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A Functional Role of IB- in Endothelial Cell Activation¹

Martin Spiecker^*, Harald Darius^* and James K. Liao^2,

^* Vascular Medicine and Atherosclerosis Unit, Cardiovascular Division, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and Medical Clinic II, Johannes Gutenberg University, Mainz, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The NF-B inhibitor IB- is a new member of the IB protein family, but its functional role in regulating NF-B-mediated induction of adhesion molecule expression is unknown. In vascular endothelial cells, IB- associates predominantly with the NF-B subunit Rel A and to a lesser extent with c-Rel, whereas IB- and IB-ß associate with Rel A only. Following stimulation with TNF-, pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine, and dexamethasone prevented IB kinase-induced IB-, but not IB-ß or IB- phosphorylation and degradation. Since the activation of NF-B is required for the induction of adhesion molecule expression, we examined the role of IB- in the transactivation of promoters from VCAM-1, ICAM-1, and E-selectin. Using reporter gene constructs of adhesion molecule promoters, PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 promoter activity. Subcloning of B cis-acting elements of VCAM-1, E-selectin, and ICAM-1 into a heterologous promoter construct revealed that PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 B promoter activity. By electrophoretic mobility shift assay, NF-B heterodimers containing c-Rel specifically bind to the B motif in the ICAM-1, but not VCAM-1 or E-selectin promoter. Indeed, overexpression of c-Rel induced ICAM-1 B promoter activity to a greater extent than that of E-selectin and overexpression of IB- inhibited ICAM-1 and VCAM-1 promoter activity in endothelial cells. These findings indicate that c-Rel-associated IB- is involved in the induction of ICAM-1 expression.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The NF-B/Rel family of transcription factors regulates the expression of many genes in eukaryotic cells in response to a variety of extracellular stimuli (reviewed in Refs. 1, 2, 3, 4). Members of the Rel protein family form homodimeric or heterodimeric cytosolic complexes constituting the NF-B family of transcription factors. The Rel proteins can be further subdivided into two groups based on their structure and function. The first group consists of p65 (Rel A), c-Rel, and Rel B which contain transcriptional activation domains necessary for gene induction (5). The second group consists of p105 and p100 which upon proteolytic processing give rise to p50 (NF-B1) and p52 (NF-B2), respectively (6, 7). The activation of NF-B involves the nuclear translocation of Rel protein dimers. With the exception of Rel B, which cannot form homodimers (8), members of both groups can bind in a tissue-specific manner as homodimers or heterodimers to B enhancer elements of target genes.

Several distinct IB³ proteins have been shown to bind and retain NF-B in the cytoplasm, thereby rendering NF-B inactive by masking its nuclear localization sequence (3, 9, 10, 11). Thus, the activation of NF-B involves the phosphorylation and degradation of IB proteins by IB kinases (IKK) and 26S proteasomes, respectively (12, 13, 14, 15). Following cytokine stimulation, the IKK complex is activated by NF-B-inducing kinase (16, 17) and consists of at least three subunits: IKK-, -ß, and - (18, 19, 20, 21). The IKK specifically phosphorylates the serine residues 32 and 36 of IB- (17, 18), which targets IB- for ubiquitination and rapid degradation by the 26S proteasome (22). The IKK also phosphorylates comparable serine residues on two other members of the IB family, IB-ß and IB-, but the subsequent degradation and resynthesis of these IBs are not completely understood. Whereas IB- is rapidly degraded and resynthesized (2, 23), the resynthesis of IB-ß is greatly delayed, which may contribute to the persistent activation of NF-B occurring via IB-ß degradation (24, 25).

A novel member of the IB family, IB-, has been recently described (26, 27, 28) and is involved in the regulation of c-Rel containing NF-B complexes (26). Compared with IB- and IB-ß, the IB- possesses different patterns of basal mRNA expression and its association with different Rel proteins occurs in a cell-specific manner (26, 27, 28). The purpose of this study, therefore, was to determine whether IB- has any functional importance in the induction of cellular adhesion molecule expression in human vascular endothelial cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

Medium 199 was purchased from Life Technologies (Gaithersburg, MD). FCS was purchased from Atlanta Biologicals (Norcross, GA). Endothelial cell growth factor was obtained from Pel-Freez Biological (Rogers, AK). Collagenase CLS 2 and trypsin TRL3 were obtained from Worthington Biochemicals (Freehold, NJ). Recombinant TNF- was purchased from Endogen (Cambridge, MA). Pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine (NAC), heparin, glutathione, alkaline phosphatase-conjugated secondary Ab, p-nitrophenyl phosphate disodium, and alkaline buffer solution were purchased from Sigma (St. Louis, MO). The proteasome inhibitor MG132 (Z-Leu-Leu-Leu-H) was purchased from Calbiochem (San Diego, CA). Rabbit polyclonal affinity-purified rabbit Abs to IB-/MAD-3, IB-ß, IB-, p50, Rel A, Rel B, c-Rel, and IKK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The IB- antiserum 812 and the IB- expression plasmid were generously provided by Nancy Rice (National Cancer Institute, Frederick, MD). The HRP-linked anti-rabbit IgG Ab (from donkey) and the enhanced chemiluminescence Western blotting kit were purchased from Amersham (Arlington Heights, IL). Polyvinylidene fluoride transfer membranes were purchased from Millipore (Bedford, MA). The micro-bicinchoninic acid (BCA) protein assay was obtained from Pierce (Rockford, IL). Low m.w. protein standards were purchased from Bio-Rad (Hercules, CA).

Cell culture

Human saphenous vein endothelial cells were isolated and cultured in a growth medium containing medium 199, 5% FCS, 50 µg/ml of endothelial cell growth factor, 100 µg/ml of heparin sulfate, 100 U/ml penicillin, and 100 µg/ml streptomycin as described previously (29). All cell cultures were incubated at 37°C in a 5% CO₂ atmosphere. Endothelial cells were identified by their typical morphological pattern (cobblestone morphology) and by immunostaining of representative plates for von Willebrand factor related-Ag as described elsewhere (30). Only confluent monolayers up to the third passage were used. Cellular viability was determined by cell count, cellular morphology, and trypan blue exclusion (29). For transient transfection studies, bovine aortic endothelial cells of less than four passages were cultured in DMEM with 10% FCS, penicillin, and streptomycin.

Cell surface enzyme immunoassays

Cells were incubated with murine mAbs against human VCAM-1(E1/6), human ICAM-1(HU5/3), or human E-selectin (HU 18/7) for 2 h. All primary Abs (mouse IgG) were obtained from Michael A. Gimbrone, Jr. (Brigham and Women’s Hospital, Boston, MA). Cell monolayers were then incubated with biotinylated horse anti-mouse IgG Ab (1:1000 dilution; Vector Laboratories, Burlingame, CA) for 1 h followed by incubation with streptavidin-alkaline phosphatase (Zymed, South San Francisco, CA) for 30 min. Washing three times with PBS was performed after each incubation step. Cells were treated with p-nitrophenylphosphate (PNPP, 1 µg/ml) for 30 min at 22°C, and absorbance was measured at 410 nm using cell monolayers without primary Ab as a blank. Integrity of the monolayers was checked before analysis. Each experiment was performed in quadruplicate.

Electrophoretic mobility shift assay

Nuclear extracts were prepared as described previously (30). Oligonucleotides corresponding to the B sequences in the human VCAM-1 promoter (5'-CCTGGGTTTCCCCTTGAAGGGATTTCCCTCC-3'), ICAM-1 promoter (5'-TTAGCTTGGAAATTCCGGAGC-3'), and E-selectin promoter (5'-AGCTTAGAGGGGATTTCCGAGAGGA-3') were synthesized (Integrated DNA Technology, Coralville, IA), annealed, radiolabeled with [-³²P]ATP and T₄ polynucleotide kinase (New England Biolabs), and purified by PAGE or column (Sephadex G-50; Pharmacia, Piscataway, NJ). Nuclear extracts (5–10 µg) were added to ³²P-labeled oligonucleotides (20,000 cpm, 0.2 ng) in a buffer containing 2 µg poly(dI · dC) (Boehringer Mannheim, Indianapolis, IN), 0.5 µg/µl BSA, 10 mM Tris-HCl (pH 7.5), 25 mM NaCl, 50 mM MgCl₂, 1 mM DTT, 1 mM EDTA, and 5% glycerol (total volume of 20 µl). DNA-protein complexes were resolved on 6% nondenaturing polyacrylamide gel electrophoresed at 12 V/cm for 3 h in low ionic strength buffer (0.5x Tris-boric acid-EDTA) at 4°C. For supershift assays, 1 µg of the indicated Ab was added to the nuclear extracts for 15 min before the addition of radiolabeled probe. To determine the specificity of shifted bands, excess unlabeled oligonucleotide (10–20-fold excess) was added directly to the nuclear extracts for 10 min before addition of corresponding radiolabeled probe. Gels were dried and autoradiography was performed at -80°C.

Western blotting

Conditioned endothelial cells were rinsed twice with ice-cold PBS before addition of the lysis buffer (100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 1 mM sodium orthovanadate, 1 mM NaF, and 1 mM PMSF) directly to the culture dishes on ice. The cell lysates were scraped, boiled, and centrifuged for 2 min at 14,000 x g. Protein concentrations were determined with the BCA method. Total cell lysates (40 and 100 µg protein) and low m.w. markers were separated by SDS-PAGE (12% running, 4% stacking).

The separated proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Immobilon P, 0.45 µm pore size; Bio-Rad, Hercules, CA) with a semidry transfer system (Bio-Rad). The blots were incubated for 1 h at room temperature in PBS buffer containing (0.1% Tween and 5% nonfat milk) before incubation with the primary Ab. After washing the membranes four times in the PBS with Tween 20 buffer, a HRP-coupled secondary Ab (1:4000) was added for 30 min. Immunodetection was accomplished using the enhanced chemiluminescence kit (Amersham).

Immunoprecipitation

Endothelial cells were harvested by scraping in ice-cold PBS. Cellular lysates were prepared with an immunoprecipitation buffer containing 50 mM Tris (pH 8.0), 1% Ipegal, 2 mM EDTA, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF, and 10 µg/ml leupeptin, aprotinin, N-tosyl-L-phenyalanylchloromethyl ketone, and tosyl lysine chloromethyl ketone. Protein concentrations were determined by BCA assay. Lysates (200 µg protein) were incubated with specific anti-Rel protein Abs in immunoprecipitation buffer for 1 h before adding 10 µl protein A-agarose beads. Immunoprecipitates were centrifuged at 12,000 rpm for 5 min at 4°C, the supernatant was discarded, and the pellet was washed four times with 1 ml of immunoprecipitation buffer. The immunoprecipitates were then resuspended in 40 µl of electrophoresis sample buffer (125 mM Tris-HCl (pH 6.8), 20% glycerol, and 10% 2-ME), boiled for 5 min, and the supernatant was separated by SDS-PAGE (12% running, 4% stacking). Immunoblotting of coprecipitated IB was performed as described above for Western blotting.

IKK assay

The IKK assay was performed as described by Mercurio et al. (20) with some modifications. The substrates, wild-type (WT) GST-[1-54]IB-, mutant (MT) GST-[1-54, ST]IB-, WT GST-[1-44]IB-ß, and MT GST-[1-44, SA]IB-ß were previously described and generously provided as purified proteins by J. DiDonato and M. Karin (La Jolla, CA) (31). Whole-cellular extracts were prepared with a buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM ß-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 µM Na₃VO₄, 1 mM benzamidine, 2 µM PMSF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM DTT. The IKK- and -ß (IKK-, IKK-ß) were immunoprecipitated with 3 µg of a human IKK-specific Ab (Santa Cruz Biotechnology) for 1 h at 4°C from 250 µg of total cellular lysates.

The IKK-Ab complex was then precipitated with protein A-agarose and washed three times with phosphated Dulbecco’s buffer (40 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.1% Nonidet P-40, 6 mM EDTA, 6 mM EGTA, 10 mM ß-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 µM Na₃VO₄, 1 mM benzamidine, 2 µM PMSF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM DTT) and once with kinase buffer (without ATP). The purified enzymes were incubated with the indicated GST-IB fusion proteins (1 µg IB-, 3 µg IB-ß) in 15 µl kinase buffer containing HEPES (20 mM, pH 7.7), MgCl₂ (10 mM), ATP (10 µM), 3 µCi of [-³²P]ATP, ß-glycerophosphate (10 mM), NaF (10 mM), PNPP (10 mM), Na₃VO₄ (300 µM), benzamidine (1 mM), PMSF (2 µM), aprotinin (10 µg/ml), leupeptin (1 µg/ml), pepstatin (1 µg/ml), and DTT (1 mM). The reaction was terminated by the addition of 2x SDS-PAGE sample buffer and boiling for 5 min. Proteins were separated on 12% SDS-polyacrylamide gels and autoradiography of the dried gel was performed.

Construction of reporter and expression plasmids

The following B binding sites corresponding to the human ICAM-1 and VCAM-1 promoter were synthesized (IDT): ICAM-1 B (sense strand): 5'-TGGCAGGTACCTAGCTGGAAATTCCGGAGCTAGCTTGGAAATTCCGGAGCCTCGAGCGGA-3' and VCAM-1 B (sense strand): 5'-TGGCAGGTACCTGCCCTGGGTTTCCCCTTGAAGGGATTTCCCTCCCGGCTCGAGCGGAT-3prime]. To enhance the promoter activity of the ICAM-1 B construct, the ICAM-1 NF-B binding sequence (underlined) was used in duplicate. The oligonucleotides were subcloned into the luciferase reporter plasmid pGL₂ enhancer (Promega, Madison, WI) after digestion of the polylinker region with KpnI and XhoI. Both NF-B reporter gene constructs were confirmed by DNA sequencing. The empty pGL₂ enhancer vector was used for control studies. A luciferase reporter plasmid with three B sites from the E-selectin promoter and a noninducible control plasmid were previously described and kindly provided by J. Anrather (New England Deaconess Hospital, Boston, MA) (32). The c-Rel cDNA in the Rc.CMV plasmid was provided by Nancy Rice (National Cancer Institute).

Overexpression of IBs

The following expression plasmids were used: WT IB-, encoding the full-length protein, is amino terminus FLAG-tagged in pCMV4. IB- N, encoding aa 37–317, is also amino terminus FLAG-tagged in pCMV4. The IB- N is lacking the serine phosphorylation sites and is therefore resistant to degradation by the 26S proteasome. However, the protein is functional in terms of NF-B inhibition (33). WT IB-ß is amino terminus FLAG-tagged in pCMV4. WT IB-, IB- N, and WT IB-ß were kindly provided by D. Ballard (Vanderbilt University, Nashville, TN) and have been described in detail previously (33, 34). The IB- cDNA is in the pcDNA3 plasmid (Promega) and was kindly provided by Nancy Rice (National Cancer Institute).

Bovine rather than human endothelial cells were used because of their higher transfection efficiency (30). Endothelial cells (1 x 10⁶ cells) were transfected with 0.7 µg of adhesion molecule reporter plasmid, 0.3 µg of control plasmid (pRSV.ß-Gal), and 0.5 µg IB expression plasmid using the calcium phosphate precipitation method (35). Total plasmid concentration was kept constant in cotransfection studies with lower expression plasmid concentrations complemented by the addition of the corresponding empty expression vector. Preliminary results using ß-galactosidase staining indicated that cellular transfection efficiency was 12%. After 48 h, cells were treated with TNF- (200 U/ml), and cellular extracts were prepared 8 h later using a lysis buffer with 100 mM potassium phosphate (pH 7, 8) and 0.2% Triton X-100. The supernatant was obtained after centrifuging the extracts at 12,000 x g for 2 min. Luciferase and ß-galactosidase activity were measured in a Berthold luminometer using a kit (Tropix, Bedford, MA). Each experiment was performed in duplicate.

Statistics

Results from enzyme immunoassays and reporter assays are expressed as means ± SEM. Means were compared by Student’s paired t test. A confidence level of p < 0.05 was taken to represent a significant difference between two means. Multiple comparisons were done by ANOVA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Kinetics of IB degradation and resynthesis

To assess differential functions of IB-, IB-ß, and IB- in endothelial cell activation, the time course of TNF--induced protein degradation and resynthesis was studied. Upon stimulation with TNF- (200 U/ml), IB- was completely degraded in whole-cell lysates within 15 min and was resynthesized after 60 min (Fig. 1). With continuous cytokine stimulation, the level of IB- protein expression was still below baseline at 12 h. In contrast, IB-ß was degraded within 30–60 min and not resynthesized until after 12 h. Similarly, the IB- protein was degraded at 15–30 min following TNF- stimulation. The IB- protein, however, reappeared after 6 h and the levels remained below baseline after 12 h. Therefore, the pattern of IB- degradation and resynthesis following TNF- stimulation differs from that of IB- and IB-ß in endothelial cells.

View larger version (69K): [in this window] [in a new window]   FIGURE 1. Comparative kinetics of IB protein degradation and resynthesis in vascular endothelial cells. Immunoblots of total cell lysates from endothelial cells stimulated with TNF- (200 U/ml, 0–12 h) were performed with Abs directed against IB-, -ß, and -. Experiments were performed four times with similar results.

Differential inhibition of IB protein phosphorylation and degradation

To further investigate differences in the regulation of IB proteins, the effect of several NF-B inhibitors on TNF--induced IB degradation was investigated. IB- was investigated at 30 min and IB-ß and IB- were investigated at 60 min after TNF- stimulation. As expected from previous studies, TNF--induced degradation of IB- protein was inhibited by the 26S proteasome inhibitor MG132, PDTC, NAC, sodium salicylate (NaS), and dexamethasone (Fig. 2). Although dexamethasone is not known to inhibit IKK activity, its ability to prevent the disappearance of IB- may be related to its ability to induce IB-. However, only MG132 and NaS were able to inhibit the degradation of IB-ß and IB-. Dexamethasone, PDTC, and NAC were unable to stabilize IB-ß and IB-.

View larger version (63K): [in this window] [in a new window]   FIGURE 2. Differential effects of NF-B inhibitors on TNF--induced IB protein degradation. Immunoblots of total cell lysates from endothelial cells stimulated with TNF- (200 U/ml) in the presence or absence of MG132 (10 µM), PDTC (200 µM), NaS (20 mM), dexamethasone (Dex, 1 µM), or NAC (30 mM) for 1 h. IB- was determined 30 min and IB-ß and IB- were determined 60 min after TNF- stimulation. Three separate experiments yielded similar results.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. IKK assay showing TNF- (200 U/ml, 8 min)-induced phosphorylation of IB-GST fusion proteins: WT IB- and IB-ß, MT IB- (residues 32 and 36 S > T) or IB-ß (residues 19 and 23 S > A) with or without PDTC (200 µM). Three separate experiments yielded similar results. The IKK Ab used recognized both IKK- and -ß.

Association of IB and regulation of adhesion molecule promoters by specific NF-B complexes

Different associations of IB proteins with NF-B subunits have been described in different cell lines (24, 26, 27, 28, 34). To determine the specificity of IB-, IB-ß, and IB- association with the NF-B subunits, Rel A, p50, c-Rel, and Rel B in vascular endothelial cells, we immunoprecipitated Rel proteins followed by immunoblotting for IB-, IB-ß, and IB- (Fig. 4). Western blotting with Abs against the IB proteins revealed an association of all three NF-B inhibitors, IB-, IB-ß, and IB-, with Rel A. However, only IB- coimmunoprecipitated with c-Rel whereas IB- and IB-ß did not. To determine the amount of Rel proteins that were actually immunoprecipitated, immunoblotting of the immunoprecipitate with the corresponding Rel Abs showed that comparable amounts of Rel A and Rel B were immunoprecipitated, whereas c-Rel was less abundant in the immunoprecipitate. The p50 band was not discernible due to the overlapping band for IgG heavy chain. These findings suggest that in contrast to IB- and IB-ß, IB- may differentially regulate NF-B complexes via selective association with c-Rel. We also attempted to immunoprecipitate with Abs to IB-, -ß, and - followed by immunoblotting for Rel proteins. These studies, however, were unrevealing probably due to the fact that c-Rel is not very abundant in endothelial cells. Thus, the amount of c-Rel immunoprecipitated with IB proteins was not sufficient for detection by subsequent immunoblotting.

View larger version (77K): [in this window] [in a new window]   FIGURE 4. Coimmunoprecipitation of IB proteins with Rel proteins. Rel family proteins (Rel A, p50, c-Rel, and Rel B) were immunoprecipitated followed by immunoblotting with Abs directed against IB-, -ß, - and to the corresponding Rel proteins. This is a representative blot from three to four separate experiments.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. Cell surface enzyme immunoassays showing the effect of MG132 (10 µM) or PDTC (200 µM) on TNF- (200 U/ml, 4 h)-induced adhesion molecule expression. Results are expressed as percent relative to TNF- stimulation. *, p < 0.001 compared with TNF- and **, p < 0.05 compared with control.

Differential regulation of B cis-acting elements in adhesion molecule promoters

Athough VCAM-1, E-selectin, and ICAM-1 are transcriptionally activated by Rel protein dimers, they contain distinct NF-B-binding motifs in their promoter region (39, 40). To investigate whether the regulation of ICAM-1, VCAM-1, and E-selectin is due to differences in the B cis-acting elements and NF-B subunit composition, we performed electrophoretic mobility shift assays using oligonucleotides corresponding to the specific B sites of the VCAM-1, E-selectin, and ICAM-1 promoters. Using the VCAM-1 B oligonucleotide, formation of an upper and lower complex was observed following stimulation with TNF- (Fig. 6). As indicated by Ab supershift experiments, the lower complex results from binding to p50 homodimers. The upper complex consists of the Rel A/p50 heterodimer. There is a darker lower band which shifts with anti-p50 Ab, but it is not clear whether this is a specific NF-B since PDTC, which inhibits NF-B, did not alter the intensity of this band. A similar binding pattern was observed with the E-selectin B oligonucleotide except there was no apparent p50/p50 homodimer. Thus, VCAM-1 and E-selectin B oligonucleotides bind in TNF--stimulated cells to p65/p50 homo/heterodimers that were inhibited by preincubation with PDTC (200 µM) for 1 h. In contrast, the ICAM-1 B oligonucleotide forms a complex of two bands with nuclear extracts of TNF--stimulated cells. The upper band is supershifted by an Ab directed against c-Rel, the lower band is supershifted by an Ab against c-Rel and p50, and neither shifted complexes were inhibited by PDTC. These findings suggest that the relative insensitivity of TNF--induced ICAM-1 expression to PDTC may, in part, be due to the differences in the Rel dimers which bind to the ICAM-1 B site compared with the VCAM-1 and E-selectin B site.

View larger version (54K): [in this window] [in a new window]   FIGURE 6. Electrophoretic mobility shift assays using oligonucleotides derived from the B sites of E-selectin, ICAM-1, and VCAM-1 promoter. Endothelial cells were stimulated with TNF- (200 U/ml, 1 h) in the presence or absence of PDTC (200 µM). For supershift analysis, 1 µg of specific Abs directed against the indicated NF-B subunit (c-Rel, p50, Rel A) was added 15 min before the radiolabeled oligonucleotide. NS, nonspecific band; FP, free probe.

View larger version (25K): [in this window] [in a new window]   FIGURE 7. A, Transient transfection of heterologous promoter containing B sites of E-selectin, ICAM-1, and VCAM-1 promoter linked to a luciferase reporter gene. Endothelial cells were transfected with the indicated luciferase reporter plasmid (0.7 µg) and RSV.ß-Gal expression plasmid (internal control, 0.3 µg) before stimulation with TNF- (200 U/ml, 8 h) in the presence or absence of PDTC (200 µM) or MG132 (10 µM). *, p < 0.01 compared with control or MG132. B, Effect of c-Rel overexpression on E-selectin and ICAM-1 B promoter activity. Endothelial cells were transfected with E-selectin or ICAM-1 B promoter-luciferase construct, RSV.ß-Gal cDNA plasmid, and increasing amounts of c-Rel cDNA construct. Promoter activity was standardized to ß-galactosidase activity and expressed as multiples of basal activity (fold induction). *, p < 0.05 compared with values for E-selectin promoter activity.

Regulation of adhesion molecule promoter activity by specific IB proteins

To further characterize the functional importance of IB- in endothelial cells, we investigated the effects of IB protein overexpression on TNF--induced VCAM-1 and ICAM-1 B heterologous promoter activity. Both ICAM-1 and VCAM-1 B promoter activity were inhibited by co-overexpression with increasing concentrations of IB-, IB-ß, and IB- cDNAs (Fig. 8). The mutant IB- construct, which cannot be phosphorylated by IKKs, was 10-fold more potent than the WT IB- construct. The ICAM-1 B promoter activity was slightly more sensitive to inhibition by IB overexpression than the VCAM B promoter activity. For a given B site, inhibition of reporter gene activity was comparably inhibited by overexpression of WT IB-, IB-ß, and IB-. Therefore, in terms of IB overexpression on isolated B elements of VCAM-1 and ICAM-1 promoter, ICAM-1 B was not selectively regulated by specific IB proteins.

View larger version (23K): [in this window] [in a new window]   FIGURE 8. Effects of specific IB overexpression on ICAM-1 or VCAM-1 promoter activity. Endothelial cells were transfected with the indicated luciferase reporter plasmid (0.7 µg), RSV.ß-Gal expression plasmid (internal control, 0.3 µg), and increasing amounts of IB expression plasmids. The total amount of DNA transfected was kept constant by supplementing with empty expression plasmids. The following expression plasmids were used: WT IB- in pCMV4, MT IB- (aa 37–317) in pCMV4, WT IB-ß in pCMV4, and WT IB- in pcDNA3. Results shown are from three transfection studies for each condition.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We find that IB-ß is degraded in human endothelial cells following TNF- stimulation; a finding which is not observed in many other cell lines (25). For example, the incomplete degradation of IB-ß following TNF- stimulation in murine fibroblasts and E29.1 cells is thought to contribute to the persistent activation of NF-B following TNF- stimulation since IB-, but not IB-ß, is rapidly resynthesized following TNF- stimulation (41, 42). However, we find that the protein levels of resynthesized IB- remain below baseline levels despite autoregulatory induction of IB- following TNF- stimulation. Taken together, these findings suggest a continuous degradation of IB- in the presence of TNF- (43). Similarly, the protein level of resynthesized IB- also remains below baseline levels following TNF- stimulation, although the level of IB- mRNA is lower compared with that of IB- (27). Our results, therefore, indicate a complex regulation of NF-B which cannot be solely explained by the lack of IB-ß resynthesis.

Although IKK- and -ß are capable of phosphorylating all three IB proteins (17, 19, 44), their activities are differentially inhibited by PDTC, NAC, and dexamethasone. For example, we find that these NF-B inhibitors selectively prevented IB-, but not IB-ß and IB- degradation. This is in contrast to other studies showing that PDTC inhibits IB-ß and overexpressed IB- degradation in murine pre-B 70Z/3 cells (24, 26). However, the murine pre-B cell line exhibits a very different pattern of IB/Rel protein association compared with endothelial cells (26). In addition, other factors such as the incubation period, the concentrations of PDTC, and stimulation with bacterial LPS and not TNF- may account for this discrepancy.

Compared with NAC and dexamethasone, PDTC was unable to completely inhibit the activation of IB-ß- and IB--associated NF-B subunits. A selective association of different Rel and IB proteins along with different IB signaling pathways would allow the release of distinct Rel proteins. We find that in human endothelial cells, IB- is associated predominantly with the NF-B subunit Rel A and, to a lesser extent, with c-Rel. It is important to note that c-Rel is much less abundant in endothelial cells than in other cell lines such as Jurkat cells (43). Interestingly, in c-Rel-enriched cells such as Jurkat and THP-1, a significantly higher amount of IB- is bound to c-Rel (26). Different findings with Rel protein overexpression and nontransfected cell lines clearly show the importance of the relative abundance of Rel proteins. Thus, the findings of one cell line may not necessarily apply to another cell line.

Our hypothesis that a differential activation of Rel proteins would lead to a differential up-regulation of NF-B-dependent genes was tested using PDTC on TNF--induced adhesion molecule expression. The incomplete suppression of ICAM-1 expression in contrast to a complete suppression of VCAM-1 and E-selectin can be due to either NF-B or other transcription factors. These findings are consistent with previous findings showing that VCAM-1 is completely inhibited by PDTC (37, 38, 45). However, the effects of PDTC on TNF--induced ICAM-1 and E-selectin differ dose dependently in previous studies. For example, using 50 µM PDTC, Marui et al. (37) reported a partial inhibition of E-selectin mRNA and no inhibition of ICAM-1 mRNA in HUVECs. Ferran et al. (38) described an 80–90% inhibition of E-selectin surface expression and mRNA in porcine aortic endothelial cells with 100 µM PDTC. Weber et al. (45) found an incomplete inhibition of ICAM-1 mRNA by 100 µM of PDTC in HUVECs. These discrepancies are most likely related to the different concentration of PDTC used and the different cell lines were examined. In comparison with our results, these data show a dose-dependent effect of PDTC on ICAM-1 and E-selectin expression. VCAM-1 is most effectively inhibited by PDTC. Although IB- degradation and the ICAM-1 B interaction with c-Rel are not inhibited by PDTC, there is an incomplete inhibition of ICAM-1 B reporter gene activity and ICAM-1 cell surface expression by PDTC. This might be related to possible modulation of NF-B transcriptional activity by PDTC independent from DNA binding. For example, phosphorylation of Rel A regulates transcriptional activation independent from IB proteins (46, 47, 48).

Endothelial cell adhesion molecules are transcriptionally regulated by multiple transcription factors (39). NF-B is essential for all three adhesion molecules. Additionally, interactions between transcription factors modulate the cytokine-induced transcriptional activation. Inhibition of other transcription factors in the VCAM-1 or E-selectin promoter region that are not required for ICAM-1 induction might explain the differential inhibition by PTDC. However, mutation of either of the VCAM-1 NF-B binding sites abolishes the TNF-induced transcriptional activation (49). Therefore, the absence of functionally active RelA/p50 heterodimers in the nucleus is sufficient to explain the unresponsiveness to cytokine stimulation following preincubation with PDTC. Using exclusively the specific NF-B sites for reporter gene plasmids does not allow to study interactions between transcription factors, but allows conclusions on the functional importance of specific Rel protein dimers. We find that PDTC has differential effects on the activation of Rel protein dimers. The specific association of IB-/c-Rel along with the differential regulatory effect of exogenous NF-B inhibitors show that these specific Rel protein/IB protein interactions might be important for the differential regulation of NF-B activity.

The results of the cotransfection study suggest an inhibitory effect of WT IB- comparable to IB-ß and IB-. Although the highest concentration (500 µg) of IB- and IB-ß inhibited the ICAM-B construct more effectively than the VCAM-B construct, IB proteins are to a certain degree redundant. This is probably related to the fact that all three IB proteins are associated most prominently with Rel A in vascular endothelial cells. Interestingly, IB- expression is up-regulated in fibroblasts derived from IB--deficient mice (26). Additionally, overexpression of IB proteins results in loss of normal transcription factor/inhibitor environment which consecutively might abolish selective NF-B regulation.

In summary, we find that the regulation of adhesion molecule expression, particularly of ICAM-1, is due, in part, to specific interaction of Rel dimers with distinct IB proteins. It remains to be determined whether association of Rel proteins with specific IB proteins make the IB proteins less susceptible to phosphorylation by IKK.

	Acknowledgments

 
We thank I. Lorenz for technical assistance, J. DiDonato, and M. Karin for GST-IB and GST-IB-ß, N. Rice for IB- Ab and c-Rel and IB- expression plasmid, D. Ballard for IB- and -ß expression plasmids, and J. Anrather for the E-selectin B luciferase plasmid.

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health (HL-52233 and HL-48743) and Deutsche Forschungsgemeinschaft. M.S. was a recipient of the Feodor Lynen Fellowship (Alexander von Humboldt-Stiftung). J.K.L. is an Established Investigator of the American Heart Association.

² Address correspondence and reprint requests to Dr. James K. Liao, Department of Medicine, Cardiovascular Division, 221 Longwood Avenue, LMRC-322, Boston, MA 02115. E-mail address:

³ Abbreviations used in this paper: IB, inhibitor B; IKK, IB kinase; PDTC, pyrrolidine dithiocarbamate; NAC, N-acetylcysteine; NaS, sodium salicylate; BCA, bicinchoninic acid; WT, wild type; MT, mutant; PNPP, p-nitrophenylphosphate.

Received for publication July 29, 1999. Accepted for publication January 4, 2000.

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