©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell Type-specific Transactivation of the VCAM-1 Promoter through an NF-B Enhancer Motif (*)

Mushtaq Ahmad , Nobuyuki Marui (§) , R. Wayne Alexander , Russell M. Medford (¶)

From the (1) Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cytokine activation of vascular cell adhesion molecule-1 (VCAM-1) gene expression by endothelial cells is an important feature in a variety of vascular inflammatory responses. Cytokines transcriptionally activate the VCAM-1 promoter in endothelial cells at least in part through two closely linked NF-B enhancer motifs, L-R (positions -77 and -63). However, cytokine activation of the dimeric NF-B transcriptional factor (p50+p65 subunits) occurs in almost all cell types, whereas VCAM-1 gene expression exhibits a cell type-specific pattern of expression. Tumor necrosis factor- markedly transactivated a transiently transfected minimal L-R motif-driven VCAM-1 promoter, p85VCAMCAT, in passaged human vascular endothelial cells but not in the human epithelial cell line, HeLa suggesting that cell type-specific factors may function through the L-R motif. Both cell types exhibited similar inductions of NF-B DNA binding activity and transcriptional activity. However, co-transfection of HeLa cells with p65 and p50 expression vectors demonstrated that the minimal VCAM-1 promoter was effectively transactivated by p65 alone but that additional co-expression of p50 blocked this activity. Furthermore, cytokine activation of the minimal VCAM-1 promoter in HeLa cells was recovered by inhibition of p50 expression using antisense oligonucleotide. These studies suggest that the NF-B(p50+p65 heterodimer) does not support transactivation of the VCAM-1 promoter with the p50 subunit potentially playing a significant inhibitory role in suppressing cytokine activation of VCAM-1. In addition, p65 associated transcriptional factors other than NF-B may serve as positive, cytokine-inducible, cell type-specific regulators of VCAM-1 gene expression.


INTRODUCTION

Vascular cell adhesion molecule-1 (VCAM-1) () is an inducible cell surface protein of vascular endothelial cells that mediates the adhesion of mononuclear leukocytes to endothelial cells in response to a wide variety of inflammatory signals (1, 2) . Endothelial expression of VCAM-1 is observed in early atherosclerotic lesions of the vessel wall, as well as other inflammatory processes, suggesting the importance of VCAM-1 in these disease states (3) . However, the molecular mechanisms regulating VCAM-1 gene expression are not well understood. The human VCAM-1 promoter contains two closely linked B-like elements, L -R, separated by five nucleotides, at positions -77 and -63 relative to the transcription start site, respectively (4) . R but not L completely conforms to the B consensus sequence for the binding of the inducible, transcriptional regulatory factor NF-B (5) . Yet, both of these sequences are necessary for the induction of VCAM-1 promoter by tumor necrosis factor (TNF-) in endothelial cells (4, 6, 7) .

The role of the NF-B transcriptional factor in transactivating VCAM-1 in response to cytokine stimulation of endothelial cells is not fully understood. NF-B is a member of the Rel family of transcriptional regulatory proteins and consists of two distinct polypeptides of 50 and 65 kDa, termed p50 and p65. In most unstimulated cell types, NF-B is complexed with an inhibitory protein, IB, in a non-DNA-binding form that is localized to the cytosol (8) . Stimulation of cells with cytokines (9) and several other activators (5, 10) results in the release of NF-B from IB and its translocation into the nucleus where it transcriptionally regulates the expression of a wide variety of genes through specific B enhancer motifs (5, 11) . An important feature of the Rel family is its ability to form a wide range of homodimers ( e.g. p65 and p50) as well as heterodimers not only within the Rel family, but also with other classes of transcriptional factors (12, 13, 14) . These complexes exhibit significant differences in their binding affinity for, and transactivation through, several B DNA motifs. Recently, it has been shown that the p65 subunit of NF-B can form homodimers that can also function as transactivators (15, 16, 17, 18, 19, 20) . Studies with the p50 subunit have shown that it can also act as a transactivator in vitro (20, 21) as well as in yeast (22) , although this has not yet been observed in mammalian cells (16, 17, 19, 23) . Thus, cell type-specific differences in the activation of this family of NF-B-like DNA-binding proteins may play an important role in regulating VCAM-1 gene expression through its L-R motif.

Two observations suggest that transcriptional regulatory factors other than the NF-B heterodimer itself may mediate transactivation through the VCAM-1 L-R elements. First, the L-R elements bind at least two different NF-B-like binding proteins (4) . Second, TNF- transactivates the VCAM-1 promoter through the L-R elements in a cell type-specific manner. Non-endothelial cell lines such as Jurkat and HeLa activate NF-B when stimulated with cytokine TNF- as well as other activators (5, 10) . However, although active in TNF--stimulated human vascular endothelial (HUVE) cells, constructs of the VCAM-1 promoter containing L-R are not transactivated in the TNF--stimulated T-cell line Jurkat (4) . This raises the possibility that instead of NF-B, other homo- or heterodimers of members of NF-B/Rel family may be involved in activating the VCAM-1 promoter in TNF--stimulated HUVE cells.

To explore the relative roles of NF-B and NF-B-like factors in regulating VCAM-1 gene transcription through the L-R motif, we have characterized the ability of these factors to mediate transcription of the VCAM-1 promoter through cytokine activation of endogenous NF-B as well as to reconstituted homo- and heterodimers of p50 and p65 using expression vectors in HeLa cells. Our studies suggest that while Rel family members, such as the p65 homodimer, are potent transactivators, the NF-B (p50+p65 heterodimer) does not effectively transactivate the VCAM-1 promoter through the L-R motif. DNA binding studies suggest that TNF- induces NF-B (p50+p65 heterodimer) and p65 homodimer or homodimer-like protein both in HUVE and HeLa cells. However, functional p65 homodimer was mainly present in HUVE cells. Thus, VCAM-1 transactivation through L-R may be a function of differential activation of NF-B (p50+p65 heterodimer) relative to other members of the Rel family, such as p65 homodimers.


MATERIALS AND METHODS

Cell Culture

HeLa cells (CCL2) were obtained from American Type Culture Collection (Rockville, MD) and maintained in minimal essential medium supplemented with 10% fetal bovine serum (Irvine Scientific, Santa Ana, CA). HUVE cells were purchased from Clonetics (San Diego, CA) and were cultured in M199 medium supplemented with 20% fetal bovine serum, 16 units/ml heparin (ESI Pharmaceuticals, Cherry Hill, NJ), 50 µg/ml endothelial cell growth supplement (Collaborative Research Incorporated, Bedford, MA), and 25 m M HEPES buffer. The medium for both cells contains 2 m M L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin. HUVE cells were grown on tissue culture plates coated with 0.1% gelatin and were used within the first 6 passages. Human recombinant TNF- was obtained from Boehringer Mannheim. All other reagents were of reagent grade.

CAT Assay

One day prior to transfection, HeLa and HUVE cells were split at the ratio that would give 60-70% confluence. The transfection was done by the calcium phosphate co-precipitation technique. For HeLa cells, 5-10 µg of reporter plasmids were used. For HUVE cells, 30 µg of reporter plasmids were transfected as described previously (24) . The promoterless plasmid, poLUC (25) , was used for adjusting the amount of transfected DNA. The cells were harvested and cell extracts were prepared by three cycles of rapid freeze-thaw in 0.25 M Tris, pH 8.0. Protein content was determined using the Bradford (26) technique. The same amounts of proteins were assayed for CAT activity according to standard protocols (27) . The CAT activity was expressed as percent of chloramphenicol converted to acetyl chloramphenicol. Acetylated and unacetylated forms of chloramphenicol were separated on thin layer chromatography and their amounts were determined after scraping by counting the radioactivity of the respective bands in scintillation vials. Each assay was performed in duplicate or triplicate and the results reported are the average of at least two separate experiments.

Expression Vectors and CAT Reporter Genes

The eukaryotic expression vectors CMV-p50, CMV-p65, CMV-p50/65, and CMV-rel/p65 contain the respective cDNAs cloned between a cytomegalovirus (CMV) promoter, -globin intron and simian virus 40 poly(A) signal (17) . CMV-p50/65 encodes a chimeric protein consisting of the DNA binding domain of p50 (amino acids 1-370) and the transactivation domain of p65 (amino acids 309-550) (28) . The reporter plasmid, p(HIVB)CAT, contains four tandem copies of the B DNA sequences cloned upstream of the human immunodeficiency virus type-1 (HIV-1) long terminal repeat and fused to the coding region of the bacterial CAT gene (28) . The sequence of the B motif of p(HIVB)CAT is 5`-GGGGACTTTCC-3`, and is identical to the B sequence found in the mouse immunoglobulin gene promoter (IgB). These vectors were generous gifts of Dr. C. Rosen (Human Genome Sciences, Rockville, MD). The reporter gene, p85VCAMCAT, contains coordinates -85 to +12 of the human VCAM-1 promoter (4) . The L-R driven heterologous promoter pTA(-77/-63)CAT has been described previously (4) . These reporter genes were generous gifts of Dr. D. Dean (Washington University, St. Louis, MO).

Nuclear Extracts Preparation

Confluent HUVE and HeLa cells were exposed to TNF- (100 units/ml) for 1-3 h. Nuclear extracts were prepared by a modification of the method of Dignam et al. (29) . Briefly, after washing with phosphate buffered saline, cells were centrifuged and the cell pellet suspended in 500 µl of buffer A (10 m M HEPES, pH 7.9, 1.5 m M MgCl, 10 m M KCl, and 1.0 m M dithiothreitol). After recentrifugation, the cells were resuspended in 80 µl buffer A containing 0.1% Triton X-100 by gentle pipetting up and down. After incubating for 10 min at 4 °C, the homogenate was centrifuged and the nuclear pellet was washed once with buffer A and resuspended in 70 µl of buffer C (20 m M HEPES, pH 7.9, 25% (v/v) glycerol, 0.42 M NaCl, 1.5 m M MgCl, 0.2 m M EDTA, 1 m M dithiothreitol). This suspension was incubated for 30 min at 4 °C followed by centrifugation at 20,000 g for 10 min. The resulting supernatant (nuclear extract) was stored at -70 °C. Protein concentrations were determined by the Bradford (26) method. To minimize proteolysis, all buffers contained 1.0 m M phenylmethylsulfonyl fluoride, aprotinin (10 µg/ml), leupeptin (10 µg/ml), and antipain (10 µg/ml).

Gel Shift Assays

The oligonucleotide containing L-R of the VCAM-1 promoter (VCAM-1 wild type oligo) was synthesized. Its sequence is as follows: 5`CTGCCCTGGGTTTCCCCTTGAAGGGATT-TCCCTCCGCCTCTGCAACAAGCTCGAGATCCTATG-3`. The sequences of L and R are underlined with a single line. The double underlined sequences represent an unrelated tail sequence added to serve as a template for synthesis of the double-stranded DNA. To prepare double-stranded DNA, first an oligonucleotide 5`-CATAGGATCTCGAGC-3` (complementary to the 3`-unrelated tail, double underlined sequence) was annealed to VCAM-1 wild type oligo. The second strand was extended with DNA polymerase (Klenow fragment) in a reaction mixture containing 50 µCi of [P]dCTP and 0.5 m M of cold dATP, dGTP, and dTTP. The reaction was followed by the addition of 0.5 m M cold dCTP to insure completion of the second strand. Unincorporated nucleotides were removed by column chromatography over a Sephadex G-50 column. The DNA binding reaction was performed at 30 °C for 15 min in a volume of 20 µl, which contained 225 µg/ml bovine serum albumin, 1.0 10cpm of P-labeled probe, 0.1 µg/ml poly(dI-dC), and 15 µl of binding buffer (12 m M HEPES pH 7.9, 4 m M Tris, 60 m M KCl, 1 m M EDTA, 12% glycerol, 1 m M dithiothreitol, and 1 m M phenylmethylsulfonyl fluoride). After the binding reaction, the samples were subjected to electrophoresis in 1 Tris-glycine buffer using 4% native polyacrylamide gels.

Antibody p50 was a generous gift of Dr. C. Rosen (Human Genome Sciences Inc., Rockville, MD) and was characterized to completely shift both the p50 homodimer and the NF-B(p50+p65 heterodimer) in gel shift assays (data not shown). Antibodies against p65, c-Rel (raised against a peptide in the carboxyl terminus region), and RelB were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. Bacterially expressed proteins, p50 and truncated p65 (containing amino acids 1-309), were prepared and purified as described previously (17) . Due to the low amounts of available purified proteins, the concentration of the purified proteins was roughly estimated from the concentration of proteins before dialysis, the final step of purification. The homo- and heterodimers of p50 and truncated p65 were prepared by incubating them alone or in combination with each other for one hour at 37 °C as described earlier (20) except that DNA binding buffer was used for dimerization. These dimers were used for DNA binding studies. The purified proteins were generous gifts of Dr. C. Rosen (Human Genome Sciences Inc., Rockville, MD).

The sequences of phosphorothioate antisense p50, p65 (30) , and the unrelated antisense (intercellular adhesion molecule-1) (31) were 5`-TGGATCATCTTCTGCCATTCT-3`, 5`-GGGGAACAGTTCGTCCATGGC-3` and 5`-CCCCCACCACTTCCCCTCTC-3`, respectively. These sequences have been used previously to inhibit the translation of the respective mRNAs (30, 31) .


RESULTS

TNF- Activates NF-B-mediated Transcription in Both HUVE and HeLa Cells but Induces the VCAM-1 Promoter Only in HUVE Cells

To explore the mechanisms underlying cell type-specific expression of VCAM-1, we chose to compare the activation of the VCAM-1 promoter in passaged HUVE cells that markedly induce VCAM-1 expression in response to cytokines (4) with the epithelial cell line, HeLa, that does not express VCAM-1 at either the mRNA or protein levels (data not shown). This lack of VCAM-1 expression in HeLa cells occurs despite its well described NF-B activation in response to several cytokines and to the phorbol ester phorbol 12-myristate 13-acetate (32, 33, 34, 35) . HeLa and HUVE cells were transfected with p85VCAMCAT, a deletion construct of the human VCAM-1 promoter (coordinates -85 to +12) that contains the L-R elements and has been previously characterized as the minimal TNF--inducible VCAM-1 promoter (4) . To assess for functional NF-B, both cell types were also transfected in parallel with p(HIVB)CAT, an NF-B-responsive promoter construct containing a tetramer of canonical B elements, p(HIVB)CAT, well characterized to be activated by NF-B(p50+p65 heterodimers) (28) .

As expected, TNF- markedly induced p(HIVB)CAT in both HUVE (Fig. 1 B, lanes 1 and 2) and HeLa cells (Fig. 1 A, lanes 1 and 2), demonstrating functional activation of NF-B. TNF- also markedly induced p85VCAMCAT activity in HUVE cells (Fig. 1 B, lanes 3 and 4). In contrast, TNF- caused little or no activation of p85VCAMCAT (Fig. 1 A, lanes 3 and 4) in HeLa cells. Similar results were obtained when the TNF- concentration was increased up to 1000 units/ml (data not shown). Similar to studies in Jurkat T cells (4) , these results suggest that activated NF-B in TNF--stimulated HeLa cells is not sufficient to effectively transactivate the minimal inducible VCAM-1 promoter, containing the L-R elements.


Figure 1: TNF- is unable to induce p85VCAMCAT in HeLa cells. A, HeLa cells were transfected with 10 µg of p85VCAMCAT or p(HIVB)CAT as described under ``Materials and Methods.'' After overnight transfection, the cells were stimulated with TNF- (200 units/ml) for 16-24 h, and CAT activity was determined. B, HUVE cells were transfected with 30 µg of p85VCAMCAT or p(HIVB)CAT. After transfection the cells were stimulated with TNF- (200 units/ml) for 16-24 h, and CAT activity was determined.



Co-expression of the p65 Subunit of NF-B Potently Transactivates the VCAM-1 Promoter in HeLa Cells

As an endogenous activation of NF-B in HeLa cells appeared not to be effective, we undertook to determine which members of the NF-B/Rel family could transactivate the VCAM-1 promoter through the L-R elements. In functional reconstitution experiments Rel-like subunits were co-transfected into HeLa cells with p85VCAMCAT or p(HIVB)CAT (a p65 homodimer and NF-B (p50+p65 heterodimer) driven reporter gene) as a control. These included expression vectors for the p50 and p65 subunits of NF-B as well as for the chimeric protein, p50/65, that contains the DNA binding domain of p50 and transactivation domain of p65 and is a functional mimic of NF-B (p50+p65 heterodimer) (28) . As shown in Fig. 2 B, co-expression of the p65 subunit markedly transactivated p85VCAMCAT (7) . In contrast, co-expression of the chimeric protein (p50/65) failed to significantly induce p85VCAMCAT. Similarly, p50 did not induce p85VCAMCAT. However, both p65 and the chimeric protein p50/65 potently induced p(HIVB)CAT (Fig. 2 A) (28) . p50 did not induce p(HIVB)CAT. These data suggest that expression of p65 either as a homodimer or in combination with a component distinct from p50 already present in HeLa cells is sufficient to transactivate the minimal inducible VCAM-1 promoter containing the L-R elements.


Figure 2: The p65 subunit of NF-B is a potent activator of the VCAM-1 promoter. HeLa cells were transfected with 10 µg of p85VCAMCAT ( Panel B) or p(HIVB)CAT ( Panel A) along with 5 µg of the expression vectors encoding p65 and p50, and the chimeric protein p50/65 as described under ``Materials and Methods.'' The CAT activity was determined using equal amount of protein. The CAT activity of p85VCAMCAT ( Panel B) and p(HIVB)CAT ( Panel A) under different conditions is expressed as percentage of acetylated chloramphenicol.



Reconstitution of a Functional NF-B (p50+p65 Heterodimer) in Vivo Enhances p(HIVB)CAT but Blocks p85VCAMCAT

To further elucidate the relative roles of p65 and the NF-B (p50+p65 heterodimer) on transactivation of the VCAM-1 promoter, functional NF-B (p50+p65 heterodimer) was reconstituted in HeLa cells by co-transfection of the expression vectors encoding p50 and p65. Recent studies have shown that the NF-B(p50+p65 heterodimer) can be efficiently formed when expression vectors of both p50 and p65 are co-transfected in different cell lines. This has been shown by gel mobility shift assays and by increases in the transcriptional activation of p(HIVB)CAT reporter genes (16, 17, 18, 19) . Studies in vitro have also suggested that the p50+p65 heterodimer is preferentially formed when purified proteins of p50 and p65 are incubated together (20) .

We first determined the concentration of p65 that was required for the maximal activation of p85VCAMCAT. p85VCAMCAT was transfected into HeLa cells along with different concentrations (0.1-10 µg) of the p65 expression vector. Increasing amounts of p65 activated p85VCAMCAT in a dose-dependent manner (7) (data not shown). The amount of p65 required for the maximal CAT activity was approximately 5 µg. Further increase in the amount of expression vector did not significantly increase or decrease CAT activity. Based on these studies, the concentration of p65 expression vector used for the activation of p85VCAMCAT was 5 µg or lower. To generate NF-B (p50+p65 heterodimer), HeLa cells were transfected with a constant amount of p65 and increasing amounts of p50 expression vectors. The effect of the co-expressed p50+p65 heterodimers on promoter function was assessed in p85VCAMCAT. The functional formation of the p50+p65 heterodimer and its ability to drive a canonical B motif was assessed using p(HIVB)CAT.

The promoters CAT activities obtained as a result of these mixing reconstitution experiments were determined and plotted as a function of the ratio of transfected p50 and p65 subunit expression vectors (Fig. 3 A). As expected, based on the predicted formation of p50+p65 heterodimers, p65-mediated activation of p(HIVB)CAT was significantly enhanced using a p50:p65 transfection ratio of 0.25 to 0.5. Further increase in the amount of transfected p50 inhibited activation of p(HIVB)CAT, perhaps by the formation of p50 homodimers. In dramatic contrast, under similar conditions, p65-mediated activation of p85VCAMCAT was almost completely inhibited by the presence of any p50, with inhibition occurring in response to a p50:p65 transfection ratio as low as 0.2. p(HIVB)CAT was activated at the same ratio. This suggests that the formation of NF-B(p50+p65 heterodimers) blocked the p85VCAMCAT activation mediated by p65. These results suggest that under conditions that transactivate a canonical NF-B driven promoter, reconstitution of a functional NF-B heterodimer inhibits p65-mediated transactivation of the VCAM-1 promoter.

To confirm that the L-R elements were the sequences in the VCAM-1 promoter that were responsible for the observed responses to transfected p65 and p50, the L-R driven heterologous promoter CAT construct, pTA(-77/-63)CAT, was used in co-transfection studies. pTA(-77/-63)CAT contains the L-R enhancer elements driving an SV40 promoter and is transactivated by TNF- in HUVE cells (4) . HeLa cells were transfected with 10 µg of pTA(-77/-63)CAT and cotransfected with p65 and p50. As expected, transfection of p65 alone (5 µg) resulted in 30-fold induction of pTA(-77/-63)CAT above the low background (Fig. 3 B, left panel). Co-expression of p50 and p65 at the ratio of 0.5 resulted in almost complete inhibition of p65-mediated transactivation of pTA(-77/-63)CAT (Fig. 3 B, left panel), whereas p(HIVB)CAT activity was enhanced (Fig. 3 B, right panel). This suggests that reconstituted functional NF-B (p50+p65 heterodimer) blocks the p65-mediated transactivation through the L-R elements of both the heterologous and homologous VCAM-1 promoters.


Figure 3: The concentrations of p50 which stimulate the p65-mediated activation of p(HIVB)CAT inhibit the p65-mediated activation of p85VCAMCAT. A, relative CAT activity of p(HIVB)CAT and p85VCAMCAT at different ratios of co-transfected p50 and p65. HeLa cells were transfected with 10 µg of p(HIVB)CAT or p85VCAMCAT along with a constant amount of p65 and increasing amounts of p50. To control transfection efficiencies, the total amount of transfected DNA was kept constant by using poLUC, a promoterless plasmid. The CAT assays were performed by using the same amount of protein. The percentage of the C-labeled chloramphenicol converted to its acetylated form was determined and plotted against the ratio of co-transfected p50 and p65 (p65 activation of the reporter genes = 100%). The transactivation of p(HIVB)CAT was determined by using 2 µg of p65 and 0, 0.5, 1.0, 2.0, and 5 µg of p50, respectively. The transactivation of p85VCAMCAT was determined by using 5 µg of p65 and 0, 1.0, 5.0, and 10 µg of p50, respectively. B, the concentrations of p50 which stimulate the p65-mediated activation of p(HIVB)CAT inhibit the p65-mediated activation of pTA77/63CAT. HeLa cells were transfected with 10 µg of pTA77/63CAT ( left panel) or p(HIVB)CAT ( right panel) along with p65 and/or p50. To control transfection efficiencies, the total amount of transfected DNA was kept constant by using poLUC, a promoterless plasmid. The CAT assays were performed by using the same amount of protein. Columns 1 and 4 show the basal levels of the respective CAT reporter gene. Columns 2 and 5 show the CAT activity mediated by p65 (5 µg). Columns 3 and 6 show the CAT activity mediated by the combination of p65 (5 µg) and p50 (1.0 µg).



To determine which part of the p50 protein was responsible for the negative effect on the transactivation potential of p65 within the NF-B(p50/p65 heterodimer) bound to L-R, functional reconstitution experiments were performed using expression vectors of p65 and chimeric protein p50/65. HeLa cells were transfected with p85VCAMCAT along with expression vectors encoding p65 and/or chimeric protein p50/65. As expected, p65 (5 µg) potently induced the CAT activity of p85VCAMCAT (data not shown). Co-expression of 1 µg of p50/65 did not affect the transactivation potential of p65 (5 µg). However, the co-expression of 5 µg of p50/65 enhanced nearly 2-fold the p65(5 µg)-stimulated CAT activity of p85VCAMCAT(data not shown). These results suggest that by replacing the carboxyl terminus of p50 with the activation domain of p65, the inhibitory effect of p50 on the transactivation potential of p65 bound to L-R is lost.

p50 and p65 Homodimers and the NF-B Heterodimer Directly Bind to the L-R Motif in Vitro

To establish whether p50 and p65 homo- and heterodimers could directly bind to the VCAM-1 L-R motif, homo- and heterodimers were prepared by incubating purified recombinant p50 and p65 proteins, either alone or mixed together, for one h at 37 °C (20) . Gel mobility shift assays were performed using P-labeled double stranded DNA containing L-R elements (VCAM wild type probe) (see ``Matrials and Methods''). p65 is a truncated protein that contains amino acids 1-309, and is capable of both dimerization (17) and of binding to DNA (15, 36, 37) . As shown in Fig. 4( lanes 1-5), p65 homodimers were able to bind to the VCAM wild type probe in a dose dependent manner. However, the amount of p65 that bound was substantially less than the amount of p50+p65 heterodimer or p50 homodimer that bound. The p50+p65 heterodimer that was generated by incubating a constant amount of p65 (400 pg) with increasing amounts of p50 (40-800 pg) also showed binding to the VCAM wild type probe. As the concentration of p50 exceeded that of p65, a second band appeared in the gel shift assay that corresponded to p50 homodimers ( lane 10). As p50 homodimers appeared, there was no decrease in the intensity of p50+p65 heterodimer binding. The complex of p50 homodimer with VCAM wild type probe is shown in lane 11. The mobility of the complex of the p50+p65 heterodimer was intermediate between the complexes of p50 and p65 homodimers, the p65 homodimer complex migrated fastest. These mobilities are consistent with the mobilities of the complexes of homo- and heterodimers of p50 and p65 (1-309) with oligonucleotide containing canonical B motif (17, 28) . Interestingly, two bands, which corresponded to the p50 homodimer and the p50+p65 heterodimer, co-existed in lane 10. At high concentrations of p50 and p65, small amounts of complexes with mobility lower than that of the p50 homodimer were also detectable ( lanes 6-10). These studies demonstrate that the homo- and heterodimers of p50 and p65 all bind to L-R, although the levels of p65 homodimer binding are lower than the levels of p50+p65 heterodimer or p50 homodimer binding.


Figure 4: The homo- and heterodimer of purified recombinant p50 and p65 proteins bind to VCAM-1 wild type probe containing L-R. The homo- and heterodimers of p50 and p65 were prepared by incubating the purified p50 or p65 proteins alone or in combination with each other for one hour at 37 °C and their binding to L-R was monitored by using P-labeled VCAM-1 wild type probe in gel shift assays. Lanes 1, 2, 3, 4, and 5 show the binding of 40, 100, 200, 400, and 800 pg of p65 to the VCAM wild type probe, respectively. Lanes 6, 7, 8, 9, and 10 had a constant amount (400 pg) of p65 and increasing amounts of p50 (40, 100, 200, 400, and 800 pg), respectively. Lane 11 had 400 pg of p50. The complexes of homo- and heterodimers of p50 and p65 with VCAM wild type probe are marked on the right side of the figure. A shorter exposure of lanes 9, 10, and 11 is shown in a box on the right side.



TNF- Induces p65-like Homodimers and NF-B (p50+p65 Heterodimers) in HUVE and HeLa Cells

The expression of NF-B/Rel proteins was determined in both HeLa and HUVE cells by gel mobility shift assay. As shown in Fig. 5 A ( lanes 1 and 3), the nuclear extract of uninduced HUVE cells has no basal level of L-R-binding nuclear proteins compared with that of the uninduced HeLa cells. TNF- induces L-R-binding nuclear proteins (DNA-protein complex A1) in HUVE and in Hela cells ( lanes 2 and 4). However, the concentration of A1 in TNF--activated HUVE cells was less than half that of HeLa cells ( lanes 2 and 4). The other two complexes, A2 and A3, in TNF--activated HUVE ( lane 2) and HeLa cells ( lane 4) were minor compared with the complex A1. These results suggest that the major protein component of the complex A1 may not be the transactivator protein, since it is induced in large amounts in TNF--activated HeLa cells where the VCAM-1 promoter and gene is not induced.


Figure 5: TNF- induces homo-and heterodimers of p50 and p65 in HUVE and HeLa cells and relatively higher concentrations of NF-B/rel proteins in HeLa cells. A, L-R binding nuclear protiens of TNF--activated HeLa and HUVE cells were compared using equal amounts of nuclear extract (2 µg of protein) ( lanes 1-4). L-R binding nuclear proteins of TNF--activated HUVE cells ( lane 5). Super shift with antibodies against p50, p65, c-Rel(c), and RelB is shown in lanes 6-9, respectively. Specific complexes A1, A2, and A3, and nonspecific ( NS) and free probes are marked. L-R-binding nuclear proteins of TNF- activated HeLa cells ( lane 10). Super shift with antibodies p50, p65, c-rel(c) (raised against a peptide in the carboxyl terminus of c-Rel), and RelB is shown in lanes 11 to 14, respectively. Specific complexes A1 and A2, NS, and free probe are marked. B, Ig/B binding nuclear proteins of TNF--treated HUVE and HeLa cells. Labeled Ig/B probe without nuclear proteins ( lane 1). Lanes 2-6 and 7-11 contain HUVE and HeLa cell nuclear extract (2 µg each lane), respectively. The volume of each antibody used was 1 µl. TNF--activated Ig/B binding nuclear proteins of HUVE ( lane 2) and HeLa ( lane 7) cells and their super shift with antibodies against p50, p65, and p49 are shown.



To further characterize the proteins in complexes A1-A3, supershift assays were performed using antibodies of NF-B/Rel proteins. The antibodies against p50 (Abp50) and p65 (Abp65) recognized their epitopes in NF-B (p50+p65 heterodimer) as well as in the homodimers of their respective subunits (data not shown). The binding of Abp65 to the NF-B (p50+p65 heterodimer) resulted in a decrease in the binding of the NF-B to L-R. As shown in Fig. 5A ( lane 5), TNF--activated HUVE cells contain the major complex, A1, and the minor complexes, A2 and A3. The complex A1 is shifted by both antibodies against p50 and p65 suggesting that the major part of the complex A1 is NF-B (p50+p65 heterodimer) ( lanes 6 and 7). Because Abp50 could not completely shift complex A1, this suggested that complex A1 also contains p65 homodimer or a heterodimer of p65 with a protein other than p50. The TNF--activated HUVE cells did not contain any L-R-binding nuclear c-Rel or RelB proteins ( lanes 8 and 9).

To determine whether p65 homodimer was specifically present in TNF--activated HUVE cells, the L-R binding nuclear proteins of TNF--activated HeLa cells were also characterized using antibodies of NF-B/Rel proteins. As shown in Fig. 5 A, lane 10, TNF- induces a major complex, A1, and a minor complex, A2. All of the complex A1 was shifted by antibody Abp65, and the major part of it by Abp50, suggesting that the complex A1 was mixture of NF-B (p50 + p65 heterodimer) and p65 homodimer or p65 heterodimer with a protein other than p50 ( lanes 11 and 12). The nuclear extract of HeLa cells did not contain any detectable amount of c-Rel or RelB ( lanes 13 and 14). Although TNF- induces homo- and heterodimers of p50 and p65 in both HUVE and HeLa cells, it induces lower concentrations of NF-B (p50+p65 heterodimer) in HUVE compared with HeLa cells.

Similar results were obtained when Ig/B was used as a probe, instead of L-R. As shown in Fig. 5B, Ig/B made a major complex A1 that had intensity approximately 2-fold higher in HeLa ( lane 7) compared with HUVE ( lane 2) cells. The complex A1 supershifted completely when the antibodies Abp50 and Abp65 were used together ( lanes 5 and 10), and it disappeared instead of supershifting when Abp65 was used alone, likely due to loss in the DNA binding ability of the NF-B proteins ( lanes 4 and 9). The antibody Abp50 also shifted the major part of complex A1 ( lanes 3 and 8). However, Abp50 was unable to shift a small part of the complex A1. The antibody Abp49 did not shift any part of the complex A1 ( lanes 6 and 11). These results also suggest that the complex A1 consists of p50+p65 heterodimers and p65 homodimers or p65 heterodimers with a protein other than p50 and, also, that the major part of the complex A1 is the p50+p65 heterodimer.

TNF- Can Activate p85VCAMCAT in HeLa Cells when Co-transfected with Antisense p50

To assess the role of endogenous p50 in the TNF--mediated regulation of p85VCAMCAT, HeLa cells were co-transfected with p85VCAMCAT and phosphorothioate antisense oligonucleotides of p50 or p65 or an unrelated antisense (intercellular adhesion molecule-1) and treated with TNF-. After 16-24 h the cells were assayed for CAT activity. As expected, p85VCAMCAT had a low level of activity and TNF- was unable to effectively induce the CAT activity (Fig. 6). However, in HeLa cells co-transfected with antisense p50, TNF- was able to induce p85VCAMCAT severalfold above the low level of background. The co-transfection of the p65 or the unrelated antisense did not improve the ability of TNF- to induce p85VCAMCAT. These results suggest that in TNF--treated HeLa cells, endogenous p50 inhibited the ability of TNF- to induce p85VCAMCAT.


Figure 6: Co-transfected antisense p50 renders p85VCAMCAT inducible by TNF- in HeLa cells. HeLa cells (30-40% confluent) were transfected with 10 µg of p85VCAMCAT and 100 n M of phosphorothioate antisense p50 or unrelated antisense (intercellular adhesion molecule-1) or p65 using calcium phosphate precipitation method. The cells were also transfected only with p85VCAMCAT. After overnight transfection, the cells were maintained in fresh medium for another 24 h. The cells were then treated with TNF- and harvested for CAT assays 16-24 h later. Equal amounts of protein (50 µg) were used in each CAT assay. The CAT activity is presented as percentage of chloramphenicol converted to acetyl chloramphenicol. These results are average of three independent experiments each performed in duplicate.




DISCUSSION

We investigated the transcriptional regulatory mechanisms underlying the cell type-specific activation of the VCAM-1 promoter by the cytokine TNF-. Prior studies established that TNF- induces the VCAM-1 promoter in HUVE cells but not Jurkat T cells through two closely linked B-like elements, L -R, located at positions -77 and -63 relative to the transcriptional start site, respectively (4) . We confirmed these findings in HUVE cells and also established that the VCAM-1 promoter is not transactivated by TNF- in another non-endothelial cell type, HeLa. Our data strongly suggest that, while TNF- markedly activates NF-B in both cell types (4, 5, 6, 10, 38, 39) , NF-B itself does not effectively transactivate the VCAM-1 promoter through these L-R elements. Importantly, the p50 subunit itself may function as a negative transcriptional regulator of this promoter. Thus, cytokine inducible transactivation of the VCAM-1 promoter, and by extrapolation the VCAM-1 gene, may be a function of negative (p50 associated) and positive (p65 associated) transcriptional factors that are activated in a cell type-specific manner.

The inability of NF-B to effectively transactivate the VCAM-1 promoter through the L-R elements was established by three functional experimental observations: 1) activation of endogenous NF-B in HeLa cells by TNF- markedly induced a transiently transfected, NF-B driven reporter gene, p(HIVB)CAT, but failed to activate p85VCAMCAT, a minimal, L -R containing the VCAM-1 promoter construct, that was induced by TNF- in HUVE cells; 2) co-expression of a p50/65 chimeric protein, that has the p50 DNA binding and p65 transactivation properties of NF-B, failed to effectively transactivate p85VCAMCAT in HeLa cells; and 3) transactivation of p85VCAMCAT in HeLa cells was dramatically inhibited under conditions of co-expression of p50 and p65, and hence NF-B (p50+p65 heterodimer) formation, whereas this coexpression markedly transactivated an NF-B driven p(HIVB)CAT reporter gene. In this functional context, we also established that the VCAM-1 L-R elements could bind the homodimers of p50 and p65 as well as NF-B prepared by heterodimerization of purified, recombinant p50 and p65 proteins. Taken together, these findings suggest that the binding of NF-B in vivo to L-R is not sufficient for the activation of p85VCAMCAT. Although the mechanism mediating this effect is not fully understood, a specific conformation of NF-B after binding to the motif may be necessary. Such a hypothesis was proposed for the in vitro transcriptional activation by p50 of the CAT reporter gene containing the B motifs of H-2K gene (20) . In this study, binding of p50 to a B motif in a chymotrypsin-resistant conformation correlated with its ability to transactivate. Thus, the different conformations of NF-B bound to different B motifs determine NF-B's functional role as a transcription activator.

In our in vivo reconstitution experiments, p50 negatively controlled the ability of p65 to transactivate the VCAM-1 promoter through the L-R elements. This is suggested by the activation of p85VCAMCAT and L-R-driven heterologous promoter, pTA(-77/-63)CAT, by p65 but not NF-B. In contrast, p50 does not inhibit the transactivation potential of p65 within the canonical IgB element until excess p50 is expressed (16, 17, 18, 19) . Indeed, it has been suggested that within NF-B, the function of p50 is to improve the DNA binding of p65, which is otherwise a weak binder (40) . Consequently, p65 can act as a more effective transactivator within NF-B. Furthermore, the transactivating activity of NF-B through various B motifs led to a model in which the transactivation domain of p65 is always exposed and available for transactivation, either as a homodimer or as the p50+p65 heterodimer and regardless of the B motif to which it is bound (20) . Our results suggest that within L-R-bound NF-B, p50 does not function as a helper subunit but may actively inhibit the transactivation potential of p65. The enhancement by co-expressed chimeric p50/65 of p65-mediated transactivation of 85VCAMCAT (data not shown) suggests that a carboxyl terminus domain of p50 was responsible for inhibiting the transactivation potential of p65 within NF-B bound to L-R. This would suggest that the transactivation domain of p65 in NF-B can either be sequestered or exposed, depending on the B motif to which NF-B is bound.

By co-expression studies, we have established that the p65 subunit alone is a potent transactivator of the VCAM-1 promoter. That p65 mediates this effect through a p65 homodimer is suggested by two experimental results: 1) when the DNA binding domain of p65 was replaced with the DNA binding domain of p50, the p65-mediated activation of p85VCAMCAT was either reduced to minimal level or lost and 2) in vivo reconstitution of the p50+p65 heterodimer inhibited p65-mediated transactivation of the minimal VCAM-1 and L-R driven heterologous promoters. These results suggest that for transactivation through L-R, both the p65 transactivation domain and the p65 DNA binding domain were necessary. Although our studies suggest that differential transactivation of L-R is mediated through direct interactions with the p65 homodimer, they do not rule out the possibility that p65 mediates transactivation through an indirect mechanism. This might include the complexing of p65 with an endogenous rel-like or other transactivating factor expressed in HeLa cells distinct from p50 or c-Rel (41) . Finally, co-expression of transcription factors could modulate the de novo expression of other transcriptional factors during the course of the transient DNA transfection. Only through defined, reconstituted in vitro transcriptional assays will these issues be definitively resolved.

To our knowledge, there is no other example to date of a naturally occurring B motif that binds to both the homo- and heterodimers of p50 and p65 and is specifically activated by p65 homodimer and not by NF-B. Synthetic and natural B-like motifs conferring differential transactivation have been identified (28, 42) . In vitro synthesized B motifs were isolated, using p65 as a binding protein, from a randomly prepared pool of B-like DNA sequences using polymerase chain reaction (28) . Several of these sequences, such as 65-2, specifically bind p65 homodimer but not NF-B (p50+p65 heterodimer) in vitro. Consistent with this pattern of DNA binding, this enhancer element confers p65, but not NF-B, transactivation to a heterologous promoter. However, this differs significantly from transactivation mediated through L-R. Although a detailed kinetic and affinity study was not performed, our in vitro binding assays demonstrated that the L-R motif bound both p65 homodimers as well as the NF-B (p50+p65 heterodimer). This suggests that the selective activation mechanism found in L-R is different than that found in the selective p65-specific B motifs that have been described (28) .

It is likely that interactions between the transcriptional factors bound to the L and R elements define the selective transactivation response to p65 and NF-B. The nature of these interactions is suggested by a comparison of these B-like motifs with other natural and synthetic B elements. Some of the synthetic p65 selected sequences (28) are either identical or similar to L or R. Structurally, R is identical to 65-4 (28) and the B motif of the promoter of the plasminogen activator gene (43) . This suggests that R should function as a canonical B enhancer element. In contrast, L is not identical to any known canonical B sequence and thus may represent a new class of B binding motifs. Indeed, the R and L elements each exhibited distinct protein binding activities, suggesting that the L-R bound protein complex consists of multiple, and likely interacting, transactivation factors (4) . Definition of the mechanism by which L-R confers specificity on L-R-driven promoters requires further understanding of binding and functional properties of the individual B elements and is under investigation. Nevertheless, the activation of L-R by p65 but not NF-B suggests that the induction of the VCAM-1 promoter by TNF- in endothelial cells is not mediated through NF-B.

Characterization of the L-R binding nuclear proteins of TNF--activated HUVE and Hela cells also suggests that NF-B is not a transactivator of the VCAM-1 promoter. Instead, high concentrations of NF-B can be correlated with suppression of the VCAM-1 promoter. In our studies, although TNF- induced NF-B in HeLa cells approximately 2-fold more than in HUVE cells, it was unable to induce the VCAM-1 gene in HeLa cells. Along with NF-B, TNF- also induced p65 both in HeLa and HUVE cells. From these studies we conclude that both p65 and NF-B play an important role in the regulation of the VCAM-1 gene. Although p65 can specifically transactivate the VCAM-1 promoter, changes in the concentration of NF-B can modify the transactivation mediated by p65.

The results of the supershift assay suggested that in HUVE and HeLa cells TNF- activated both homo- and heterodimers of p50 and p65, although the concentration of L-R binding NF-B/Rel proteins was higher in HeLa cells. The major part of the NF-B/Rel proteins was the p50+p65 heterodimer. These results also suggest that the lower expression of p50 compared with p65 gene in HUVE cells is one of the mechanism by which TNF- can induce VCAM-1 in this cell type. Whether this is a general characteristic of other endothelial cells needs to be determined.

Our results employing co-transfection of p50 and p65 expression vectors suggested that the NF-B heterodimer (p50+p65) does not transactivate the minimal VCAM-1 promoter p85VCAMCAT. By extension, these results suggest that p50 may also function as a negative regulator of VCAM-1 promoter activity in HeLa cells. A similar role of p49, the processed product of p100, similar to p50, has been suggested in the regulation of IL-6 promoter (44) . To assess this proposed functional role of the endogenous p50 subunit, we used an antisense oligonucleotide directed against p50 (ASp50). In HeLa cells, we demonstrated that despite marked activation of NF-B-mediated transcriptional activity, the minimal VCAM-1 promoter p85VCAMCAT was not significantly induced by the cytokine TNF-. Strikingly, co-transfection of ASp50, but not an unrelated antisense or p65 oligonucleotides, conferred cytokine inducibility to the VCAM-1 promoter. This suggests that the cell type-specific pattern of cytokine activation of the VCAM-1 promoter, and by extrapolation the VCAM-1 gene, may be regulated through a p50 subunit dependent mechanism. While it is tempting to speculate that the ASp50 functions by altering the relative proportion of p50 in the L-R complex, we have not directly assessed this in these studies and thus cannot rule out an indirect effect of p50 on the expression of other transcriptional factors.

In conclusion, we have utilized both overexpression and targeted inhibition strategies to develop a model of cell type-specific VCAM-1 promoter transactivation as a function of the relative level of p50 versus either a p65 homodimer or p65 heterodimer with a protein other than p50. This model is consistent with the recently proposed model that also suggests that the expression of distinct NF-B/Rel transcription factors are responsible for cell type-specific and inducible gene activation (45) . Recent demonstration of the physical interaction between p65 and non-Rel factors suggest that non-Rel factors may also be involved in the tissue specific regulation of VCAM-1 gene. A detailed analysis of the NF-B/Rel proteins and their interacting non-Rel factors in endothelial cells will be required to identify the exact nature of the endogenous transactivator of VCAM-1 gene expression and its relationship to the p50 mediated inhibitory factors.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Research Grant PO1-HL48667 (to R. M. M. and R. W. A.) and an American Heart Association Grant-in-Aid (Georgia Affiliate) (to R. M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The second author contributed equally to this paper.

Established Investigator of the American Heart Association. To whom correspondence should be addressed: Division of Cardiology, Dept. of Medicine, Emory University, 1639 Pierce Dr., P.O. Drawer LL, Atlanta, GA 30322. Tel.: 404-727-3106; Fax: 404-727-3330; e-mail: rmedfor@eagle.cc.emory.edu.

The abbreviations used are: VCAM, vascular cell adhesion molecule; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; HUVE, human umbilical vein endothelial; TNF, tumor necrosis factor.


ACKNOWLEDGEMENTS

We are indebted to Dr. Margaret Offermann (Winship Cancer Center, Emory University) for critical reading of the manuscript and extensive discussions and to Dr. Charles Kunsch (Human Genome Sciences, Rockville, MD) for his many helpful insights and comments. We are thankful to Lynn Olliff for technical support in preparing plasmids and to Kate W. Harris for editing the manuscript.


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