induced NF-κB activity by rapamycin is secondary to increased nuclear uptake and DNA binding of NF-κB. Analysis of nuclear extracts by Western blotting showed that thrombin induced nuclear translocation of p65/RelA in a time-dependent manner. Accumulation of p65/RelA in the nucleus began at 1 h, peaked at 2 h, and declined at 4 h after thrombin challenge (Fig. 4⇓A). Interestingly, inhibition of mTOR by rapamycin resulted in a rapid and persistent nuclear localization of RelA/p65; increase in nuclear p65/RelA was detected as early as 10 min, peaked at 2 h, and sustained up to 4 h after thrombin challenge (Fig. 4⇓A). In related studies, we evaluated the DNA binding function of the nuclear NF-κB. EMSA revealed that inhibition of mTOR resulted in an early onset and prolonged DNA binding activity of NF-κB in response to thrombin challenge (Fig. 4⇓B; compare lanes 2 and 5 with lanes 7 and 10).
Inhibition of mTOR induces a rapid and persistent IκBα phosphorylation by augmenting thrombin-induced IKK activation
We evaluated the effect of mTOR inhibition on thrombin-induced phosphorylation of IκBα on Ser32 and Ser36, a requirement for IκBα degradation-dependent NF-κB translocation and DNA binding in the nucleus (37, 38). Phosphorylated form of IκBα was detected within 30 min, peaked at 2 h, and declined by 4 h after thrombin challenge (Fig. 5⇓A). Inhibition of mTOR altered the kinetics of thrombin response such that IκBα phosphorylation occurred within 10 min, peaked between 1 and 2 h, and remained significantly elevated at 4 h after thrombin challenge (Fig. 5⇓A). We next determined the effect of inhibition of mTOR on thrombin-induced IκBα degradation. We observed an early onset of thrombin-induced IκBα degradation after mTOR inhibition (Fig. 5⇓B), consistent with rapid phosphorylation of IκBα by thrombin in rapamycin-pretreated cells (Fig. 5⇓A). However, in contrast to its effect on IκBα phosphorylation, inhibition of mTOR did not show prolonged IκBα degradation by thrombin (Fig. 5⇓). Because Ser32 and Ser36 phosphorylation of IκBα is catalyzed by IKK complex (39, 40, 41), we addressed the possibility that rapamycin potentiates IκBα phosphorylation by augmenting IKK activation. We performed an in vitro kinase assay in which GST-IκBα was used as an exogenous substrate to determine IKK activity. The IKK immunoprecipitates from thrombin-treated cells showed increased phosphorylation of GST-IκBα compared with the immunoprecipitates from control cells (Fig. 6⇓), indicating the activation of IKK by thrombin. Pretreatment of cells with rapamycin potentiated thrombin-induced phosphorylation of GST-IκBα (Fig. 6⇓). We observed that a low concentration (5 ng/ml) of rapamycin was sufficient to augment the IKK activity, consistent with the effects of rapamycin on NF-κB activity and transcription of ICAM-1 and E-selectin genes after thrombin challenge of endothelial cells (Fig. 2⇑C).
The present study describes a novel function of mTOR in the regulation of ICAM-1 gene expression in endothelial cells. The data are consistent with a role of mTOR as a “speed breaker” in the pathway to NF-κB activation, an essential event in the mechanism of ICAM-1 expression in endothelial cells (6, 42). We provide evidence that thrombin induces activation of mTOR in endothelial cells and that inhibition of mTOR potentiates thrombin-induced ICAM-1 expression. This effect of mTOR inhibition is secondary to augmented NF-κB activity. We further show that inhibition of mTOR enhances thrombin-induced NF-κB activity by potentiating the activation of IKK.
We used rapamycin to address the regulatory function of mTOR in thrombin-induced ICAM-1 expression in endothelial cells. We found that pretreatment of cells with rapamycin augmented thrombin-induced ICAM-1 protein expression and that this effect of rapamycin was secondary to increased expression of ICAM-1 mRNA. Like its effect on ICAM-1, rapamycin also enhanced the expression of E-selectin mRNA by thrombin. The finding that inhibition of mTOR augmented thrombin-induced ICAM-1 as well as E-selectin mRNA expression led us to examine the effect of rapamycin on thrombin-induced NF-κB activity. Results showed that thrombin-induced NF-κB-dependent reporter activity was significantly increased in cells pretreated with rapamycin. Our observation that rapamycin was effective in inhibiting phosphorylation of p70 S6 kinase induced by thrombin and also by phorbol ester, a known activator of mTOR (34), indicated that the effect of rapamycin on NF-κB activity and ICAM-1 expression is likely due to inhibition of mTOR signaling. To confirm this, we assessed the effect of over-expression of mTOR on NF-κB activity. We found that over-expression of mTOR inhibited NF-κB-dependent reporter activity induced by thrombin. Taken together, these data indicate the involvement of mTOR in modulating thrombin-induced NF-κB activity and the resultant ICAM-1 expression in endothelial cells.
We addressed the mechanism by which mTOR regulates NF-κB activity following thrombin challenge of endothelial cells. As transactivation of genes by NF-κB requires its translocation and DNA binding in the nucleus secondary to phosphorylation and degradation of IκBα in the cytoplasm (13, 14, 37, 38), we determined whether inhibition of mTOR augmented NF-κB activity by influencing these events. Interestingly, we found that inhibition of mTOR accelerated and stabilized thrombin-induced nuclear translocation of RelA/p65 and DNA binding activity of NF-κB. Consistent with these data, inhibition of mTOR induced an early onset of thrombin-induced phosphorylation of IκΒα on Ser32 and Ser36 and, consequently, its degradation by the 26 S proteasome. In addition, we found that inhibition of mTOR resulted in stabilization of IκΒα phosphorylation induced by thrombin. However, in contrast, we did not observe prolonged loss of IκΒα following thrombin challenge of rapamycin-treated cells. We believe that the effect of mTOR inhibition on IκΒα degradation at later time points (2 and 4 h; Fig. 5⇑B) is likely masked by the early de novo synthesis of IκΒα as a result of accelerated NF-κB activation by thrombin in rapamycin-treated cells. Given the essential role of IKK in Ser32 and Ser36 phosphorylation of IκΒα (39, 40, 41), we determined whether the potentiating effect of mTOR inhibition on IκBα phosphorylation is secondary to augmented activation of IKK. In vitro kinase assay showed that inhibition of mTOR following exposure of cells to rapamycin indeed potentiated the activation of IKK in response to thrombin challenge.
The efficient and stable activation of NF-κB following mTOR inhibition is consistent with a recent report by Schmidt et al. (43) showing a rapid and persistent activation of NF-κB. These studies demonstrate that MEK kinase (MEKK)3 regulates the rapid activation of NF-κB by activating IKKβ to associate with IκBα-NF-κB complex whereas MEKK2 controls a delay in the activation of NF-κB by directing IKKβ to associate with IκBβ-NF-κB complex after cytokine stimulation (43). It remains to be determined whether thrombin controls the rapid and sustained activation of NF-κB by MEKK2/3-dependent mechanisms and whether mTOR inhibition augments thrombin-induced NF-κB activity by promoting MEKK2/3 activation.
We have previously shown that activation of Gαq and dissociation of Gβγ complex after thrombin stimulation of protease-activated receptor-1 results in parallel activation of the protein kinase C (PKC)-δ and PI3K-dependent pathways (12). These pathways converge at Akt to activate IKK and induce NF-κB activation and ICAM-1 transcription in endothelial cells (12). Because Akt lies upstream of mTOR to regulate its function (21), it may be that activation of Akt by both PI3K and PKC-δ leads to activation of mTOR in addition to IKK. We postulate that activated mTOR serves two functions: 1) it rapidly interacts, directly or indirectly, with IKK to down-regulate its activity; and 2) activated mTOR also associates with PKC-δ, and this interaction decreases PKC-δ activity in a negative feedback manner. In support of this model, we have found that thrombin induces association of mTOR with PKC-δ in a rapamycin-sensitive manner (M. Minhajuddin, unpublished results); however, it remains to be determined whether mTOR also similarly interacts with IKK. The ability of PKC-δ to associate with mTOR is consistent with previous studies (44). Because PKC-δ, a critical activator of NF-κB (11, 45, 46), regulates thrombin signaling of Akt/IKK activation (12), future studies will address the possibility that association of mTOR down-regulates PKC-δ activity and thereby PKC-δ-dependent Akt/IKK activation and that inhibition of mTOR relieves both PKC-δ-dependent and -independent suppression of IKK activity resulting in augmented NF-κB activation and ICAM-1 expression. Because Akt also plays an important role (F. Fazal, unpublished results) but not PKC-δ (47) in TNF-α-induced NF-κB activity and ICAM-1 expression, the proposed model is consistent with the requirement of higher concentration of rapamycin to augment the TNF-α response.
In summary, the present study establishes a novel function of mTOR in down-regulating ICAM-1 expression by interfering with the activation of IKK after thrombin stimulation of endothelial cells. The crucial role of IKKβ in promoting cell growth and tumorigenesis (24) and also in linking inflammation to tumorigenesis (25) suggests that the negative regulation of IKK activity by mTOR may be a mechanism of ensuring proper cell growth. Our findings assume added importance as ICAM-1 is implicated in the pathogenesis of allograft rejection (6, 7, 8, 9), and the rapamycin is used to prevent rejection of organ grafts (20, 23).
We are grateful to David Sabatini for kindly providing the mTOR construct. We are thankful to Shahab Uddin for helpful discussions.
The authors have no financial conflict of interest.
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 Grant HL67424.
↵2 Current address: Department of Medicine, University of Illinois, Chicago, IL 60612.
↵3 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:
↵4 Abbreviations used in this paper: IKK, IκB kinase; mTOR, mammalian target of rapamycin; LUC, luciferase; FKBP12, FK506-binding protein 12; PVDF, polyvinylidene difluoride; EBM2, endothelial basal medium 2; MEKK, MEK kinase; PKC, protein kinase C.
- Received June 24, 2004.
- Accepted February 18, 2005.
- Copyright © 2005 by The American Association of Immunologists