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The Journal of Immunology, 2000, 164: 2077-2083.
Copyright © 2000 by The American Association of Immunologists

Induction of E-Selectin Expression by Double-Stranded RNA and TNF-{alpha} Is Attenuated in Murine Aortic Endothelial Cells Derived from Double-Stranded RNA-Activated Kinase (PKR)-Null Mice1

Sudip K. Bandyopadhyay2,*,{dagger}, Carol A. de la Motte{ddagger} and Bryan R. G. Williams*

Departments of * Cancer Biology, {dagger} Urology, and {ddagger} Colorectal Surgery, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The adherence of leukocytes on the endothelium is mediated in part by the transient expression of the E-selectin adhesion molecule. Because we have previously shown that the dsRNA-activated kinase PKR mediates dsRNA induction of NF-{kappa}B, we used murine aortic endothelial (MuAE) cells isolated from wild-type and PKR-null mice to investigate the role of PKR in the induction of E-selectin expression by dsRNA (pIC) and TNF-{alpha}. E-selectin mRNA and protein expression was inducible by both pIC and TNF-{alpha} in wild-type MuAE cells, whereas induction of E-selectin expression by these agents was defective in PKR-null MuAE cells. Induction of E-selectin promoter activity and NF-{kappa}B DNA binding activity were substantially reduced in pIC- or TNF-{alpha}-treated PKR-null cells, indicating a role for PKR in both pIC and TNF-{alpha} induction of E-selectin via an NF-{kappa}B-dependent pathway. In PKR-null cells, pIC-mediated degradation of I{kappa}Bß is deficient. Activation of this pathway requires the PKR-dependent degradation of the I{kappa}Bß protein. Moreover, both phosphorylated and unphosphorylated activating transcription factor 2 DNA-binding activities were reduced in PKR-null aortic endothelial cells. These results indicate that the PKR is required for full activation of E-selectin expression by pIC and TNF-{alpha} in primary mouse aortic endothelial cells identifying activating transcription factor 2 as a new target for PKR-dependent regulation and suggest a role for PKR in leukocyte adhesion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The dsRNA-activated kinase (PKR)3 is a serine-threonine kinase that is induced by IFN and activated by dsRNA (1). Once activated, PKR phosphorylates the {alpha} subunit of the eukaryotic initiation factor eIF2, inhibiting protein synthesis (2, 3, 4, 5, 6, 7, 8, 9). PKR has both antiviral properties and growth-suppressive functions (9, 10, 11, 12) and has been implicated in cell cycle regulation (13). PKR is also a signal transducer for dsRNA and IFN-mediated IFN-regulatory factor 1 (IRF-1) and NF-{kappa}B-dependent gene transcription (14, 15, 16, 17). NF-{kappa}B activation by dsRNA is mediated by the phosphorylation of its inhibitor I{kappa}B via a PKR-dependent pathway (14, 15). In the absence of PKR, the activation of NF-{kappa}B by dsRNA is deficient, and NF-{kappa}B-dependent gene transcription is impaired. PKR is a mediator of dsRNA, TNF-{alpha}, LPS, and specific virus-induced apoptotic cell death (18, 19, 20, 21, 22, 23). Mouse embryo fibroblasts (MEFs) derived from PKR-null mice or murine cell lines expressing catalytically inactive mutant PKR are resistant to apoptosis induced by dsRNA (18, 24). This could be correlated with a failure to activate the transcription factor IRF-1 and to induce Fas (18) and Fas-associated death domain (24). PKR associates in vitro with tumor suppressor, p53, and this physical association is enhanced by IFNs (25). Defective phosphorylation of mouse p53 on Ser18 is implicated in impaired transcriptional induction of the p53-inducible genes in Pkr0/0 cells (26).

Elevated expression of adhesion molecules on vascular endothelial cells is thought to play an important role in the onset and development of inflammation (27, 28, 29). The attachment of leukocytes to endothelial cells via specific adhesion molecules is a complicated, multistep process. The initial rolling of leukocytes along the blood vessel depends on the transient interaction of leukocytes and endothelium cells (27, 28, 29, 30, 31). Cellular adhesion molecules such as VCAM-1 and E-selectin are elevated by several cytokines and dsRNA in the appropriate cells (32, 33, 34, 35). dsRNA is produced in cells as an intermediate of virus replication and is capable of inducing primary gene transcription in cell culture in the absence of protein synthesis, mimicking the induction of different genes by virus infection (36, 37, 38, 39, 40). E-selectin is expressed on endothelial cells in chronic and acute inflammation and can be induced by the inflammatory cytokines TNF-{alpha} or IL-1 at the transcriptional level (34, 41, 42). Detailed analyses of the human proximal promoter region of the E-selectin gene have revealed the importance of NF-{kappa}B and NF- endothelial leukocyte adhesion molecule 1 (ELAM-1) sequences in the activation of the promoter by IL-1 and TNF-{alpha} (43, 44, 45, 46), and both elements are required for full activation of the E-selectin gene (44, 46). Murine E-selectin is a single copy gene with 70% nucleotide sequence similarity with its human counterpart (47). The regulatory elements for transcriptional induction by TNF-{alpha} or IL-1 are conserved in both the human and mouse genes and they are likely to be similarly regulated.

In this report, we demonstrate that the induction of E-selectin mRNA and protein by pIC or TNF-{alpha} is attenuated in endothelial cells derived from PKR-null mice. This is due to an overall reduction of NF-ELAM-1 complex formation including ATF-2-NF-ELAM-1 DNA binding coupled with a reduced ability of these agents to induce NF-{kappa}B activation because of a failure to efficiently degrade I{kappa}Bß and to a lesser extent I{kappa}B{alpha} proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

All cell culture materials and oligonucleotides were obtained from Life Technologies (Gaithersburg, MD). pIC was purchased from Sigma (St. Louis, MO), and murine TNF-{alpha} and EGF were from Boehringer Mannheim (Indianapolis, IN). Radioactive [{gamma}-32P]ATP and [{alpha}-32P]dCTP were from DuPont NEN (Wilmington, DE), and 125I-streptavidin was from Amersham (Arlington Heights, IL). Abs to IkB{alpha}, IkBß, p50, p65, p52, and ATF-2 were from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-ATF-2 was from New England Biolabs (Beverly, MA), and purified rat anti-mouse monoclonal E-selectin Ab was purchased from PharMingen and biotin-conjugated affinity-pure F(ab')2 fragment goat anti-rat IgG + IgM (H + L) was from Jackson ImmunoResearch (West Grove, PA).

Culture of mouse aortic endothelial cells

Mice (4–5 mo) were sacrificed, and aortic endothelial (AE) cells were isolated from five age-matched wild-type and PKR-deficient mice as described previously (17, 48). Briefly, the aorta was opened longitudinally and rinsed in serum-free medium. The exposed inner surface was digested with 2 mg/ml collagenase in serum-free medium for 10 min at 37°C, and the endothelial cell patches were collected with a cotton swab and grown on a fibronectin-coated plate with MCDB131 medium containing 15% FBS, glutamine, penicillin-streptomycin, 90 µg/ml heparin, 10 ng/ml murine epidermal growth factor, and 1 µg/ml cortisone in 5% CO2 at 37°C cell culture incubator. Both Pkr+/+ and Pkr0/0 endothelial cells were isolated at the same time and exhibited the typical cobblestone-like morphology. The cells were subcultured by trypsinization when confluent and used for experiments within passages 3 and 5 with an equal number of cells. MEF from wild-type and PKR-null mice were prepared as described previously (13, 17).

Expression of E-selectin in AE cells

Equal numbers of Pkr+/+ and Pkr0/0 AE cells were plated in 48-well plates, and the next day cells were treated with 100 µg/ml pIC and 10 ng/ml TNF-{alpha} for 6 h in fresh medium. Cells were washed twice with medium containing 1% BSA and incubated for 1 h at 4°C with 1 µg/ml E-selectin mAb, washed three times with medium, and incubated for 30 m at 4°C with biotin-conjugated secondary Ab. Cells were washed four times and reincubated with 0.25 µCi/well 125I-streptavadin for 15 m at 4°C, washed four times, and lysed with 1% Triton X-100 for radioactive quantitation (49).

Northern blot analysis

AE cells were grown to 80% confluence in 10-cm petri dishes and placed in MCDB 131 medium with 2% serum for 16 h. After incubation in serum-free medium for 4 h, cells were treated with pIC and TNF-{alpha} for 2 h or left untreated. Total RNA was prepared (37), and 10 µg of total RNA were electrophoresed in a 1% agarose-formaldehyde gel, transferred to nylon membrane (Gene-screen) and probed with a 1.1-kb AvaI fragment of the mouse E-selectin gene (from Dr. Mark A. Labow, Hoffmann La Roche, Nutley, NJ) labeled by random priming kit to an efficiency of >108 cpm/µg DNA. Hybridization and washing were performed according to standard procedures. Blots were reprobed with actin for normalization, and results were visualized by autoradiography and normalized with actin by PhosphoImager (Molecular Dynamics, Sunnyvale, CA) quantitation.

Transient transfection and luciferase assay

The E-selectin promoter (540-bp 5'-region from the transcription start site of the E-selectin gene) luciferase reporter DNA (49) together with Rous sarcoma virus-ß-galactosidase DNA were transfected into wild-type and PKR knockout MEFs by electroporation (Life Technologies) at room temperature (50). After electroporation, cells were plated equally into six-well plates, and the next day cells were treated in serum-free medium with pIC and TNF-{alpha} for 5 h. Cells were harvested and assayed for luciferase and ß-galactosidase activities (Promega, Madison, WI) and light emission was measured by a Luminometer (model ML 2250; Dynatech Laboratories, Chantilly, VA).

Electrophoretic mobility shift assays

Synthetic dsDNA containing the {kappa}B sequences from -133 to -110 (5'-ACTCAGTGGATATTCCCAGAAAAC-3') and the NF-ELAM-1 sequences from -162 to -139 (5'-GTCTCTGACATCACTATGAAAGTG-3') of murine E-selectin gene (45, 46, 47), were annealed, labeled by kinase reaction with [{gamma}-32P]ATP, and used as probes in EMSAs. Whole cell extracts were prepared for EMSA (51) which was performed with an equal amount of protein (6 µg) as described previously (17). Supershift analysis was done by preincubating the specific Ab for 10 min at room temperature in the reaction before addition of the probe.

Western blot analysis

Cell extracts were prepared after the indicated treatments according to published procedures (52) and equal amounts of protein (20 µg) for each lane was electrophoresed on SDS-10% polyacrylamide gel and transferred to a polyvinylidine difluoride-type transfer membrane at 4°C (Immobilo-P, Millipore, Bedford, MA) by standard procedure. Blocking was performed in PBST containing 5% nonfat dry milk for 2 h at room temperature. Diluted primary Abs, IkB{alpha}, and IkBß (1:500) were incubated with membranes in 5% milk-PBS containing 0.1% Tween 20 (PBST) for 16 h at 4°C. After five washes with PBST, HRP-conjugated secondary Ab (1:5000) was incubated for 1 h at room temperature. Blots were washed four times with PBST and twice with PBS and analyzed using the ECL kit from Amersham according to their protocol.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of E-selectin protein expression by pIC and TNF-{alpha} is reduced in AE cells derived from PKR-null mice

The induction of the VCAM-1 gene in human endothelial cells by dsRNA has been reported to be correlated with the activation of PKR (33). Because E-selectin is an important leukocyte adhesion molecule potentially regulated by dsRNA during virus infection, we investigated whether PKR plays a role in its regulation by pIC. Endothelial cells (MuAE cells) derived from the aorta of genetically matched PKR wild-type (Pkr+/+) and knockout (Pkr0/0) mice were used to address this question. Wild-type MuAE cells were treated with pIC for different times, and E-selectin protein expression was determined by RIA. Induction of E-selectin protein expression was observed after pIC addition, reaching a maximum by 4 h and declining slightly by 22 h (Fig. 1GoA). Because TNF-{alpha} is a well-known inducer of E-selectin in endothelial cells (34, 42), we investigated whether there was any defect in induction of E-selectin expression in Pkr0/0 MuAE cells treated with TNF-{alpha}. Both dsRNA and TNF-{alpha} strongly induced E-selectin protein expression in Pkr+/+ AE cells compared with untreated cells, whereas induction in Pkr0/0 cells treated with either dsRNA or TNF-{alpha} was severely attenuated (Fig. 1GoB). These results indicate that PKR is essential for induction by pIC of E-selectin expression on the primary aortic endothelial cell surface. These data also suggest that PKR plays a role in the induction of E-selectin protein expression by TNF-{alpha}. Moreover, because the basal levels of E-selectin expression are reduced in Pkr0/0 cells compared with wild-type cells, we conclude that PKR is required to maintain basal levels of E-selectin expression on the cell surface.



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  		FIGURE 1. Reduced cell surface expression of E-selectin in Pkr0/0 MuAE cells. A, MuAE cells isolated from PKR wild-type mice were treated with 100 µg/ml pIC for the indicated time. B, Both Pkr+/+ and Pkr0/0 MuAE cells were treated with pIC or TNF-{alpha} for 6 h or left untreated (Con), and cell surface expression of E-selectin was measured as described in Materials and Methods. Samples were performed at least in triplicate in each experiment and representative data from one such experiment are shown.

 
Reduced expression of E-selectin mRNA in PKR-null MuAE cells induced with pIC and TNF-{alpha}

E-selectin expression induced in response to pIC or TNF-{alpha} is controlled at the transcriptional level (41, 42, 43, 49, 53). To determine whether the defect in the response of E-selectin to induction by pIC and TNF-{alpha} in Pkr0/0 AE cells was reflected at the mRNA level, wild-type and knockout cells were exposed to inducers, and E-selectin RNA levels were measured by Northern blot analysis. E-selectin steady state RNA was strongly induced (8-fold) in pIC-treated wild-type AE cells (Fig. 2GoA, lane 5) compared with the untreated cells (Fig. 2GoA, lane 2), where the expression was undetectable. TNF-{alpha} treatment also strongly induced (12-fold) E-selectin RNA expression (Fig. 2GoA, lane 6). However, in Pkr0/0 cells, pIC-induced E-selectin mRNA expression was reduced to 3-fold (Fig. 2GoB, lane 3) compared with untreated cells (Fig. 2GoB, lane 1). In TNF-{alpha}-treated cells, E-selectin mRNA expression was similarly depressed to a 4-fold induction (Fig. 2GoB, lane 4). The induction of E-selectin mRNA by TNF-{alpha} and pIC in endothelial cells is rapid and transient. Maximum expression of E-selectin usually occurs at 2 h and then declines to a basal level by about 24 h. We also did not observe the E-selectin mRNA expression at 24 h by treatments with either TNF-{alpha} or pIC in both Pkr+/+ and Pkr0/0 cells (data not shown). These results indicate a requirement for PKR in regulating the steady state levels of E-selectin mRNA.



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  		FIGURE 2. Reduced steady state mRNA expression of E-selectin in Pkr0/0 MuAE cells. A, Pkr+/+ (lanes 2, 5, and 6) and Pkr0/0 (lanes 1, 3, and 4) cells, after serum starvation for 4 h, were left untreated (lanes 1 and 2) or were treated with 100 µg/ml pIC (lanes 3 and 5) or with 10 ng/ml TNF-{alpha} (lanes 4 and 6) for 2 h. Total RNA of 10 µg for each sample was used for the Northern blot. The blot was probed with a labeled mouse E-selectin DNA. The same blot was deprobed and rehybridized with labeled actin cDNA for normalization. B, Fold induction was calculated from the PhosphoImager data of E-selectin divided by the actin values.

 
Induction of the E-selectin promoter-driven luciferase activity by pIC and TNF-{alpha} is reduced in PKR-null cells

The induction of E-selectin mRNA by dsRNA and cytokines is a transcriptional event mediated by complex elements in promoter of the E-selectin gene (41, 42, 43, 49, 53). Accordingly, we determined whether the observed defect in induction of E-selectin mRNA by dsRNA and TNF-{alpha} in Pkr0/0 cells was due to reduced transcriptional activation. Because primary AE cells are difficult to transfect, embryonic fibroblast derived from Pkr+/+ and Pkr0/0 mice were transiently transfected with an E-selectin promoter-driven reporter luciferase construct, and transfection efficiencies between the two cell types corrected by ß-galactosidase activity (see Materials and Methods). After 18 h of transfection, cells were treated with pIC or TNF-{alpha} for 5 h or left untreated, and ß-galactosidase and luciferase activities were measured from lysates. Relative luciferase activity was induced strongly after pIC and TNF-{alpha} treatment (3.4- and 6.1-fold, respectively) of Pkr+/+ cells (Fig. 3Go). In Pkr0/0 cells, basal luciferase activity was lower than Pkr+/+ cells, and pIC failed to induce (1.5-fold) the E-selectin-luciferase reporter. TNF-{alpha} induction of the E-selectin reporter was also reduced (3.9 fold) in transfected Pkr0/0 cells compared with untreated cells (Fig. 3Go). We conclude from these results that PKR plays an important role in the transcriptional induction of the E-selectin gene by both pIC and TNF-{alpha}.



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  		FIGURE 3. Reduced E-selectin-luciferase activity in Pkr0/0 cells. Both Pkr+/+ and Pkr0/0 MEFs were transiently transfected with E-selectin luciferase and Rous sarcoma virus-ß-galactosidase (ß-gal) DNAs by electroporation at room temperature. On the next day, cells were treated with 20 µg/ml pIC or with 10 ng/ml TNF-{alpha} for 5 h in serum-free medium. Unt., untreated. Relative luciferase activities were represented as indicated.

 
PKR is required for maximum activation of NF-{kappa}B-DNA binding by dsRNA and TNF-{alpha} in MuAE cells

We have previously shown that NF-{kappa}B activation by pIC in MEFs and HeLa cells requires PKR (14, 15, 16, 17). Analysis of the E-selectin promoter revealed the presence of regulatory elements for cytokine inducibility within the first 160 bp 5' from the transcriptional start site of the E-selectin gene (43, 44, 45, 46, 47). The {kappa}B elements and an ATF-like site within the regulatory elements are required for maximum transcriptional induction of the E-selectin gene by cytokines (43, 44, 45, 46). To determine whether the defect in transcriptional activation of E-selectin in MuAE cells could be attributed to a failure to induce NF-{kappa}B derived from Pkr0/0 mice, EMSAs were performed using a {kappa}B sequence derived from the mouse E-selectin promoter. Both Pkr+/+ and Pkr0/0 AE cells were treated with pIC and TNF-{alpha}, and whole cell extracts were prepared and used for EMSA. NF-{kappa}B was induced maximally by pIC after 2h in Pkr+/+ cells. TNF-{alpha} also induced NF-{kappa}B in Pkr+/+ cell extracts but less effectively than pIC. In Pkr0/0 cells, the induction of NF-{kappa}B by either pIC or TNF-{alpha} was impaired (Fig. 4GoA) as is most evident when the slowest migrating NF-{kappa}B complex in the TNF-{alpha}-treated cell extracts is compared in Pkr0/0 cells and to wild-type AE cells. We also used these 2-h-treated extracts to assay for NF-I-binding protein by EMSA, and results showed that there was no marked difference in NF-I binding among the 2-h-treated samples (Fig. 4GoB). To characterize the composition of NF-{kappa}B complexes, supershifts were performed with Abs to p50, p65, c-Rel, and p52. Because the p65 Ab supershifted all complexes and the p50 Ab reduced the intensity of faster migrating complex (Fig. 4GoC), we conclude that pIC-induced NF-{kappa}B complexes were composed of p65 and p50 heterodimers and do not involve c-Rel or p52. The results of a supershift experiment using TNF-{alpha}-treated extracts were identical with those from the pIC-treated samples (data not shown). These results show that PKR is essential for the maximum activation NF-{kappa}B in MuAE cells by pIC or TNF-{alpha}.



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  		FIGURE 4. Impaired NF-{kappa}B-DNA-binding activity in Pkr0/0 AE cells. A, Both Pkr+/+ and Pkr0/0 cells were serum starved for 15 h and treated with 100 µg/ml pIC or 10 ng/ml TNF-{alpha} for the indicated time. Equal amounts of whole cell extracts were used for EMSA assay as described in Materials and Methods. B, The same 2-h-treated extracts (used in A) were assayed for NF-I binding by EMSA (69 ). C, Super shift analyses were performed using 2-h-pIC-treated extracts after preincubation with various Abs as indicated.

 
NF-ELAM-1 complex binding to the cAMP response element (CRE)/ATF element is reduced in PKR-null MuAE cells

It has been shown that both NF-ELAM-1 and NF-{kappa}B elements are required for full E-selectin promoter activity (43, 44, 45, 46). cAMP-independent ATF family members interact with NF-{kappa}B for activation of E-selectin by different cytokines (45, 46, 47). The NF-ELAM-1 sequence (TGACATCA) differs from the CRE by one nucleotide (TGACgTCA), and in bovine aortic endothelial cells three NF-ELAM-1-DNA binding complexes have been identified as ATF-2, heterodimer of ATF-2 and c-Jun, and nonphosphorylated cAMP response element binding protein (54). To determine whether PKR plays any role in inducing the DNA-binding activity of NF-ELAM-1, EMSAs were performed with extracts prepared from both Pkr+/+ and Pkr0/0 AE cells using the NF-ELAM-1 element as a probe. Both cell types were similarly treated with pIC or TNF-{alpha} as described in Fig. 4Go. The results (Fig. 5GoA) show that DNA-binding complexes are reduced in Pkr0/0 cells compared with Pkr+/+ cells, although both pIC and TNF-{alpha} treatments increase the intensity of complexes in both cell types (Fig. 5GoA). Super shift experiments were performed with phospho-ATF-2 and ATF-2 Abs to confirm the identity of the complexes. The slower migrating complexes were completely supershifted by ATF-2 Ab, whereas only the slowest migrating complex (phosphorylated ATF-2) is supershifted by phospho-ATF-2 Ab in both cell types (Fig. 5GoB). Another DNA-binding protein, STAT6, was induced equally by IL-4 treatment in both Pkr+/+ and Pkr0/0 AE cells (data not shown). These experiments show that the NF-ELAM-1 complex and both phosphorylated and unphosphorylated ATF-2 binding with NF-ELAM-1-DNA element is reduced in Pkr0/0 cells compared with the Pkr+/+ AE cells, indicating a requirement of PKR in the activation of transcription factor binding to NF-ELAM-1.



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  		FIGURE 5. Reduced NF-ELAM-1-DNA binding complexes in Pkr0/0 AE cells. A, Cellular extracts were prepared as described in Fig. 4Go, and EMSA assays were performed with equal amount of proteins using labeled NF-ELAM-1 element as a probe. B, Super shift assays were performed after preincubation of extracts with phospho-ATF-2 or ATF-2 Abs as indicated.

 
PKR is necessary for pIC-mediated degradation of IkBß protein

We have suggested previously that PKR can act as a signal transducer for both NF-{kappa}B and IRF-1-dependent gene induction (17, 18). Activation of NF-{kappa}B by dsRNA, in vitro, is mediated by PKR via the phosphorylation of I{kappa}B (14, 15). To test whether targeted degradation of I{kappa}B occurs by a PKR-dependent pathway in MuAE cells, these cells were treated for different times with pIC and I{kappa}B{alpha} and I{kappa}Bß protein levels measured in cellular extracts by Western blot analysis. The results (Fig. 6GoA) clearly show that the complete degradation of I{kappa}Bß protein occurred by 180 min after pIC treatment. In contrast, PKR-null cells exhibited no degradation of I{kappa}Bß under the same conditions. Partial degradation of I{kappa}B{alpha} is seen at 60 min of pIC treatment in Pkr+/+ AE cells, but again in Pkr0/0 AE cells there is no pIC-induced degradation of I{kappa}B{alpha} protein (Fig. 6GoB). Hence, the full activation of NF-{kappa}B in Pkr+/+ AE cells is likely due to the combined effects of I{kappa}Bß degradation and partial I{kappa}B{alpha} degradation. In cellular extracts prepared from wild-type and Pkr-null AE cells after TNF-{alpha} treatment, the I{kappa}Bß was only partially degraded after 30 min of TNF-{alpha} treatment and levels were decreased furthers with time (Fig. 6GoC). On the other hand, I{kappa}B{alpha} was completely degraded within 15 min after TNF-{alpha} treatment in Pkr+/+ AE cells and reappeared at 120 min. In Pkr0/0 AE cells, I{kappa}B{alpha} was similarly degraded within 15 min but reappeared sooner (60 min) after TNF-{alpha} treatment (Fig. 6GoD) than in wild-type cells. Because the complete degradation of IkBß occurs at 3 h after pIC treatment in Pkr+/+ AE cells, we treated both Pkr+/+ and Pkr0/0 AE cells with pIC or TNF-{alpha} for 3 h and determined the levels of p65 in cytosolic fractions by Western blot. There was a slight increase in the amount of p65 protein in Pkr0/0 cells compared with Pkr+/+ cells (Fig. 6GoE) when controls for protein (actin) loading were taken in account. These results indicate that PKR is required for effective targeted degradation of I{kappa}Bß in MuAE cells but also contributes to a lesser extent to the targeted degradation of I{kappa}B{alpha}.



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  		FIGURE 6. Induction of degradation of I{kappa}Bß by pIC in AE cells. Both Pkr+/+ and Pkr0/0 cells were treated with 100 µg/ml pIC (A, B, and E) or with 10 ng/ml TNF-{alpha} (C, D, and E) for the indicated time, and equal amounts of extracts were used for Western blots with I{kappa}Bß (A and C) or I{kappa}B{alpha} (B and D) or p65 (E, treated for 3 h) Abs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The IFN-inducible protein kinase, PKR, has been implicated in a variety of important biological roles including translational control, antiviral activity, and cell growth inhibition. Antisense and gene knockout experiments have also implicated PKR as a signal transducer and transcriptional regulator of different NF-{kappa}B and IRF-1-dependent genes (2, 3, 4, 5, 6, 14, 15, 16, 17). dsRNA is a byproduct during virus infection, and many genes such as PKR and E-selectin are induced during viral infections as a result (4, 5, 6, 7, 30, 49). Adhesion molecules such as E-selectin are also expressed on the endothelial cell surface during inflammation resulting from virus infection or tissue injury after induction by cytokines (30, 41, 42, 49).

Here, we have investigated the role of PKR in pIC and TNF-{alpha}-mediated induction of the ELAM E-selectin. For this purpose, we isolated aortic endothelial cells from mice with a targeted deletion in the PKR gene and from wild-type mice in a matched background (16, 18). These isolated endothelial cells respond to pIC and TNF-{alpha} by expressing elevated levels of E-selectin protein as previously been reported for human endothelial cells (30, 49). However, the induction of E-selectin by pIC and TNF-{alpha} is greatly reduced in the PKR-null aortic endothelial cells, indicating that PKR is required for this process.

The lower levels of induction of E-selectin message after pIC and TNF-{alpha} treatments in Pkr0/0 cells (Fig. 2Go) compared with wild-type cells indicate a positive role of PKR in E-selectin expression. As reported earlier, up-regulation of E-selectin by pIC and TNF-{alpha} is largely accomplished at the transcriptional level (41, 42, 43, 49, 53). Transient transfection assays of E-selectin-promoter-luciferase constructs into both PKR wild-type and null MEFs show that PKR is required for the efficient transcriptional response of the E-selectin promoters to both pIC and TNF-{alpha} (Fig. 3Go). We have previously reported that PKR is an important signaling molecule in the regulation of NF-{kappa}B-dependent genes (17, 18). However, in mouse embryo fibroblasts the presence or absence of PKR does not impact on the transcriptional response to TNF-{alpha} alone (17). Clearly, the endothelial cell response to TNF-{alpha} is more dependent on PKR.

Extensive analysis of cytokines responsive elements in the human E-selectin promoter region has identified in addition to {kappa}B elements, other regulatory sequences that influence cytokine-induced promoter activity (43, 44, 45, 46). There are striking similarities in the organization of the E-selectin and IFN-ß gene promoters (55, 56). In addition to {kappa}B sites, both promoters contain CRE/ATF-like binding elements (55). Both genes are silent in the uninduced state, but on stimulation with cytokines or pIC they are strongly induced. The transcriptional activation of E-selectin by cytokines is partly mediated by the interaction of cyclic AMP-independent ATF family members with NF-{kappa}B (45). Additionally, induction of both E-selectin and IFN-ß genes requires the presence of high mobility group protein (HMG)-I(Y) (57, 58). The HMG-I(Y) protein plays an important role in the assembly and function of both gene enhanceosomes (59, 60). We reported previously that the induction of IFN-ß mRNA by dsRNA is impaired in Pkr0/0 MEFs whereas induced levels of the IFN-{gamma} mRNA by virus infection are unaltered in Pkr-null mice compared with Pkr wild-type mice (16). The reduced activation of E-selectin we observed in Pkr0/0 AE cells treated with pIC is similar to that seen in Pkr0/0 MEFs (16). Interestingly, in transient transfection assays, the murine E-selectin promoter (-383 to +80) containing the {kappa}B sequence at -133 to -110 region is inducible by IL-1, although cellular extracts from heart and lung tissues or from HUVEC treated with IL-1 do not exhibit binding activity to the murine E-selectin {kappa}B element (47). Subsequent analyses of the human E-selectin promoter region revealed that multiple NF-{kappa}B binding sites are required for TNF-{alpha} inducibility in HUVEC (44). The {kappa}B site in the murine promoter (AGAAAACTTT) differs from the human {kappa}B site (gGgAAAgTTT) by three bases but is capable of binding NF-{kappa}B in extracts from TNF-{alpha}-treated cells (47).

Although it remains unclear where PKR lies in the signaling pathway activated by pIC or TNF-{alpha} leading to NF-{kappa}B activation, our previous data showed that it is required for pIC signaling in MEFs. In accord with this, the pIC-activated NF-{kappa}B-DNA complex in Pkr0/0 AE cells was reduced compared with wild-type cells. The observation that it was not completely abrogated is consistent with our previous data showing that a combination of pIC and a cytokine (IFN-{alpha} or -{gamma}) could compensate for the absence of PKR (17). TNF-{alpha} induced NF-{kappa}B activation in MuAE cells appears to be partially dependent on PKR, because the activation of NF-{kappa}B is reduced in Pkr0/0 endothelial cells. The lower level of basal E-selectin expression and promoter activity seen in Pkr-null cells compared with wild-type cells is probably due to the reduced level of active NF-ELAM-1 (Fig. 5GoA). The phosphorylation of I{kappa}B by the kinase IKK is essential for its targeting for ubiquitination, degradation allowing the activation of NF-{kappa}B (61, 62, 63, 64, 65, 66). Although PKR can phosphorylate I{kappa}B{alpha} in vitro, it is not certain that this occurs in vivo (14, 15, 33). In fact, in the MuAE cells, pIC induced only slight degradation of I{kappa}B{alpha} in wild-type cells but not in Pkr0/0 cells, implying a minor role of PKR in I{kappa}B{alpha} degradation in these cells (Fig. 6GoB). However, our data (Fig. 6GoA) clearly indicates that PKR is required and plays a major role in I{kappa}Bß degradation induced by pIC in MuAE cells because pIC fails to induce I{kappa}Bß degradation in Pkr-null cells. Previously, functional differences between the I{kappa}B{alpha} and I{kappa}Bß isoforms have been described in their degradation by inducers. Degradation of I{kappa}B{alpha} occurs earlier than I{kappa}Bß after cellular exposure to NF-{kappa}B inducers (66), and it has been proposed that I{kappa}Bß degradation coupled with newly synthesized unphosphorylated I{kappa}Bß mediates the persistent activation of NF-{kappa}B by some inducers in a cell type-specific manner (67). Recently, it has been shown that TNF-{alpha} synergizes with IFN-{gamma} to activate NF{kappa}B-DNA binding through the specific degradation of I{kappa}Bß. Expression of a dominant negative mutant of PKR or using PKR inhibitor blocked this effect (68). Similar results have been observed in Pkr-null cells (70). Taken together with results shown here, it can be concluded that the major target of PKR-dependent signaling of NF-{kappa}B is I{kappa}Bß. The role of PKR in E-selectin expression by pIC and TNF-{alpha} in primary AE cells is to modulate the degradation of IkB and to facilitate the activation of NF-ELAM-1. The mechanisms by how this is achieved are under investigation.


   Acknowledgments
 
We acknowledge the gift of mouse E-selectin cDNA from Dr. Mark A. Lobow, Hoffman La-Roche, the E-selectin luciferase construct from Dr. Paul E. Dicorleto, and NF-I oligo from Richard M. Gonostajski, Lerner Research Institute.


   Footnotes
 
1 This work was supported in part by Grant AI RO1 134039 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Sudip K. Bandyopadhyay, Department of Cancer Biology, Lerner Research Institute, NB-40, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: Back

3 Abbreviations used in this paper: PKR, double-stranded RNA-activated kinase; MuAE cells, murine aortic endothelial cells; pIC, double-stranded RNA; IRF-1, interferon-regulatory factor 1; MEFs, mouse embryo fibroblasts; ELAM, endothelial leukocyte adhesion molecule; ATF-2, activating transcription factor 2; CRE, cAMP response element; AE, aortic endothelial; HMG, high mobility group protein. Back

Received for publication August 26, 1999. Accepted for publication December 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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