Discrimination between RelA and RelB Transcriptional Regulation by a Dominant Negative Mutant of Ikappa Balpha *

Valérie Ferreira, Nadine Tarantino, and Marie KörnerDagger

From the Laboratoire d'Immunologie Cellulaire, CNRS URA 625, Bat. CERVI, Hôpital de la Pitié Salpêtrière, 83, Bd. de l'Hôpital, 75013 Paris, France

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

RelA and RelB belong to the nuclear factor-kappa B (NF-kappa B-Rel) transcription factor family. Both proteins are structurally and functionally related, but their intracellular and tissue distributions are different. In resting cells, RelB is found mostly in the nucleus, whereas RelA is sequestered in the cytosol by protein inhibitors, among which Ikappa Balpha is the dominant form in lymphocytes. Upon cellular activation Ikappa Balpha is proteolyzed, allowing RelA dimers to enter the nucleus and activate target genes. To study the selectivity of gene regulation by RelA and RelB, we generated T cell lines stably expressing a dominant negative mutant of Ikappa Balpha . We show that selective inhibition of RelA-NF-kappa B decreased induction of NFKB1, interleukin-2, and interleukin-2Ralpha genes but not c-myc. Transcription driven by the Ikappa Balpha promoter was blocked by the transgenic Ikappa Balpha ; however, wild type Ikappa Balpha was expressed in the transgenic cell clones but with much slower kinetics than that in control cells. Wild type Ikappa Balpha expression was concomitant with RelB up-regulation, suggesting that RelB could be involved in transcription of Ikappa Balpha through binding to an alternative site. These results indicate that RelB and RelA have both distinct and overlapping effects on gene expression.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Nuclear factor kappa B (NF-kappa B)1 is part of the Rel family of eukaryotic transcription factors which share structural and functional properties. Although ubiquitously expressed in higher eukaryots NF-kappa B has been intensively studied mostly in cells belonging to the immune system where it was first discovered (for review, see Refs. 1 and 2). NF-kappa B-Rel factors were shown to participate in the expression of genes essential for the immune responses and to regulate gene transcription during inflammatory reactions. The prototypical NF-kappa B is a homodimer or heterodimer composed of 50-kDa (p50) and/or 65-kDa (p65 or RelA) polypeptides. In vertebrates other members of the family are c-Rel, RelB, and p52. The tissue and cellular distribution of the three last members is more restrained than that of the prototypical NF-kappa B. For example, the expression of RelB was described as being predominant in dendritic cells from primary and secondary lymphoid organs (3-6). RelB has also been detected in other cells and tissue but in lower amounts or after specific activation. c-Rel and p52 are also expressed mainly in cells from the hematopoietic lineages. P50 and p52 are generated by proteolytic processing of precursor polypeptides (p105 (NFKB1 gene) and p100 (NFKB2 gene), respectively) (1, 7). Each member of the NF-kappa B-Rel family contains a 300-amino acid sequence called the Rel homology domain, which is critical for nuclear translocation, protein-protein interactions, and sequence-specific DNA binding. All members of the NF-kappa B-Rel family form dimers. The dimers can be classified into two pools on the basis of their intracellular localization, which is critical in regulating their activity. One pool of NF-kappa B-Rel dimers is cytosolic in the absence of cellular activators, whereas the second pool is constitutively nuclear. The intracellular location of the dimers depends on the capacity of the NF-kappa B-Rel family members to interact with ankyrin repeat-containing proteins, collectively called Ikappa B. The cytoplasmic Ikappa Bs inhibit NF-kappa B-Rel complexes by preventing both NF-kappa B-Rel nuclear translocation, and their interaction with specific decameric DNA sequences called kappa B (8, 9). Thus Ikappa Bs represent intracellular regulators of NF-kappa B activity. Several members of Ikappa B regulatory family have been characterized, including Ikappa Balpha , Ikappa Bbeta , Ikappa Bgamma , the two NF-kappa B protein precursors p105 and p100, and bcl-3. Except for bcl-3, Ikappa B molecules are mostly cytosolic, although nuclear Ikappa Balpha has been reported in cultured cells (10-12) and in vivo.2 p50 and p52 homodimers as well as RelB-p50 and RelB-p52 heterodimers do not interact efficiently with cytosolic Ikappa Bs. Consequently they are found in nuclei of cells that produce these complexes (13). Therefore their regulation should be distinct from the cytosolic forms of NF-kappa B. The p50 and p52 homodimers were reported to interact with the nuclear bcl-3. The resulting trimers seem to constitute transcriptional activators, whereas p50 and p52 homodimers are unable to enhance RNA polymerase II-driven transcription (14). In contrast to other members of the NF-kappa B family, RelB contains in its NH2-terminal domain a leucine zipper-like structure that is essential for transactivation of target genes (15). However, the regulation of RelB activity is still poorly understood.

Studies of T lymphocytes, isolated from Ikappa Balpha -deficient mice, demonstrated that the dominant Ikappa B regulator of NF-kappa B-Rel is Ikappa Balpha , the product of the MAD3 gene (16, 17). Activation of cells with adequate signals such as T cell receptor triggering, phorbol esters, interleukin 1 (IL-1), tumor necrosis factor (TNF-alpha ), and others results in Ikappa Balpha degradation by 26 S proteasomes (for review, see Ref. 7). This renders dimers, which contain RelA and c-Rel proteins, free to translocate into nuclei where they activate transcription of target genes. The molecular mechanism resulting in Ikappa Balpha proteolysis is complex and not completely elucidated. However, at least two post-translational covalent modifications have been reported to be essential for its degradation. The first critical event is phosphorylation of serines 32 and 36 in the NH2-terminal region of Ikappa Balpha , carried out by Ser/Thr kinase(s) including a multienzyme complex of 700 kDa (18). This double phosphorylation of Ikappa Balpha does not lead to dissociation from NF-kappa B, but it is prerequisite for the second modification step, which is the ubiquitination of two NH2-terminal lysines at positions 21 and 22 (19). Subsequently the phosphorylated and ubiquitinated Ikappa Balpha is proteolyzed by the 26 S-proteasome complex (20, 21).

Once released from Ikappa Balpha , NF-kappa B-Rel proteins translocate rapidly to the nucleus where they exert their regulatory functions by interacting with specific decameric kappa B sequences and the general transcription factor TFIIB (22). A plethora of genes have been shown to contain kappa B sequences in their promoters (for review, see Ref. 23). In T cells, gene products involved in cell adhesion (intercellular cell adhesion molecule-1; ICAM-1), cell growth control (IL-2, its receptor IL-2Ralpha , and c-myc), and proinflammatory mediators (IL-6, TNF-alpha ) are suspected of being transcriptionally regulated by NF-kappa B. Furthermore, viruses with T cell tropism, such as HIV, are also thought to be transcriptionally regulated by NF-kappa B proteins (24). Specific relationships between distinct NF-kappa B complexes and particular target genes are not yet understood, although preferential binding and preferential transcriptional activation efficiencies have been demonstrated by transfection experiments with discrete NF-kappa B expression vectors and distinct kappa B sequences (25-29). These observations suggest that distinct NF-kappa B-Rel complexes modulate transcription of different genes selectively.

Transient transfection assays with a mutated form of Ikappa Balpha , in which serines 32 and 36 were replaced by alanines, demonstrated that the double mutation prevented proteolytic degradation of the transgenic Ikappa Balpha by the usual NF-kappa B activators (TNF-alpha and phorbol esters) (30-32). Thus the double mutation generates a constitutive repressor of the cytosolic NF-kappa B-Rel proteins. Because the 32/36A Ikappa Balpha mutant (Ikappa Balpha 32/36A) should not affect the constitutively nuclear pool of RelB proteins, Ikappa Balpha 32/36A potentially represents a powerful and selective tool for the study of the respective roles of NF-kappa B and RelB protein complexes in gene expression. We have therefore used cell clones that express both RelA and RelB subunits for stable transfections with the Ikappa Balpha 32/36A. We report in the present publication the effects of the expression of the transgenic Ikappa Balpha on NF-kappa B-Rel activation and gene expression.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells-- The parental HPB-ALL cell line was cultured in RPMI 1640 medium containing glutamine, antibiotics, and 10% fetal calf serum.

Stable Transfections-- Expression vector pCMV- Ikappa Balpha 32/36A was made by inserting MAD3 cDNA mutant at amino acid positions 32 and 36 into the XbaI/HindIII sites of the pcDNA3 vector from Invitrogen (S32/36A mutant in 32). HPB-ALL cells were transfected with pCMV-Ikappa Balpha 32/36A and the empty pcDNA3 vector by electroporation. G418-resistant cells were cloned by limiting dilution and genotyped by Southern blotting (33), using a full-length MAD3 cDNA probe. The clones with stable integration of Ikappa Balpha 32/36A were grown in RPMI with 10% fetal calf serum in presence of 1 mg/ml G418.

Cell Extracts-- In gel shift experiments (electrophoretic mobility shift assay; EMSA) cells were incubated for the indicated periods of time with activators, and nuclear proteins were extracted as described previously (34). In Western blotting, the cytosolic extracts were obtained in the hypotonic buffer described in Ref. 34.

Western Blotting-- Equal amounts of protein (30 µg) extracted from cytoplasma of control or Ikappa Balpha 32/36A-transfected clones were fractionated on 10% polyacrylamide gels by electrophoresis in denaturing conditions, according to Porzio and Pearson (35). Proteins were electrotransferred onto polyvinylidene difluoride membranes (Millipore). The efficacy of the transfer was tested by Ponceau Red staining. The wild type and transgenic Ikappa Balpha were determined using a monoclonal antibody (MAD 10B) specific for an NH2-terminal domain of Ikappa Balpha (36). The antigen-antibody complex was revealed using horseradish peroxidase-coupled anti-mouse antibody and the Amersham enhanced chemiluminescence visualization system (ECL) kit. The autoradiography was carried out for 5 s to 10 min. For RelB Western blotting, the identical procedure was followed except that 70 µg of nuclear extracts was used. The RelB-specific antiserum was from Santa Cruz (Tebu, France), and its dilution was 1/500.

EMSA-- The EMSA was performed using 10 µg of nuclear protein extracts/incubation. The kappa B oligonucleotide used was a kind gift from Dr. Leo Lee (NCI, Frederick, MD) and corresponds to the tandem kappa B sequence (PRE) from the HIV LTR. The gel shift experiments were carried out following the procedure described in Ref. 34. To identify the PRE-binding proteins, nuclear extracts from control HPB-ALL cells and one of the stably transfected clones, the A3 clone, were incubated with 2 µl of antibodies specific for individual NF-kappa B-Rel proteins before the addition of the radiolabeled PRE oligonucleotide. All antibodies were purchased from Santa Cruz. In addition to the Santa Cruz antibodies, we also used an antiserum specific for the COOH-terminal domain of the RelA molecule (named 1226 in Ref. 37), kindly provided by Dr. Nancy Rice (NCI).

Chloramphenicol Acetyltransferase (CAT) and Luciferase (Luc) Assays-- The following vectors were used for kappa B-dependent CAT and Luc assays. The 1168hIL6Luc+ construct, which contains 534 base pairs of the human IL-6 promoter, was kindly provided by Prof. G. Haegeman (Gent University, Belgium). Dr. A. Israël (Pasteur Institute, France) provided us with the 1.2HN-Luc construct (38) containing the NFKB1 (p105) promoter region and the 0.4SK, 0.2SK, and 0.4SK68Delta kappa B Luc plasmid containing the MAD3 (Ikappa Balpha ) promoter constructs (39). The 0.4SK contains all three kappa B sites from the MAD3 promoter domain, whereas the 0.2SK contains only the proximal kappa B1 site, and the 0.4SKDelta kappa B contains only the kappa B2 and kappa B3 sites. To monitor the tranfection of the three constructs of MAD3 promoter, the p.beta gal-promoter vector (CLONTECH), which contains a functional LacZ gene downstream of the SV40 early promoter, was cotransfected, and the beta -galactosidase activity was measured by spectrophotometry in the presence of 100 nM o-nitrophenol beta -D-galactoside. The ICAM-1 promoter-Luc construct (pGL1.3) was described by Ledebur and Parks (40) and was provided by Dr. K. Roebuck (Rush, Chicago). The c-myc promoter (-2325 to +36) and c-fos promoter (-711 to +42) CAT constructs are described in Ref. 41. The LTR3 CAT-218 construct containing the 218 base pairs upstream from the transcription initiation start of the HIV 5'-LTR (42) was provided by Dr. R. B. Gaynor (UCLA, Los Angeles). Finally, Dr. G. R. Crabtree (HHMI, Stanford, CA) provided us with the IL-2 promoter-Luc construct (pCLN15Delta CX) (-326 to +45). Transient transfections of the cell clones with CAT and Luc vectors were performed by electroporation at 200 V, 500 microfarads (Bio-Rad electroporation system) with 20 µg of plasmid DNA/5·106 cells. 2 h after transfection, cells were split into two pools. One pool of cells was incubated in RPMI (untreated cells), and the other pool was incubated with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) plus 1 µg/ml phytohemagglutinin (PHA) for 24 h (activated cells). The cells were then collected, washed once with phosphate-buffered saline, and lysed by three cycles of freezing/thawing in 150 mM Tris-HCl, pH 8. Cell extracts, normalized for total protein content (43), were assayed for CAT activity using [14C]chloramphenicol (NEN Life Science Products) according to Gorman et al. (44). The chloramphenicol conversion was quantified using a BetaImager 1200 apparatus (Biospace, France). The results were expressed as percent of chloramphenicol conversion/mg of protein (relative CAT units). Transfection experiments were repeated at least three times, using two independent plasmid preparations. Luc assays were performed using the Promega luciferase assay system. The cells were lysed with 25 mM Tris phosphate, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, 1% Triton X-100, pH 7.8. The light emission was measured in a luminometer (Bio-Rad). The results were calculated as relative light units (light emission/background/mg of protein).

Northern Blotting-- cDNA probes used for Northern blotting were obtained by enzymatic digestion of the following vectors: pKH47-c-myc (PstI/EcoRI digestion generating a 1,200-base pair fragment of the c-myc cDNA), p1IL-2 (PstI/BglII digestion generating a full-length cDNA), pBRchIL6F2 (PstI digestion generating an 855-base pair fragment of the human IL-6 cDNA), p105 (HindIII/ApoI digestion generating full-length cDNA), pCMV-MAD3 (HindIII/XbaI digestion generating full-length Ikappa Balpha cDNA), pBr322-actin (PstI digestion generating full-length actin cDNA). The cDNA probes were radiolabeled using [alpha -32P]dCTP (Amersham) and the Rediprime kit (Amersham). Total cytoplasmic RNA was prepared according to a modified method of Chomczynski and Sacchi (45), using the Stratagene RNA kit. Total RNA (10-20 µg) was fractionated by electrophoresis on 0.7% agarose gels containing 2.2 M formaldehyde. Gels were blotted on Hybond N+ membranes (Amersham) according to the indications of the manufacturer. Membranes were hybridized with 32P-labeled probes in Quickhyb solution (Stratagene) according to the protocol supplied by the manufacturer, at 65 °C. Membranes were autoradiographed for 1-12 h at -70 °C with intensifying screens. Membranes were stripped by boiling in H2O and rehybridized with the beta -actin probe to normalize loading of RNA samples.

Measurement of IL-2 Production by Enzyme-linked Immunosorbent Assay (ELISA)-- Control, A3, and D7 clone cells (105 cells/condition) were activated by PMA (10 ng/ml) plus PHA (1 µg/ml) for 12 h at 37 °C. Cell supernatants were tested for IL-2 by ELISA using the Immunotech human IL-2 ELISA kit (Immunotech, France). All assays were performed in quadruplicate.

Measurement of CD25 Expression by Flow Cytometry-- Control, A3, F10, and D7 cells were activated for 24 h by PMA (10 ng/ml) and PHA (1 µg/ml). Unstimulated cells and PMA plus PHA-treated cells were tested for CD25 by flow cytometry using a phycoerythrin-conjugated human CD25-specific monoclonal antibody from Caltag Laboratories and fluorescence-activated cell sorter apparatus from Becton-Dickinson.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Characterization of Stable HPB-ALL Clones Transfected with the 32/36A Mutant Ikappa Balpha -- The parental HPB-ALL cell line is a T cell tumor producing IL-2 and IL-6 in response to T cell activators, such as phorbol esters, in the presence of a Ca2+ influx activators (PHA, ionomycin, CD3-specific antibodies, etc.). Its phenotype is close to a double positive thymocyte (CD4+/CD8+, CD1a+, CD3+). We chose this line as a model for studying the inhibition of the inducible NF-kappa B by a dominant negative form of Ikappa Balpha (Ikappa Balpha 32/36A). The stability of the integration of the mutant Ikappa Balpha was verified by Southern blotting of DNA extracted from several clones isolated by limiting dilution and cultured for 1 month in the presence of the selective antibiotic (not shown). Three clones, A3, D7, and F10, were identified as stably transfected with Ikappa Balpha 32/36A. To verify that the Ikappa Balpha cDNA was expressed in these clones, we performed Western blot analysis of the cytosolic fractions of the control and the "mutant" clones, using a monoclonal antibody specific for the NH2-terminal domain of Ikappa Balpha (36). The wild type and the mutant Ikappa Balpha are distinguishable on the basis of their electrophoretic migration because the 32/36A mutant migrates slightly slower in SDS gels (32). In the three clones that integrated Ikappa Balpha 32/36A, a slower migrating protein was specifically detected by the antibody in addition to the wild type Ikappa Balpha (Fig. 1A). Judging by the immunoblot results, the mutant and wild type Ikappa Balpha were expressed at comparable levels in clone A3 and F10, whereas in clone D7, the mutant Ikappa Balpha was more highly expressed relative to the wild type Ikappa Balpha . In control cells, only the faster migrating 36-kDa Ikappa Balpha was detected. In none of the mutant clones did expression of the transgenic Ikappa Balpha prevent constitutive production of the wild type Ikappa Balpha .


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Fig. 1.   Panel A, expression of the mutant Ikappa Balpha cDNA in the stable cell clones was controlled by Western blotting. Cytosolic proteins (30 µg/lane) from parental HPB-ALL cells (lane 1), empty vector transfected cells (lane 2), MAD332/36A transfected cells (lanes 3-5), and a false positive cell clone (lane 6) were prepared as described under "Materials and Methods" and tested for Ikappa Balpha by Western blotting using MAD B10 monoclonal antibodies. The positions of the wild type (Ikappa Balpha ) and Ikappa Balpha 32/36A (mut Ikappa Balpha ) proteins are indicated by arrows. Panel B, the capacity of NF-kappa B to translocate into nuclei in response to PMA plus PHA was analyzed by EMSA. Nuclear proteins from untreated cells (lanes 1, 3, 5, 7, 9, and 11) and from cells treated with PMA plus PHA for 1 h (lanes 2, 4, 6, 8, 10, and 12) were tested for PRE oligonucleotide binding by EMSA. The position of the retarded oligonucleotide is indicated by the arrow.

To determine whether the mutant Ikappa Balpha could inhibit translocation of NF-kappa B, we performed gel shift experiments with nuclear extracts from resting and PMA plus PHA-treated cell clones (Fig. 1B). In control cells, a 1-h PMA plus PHA activation generated a nuclear translocation of kappa B oligonucleotide-binding proteins, visible as a doublet. In contrast, in the three stable clones, neither constitutive nor inducible kappa B oligonucleotide binding activities were detected, suggesting that the mutant Ikappa Balpha prevented NF-kappa B translocation.

Ikappa Balpha 32/36A Blocks Nuclear Translocation of RelA-NF-kappa B but Not of RelB-p50-- To investigate the duration of NF-kappa B inhibition by the mutant Ikappa Balpha , we analyzed NF-kappa B nuclear translocation during a time course of PMA plus PHA treatment. In the control cells, kappa B binding activities were clearly detectable at the 3 h time point and increased in intensity up to 24 h of treatment (Fig. 2A). In the A3 clone, no significant kappa B binding activity was detected until 7 h of activation. However, by the 7 h time point, a kappa B binding activity, migrating as a doublet, was clearly detected and reached levels similar to the control by 24 h of activation (Fig. 2A). To identify the proteins in the complexes bound to kappa B oligonucleotide, we tested the abilities of antibodies specific for RelA, c-Rel, p50, and RelB to affect the EMSA patterns of control and A3 cells after 7 and 24 h of PMA plus PHA stimulation. In the absence of specific antibodies, several complexes were detectable in the control cells. The discrimination of these complexes was difficult in this type of gels; but clearly, in the A3 clone, only two bands were detectable after 7 h of cell stimulation, whereas in control cells additional, slower migrating bands, existed (see Fig. 2B and photographically enlarged view in Fig. 2C). Antibodies specific for RelA and RelB demonstrated that the two upper bands from the control cells contained RelA, whereas one of the lower bands contained RelB (Fig. 2B). p50-specific antibodies removed the two lower bands from both A3 and control cells. Thus, the upper band in A3 clone was composed of RelB-p50 dimers, whereas the lower band was the p50 homodimer. Antibodies specific for c-Rel had no effect on the kappa B-binding proteins in control or A3 cell nuclear extracts (Fig. 2), whereas they inhibited efficiently c-Rel-p50 binding in control cells (not shown). Thus, whereas in control cells both RelA and RelB dimers were detected, in the A3 clone only RelB-p50 and p50 dimers were detected. After 24 h of PMA plus PHA activation, both control and A3 nuclear extracts contained only the two faster migrating complexes (p50-p50 and RelB-p50) (Fig. 2B). These results demonstrated that in control HPB-ALL cells, the initial effect of PMA plus PHA activation led to nuclear translocation of cytosolic NF-kappa B proteins (RelA homo- and heterodimers). As expected, in the Ikappa Balpha 32/36A-transfected A3 clone, the translocation of these proteins was inhibited. Prolonged stimulation led to RelB activation in both cell clones. Furthermore, after 24 h of PMA plus PHA treatment activation of the RelA-containing complexes was also inhibited. It was not surprising to observe comparable levels of RelB in both control and A3 cell clones since RelB activation was reported to be independent of Ikappa Balpha . Similar results were obtained with the two other Ikappa Balpha 32/36A-transfected F10 and D7 cell clones (not shown). Thus, we have generated a cell system in which the prototypical NF-kappa B is inhibited selectively by Ikappa Balpha 32/36A, but activation of RelB remains potentially intact. Western blotting analysis of nuclear extracts from A3 and HPB-ALL cells further assessed the presence of RelB. RelB nuclear amounts were increased upon PMA plus PHA stimulation (Fig. 3). In addition, immunochemical analysis with RelB-specific antibodies confirmed the increase of RelB in nuclei of control cells and A3 cells after 24 h of stimulation (not shown). This is suggestive of a transcriptional, or at least, pretranslational, regulation of the RelB in PMA plus PHA-activated HPB-ALL T cells.


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Fig. 2.   Identification of kappa B-binding complexes in control and Ikappa Balpha 32/36A-expressing clones. Panel A, kinetic analysis of kappa B oligonucleotide binding activities was performed by EMSA. Equal amounts of nuclear proteins from untreated (0) or PMA plus PHA activated (from 3 to 24 h) cells were used. Control cells are pcDNA3-transfected HPB-ALL cells. Panel B, inhibition of kappa B-binding complexes with specific antibodies. Antisera specific for RelA, c-Rel, RelB, and p50 were added to the reaction mixture 45 min before the addition of the 32P-labeled PRE oligonucleotide. The nuclear proteins used were extracted from control cells (C) and the A3 cell clone activated by PMA plus PHA for 7 h (upper panel) and 24 h (lower panel). Positions of the major kappa B-binding complexes in the absence of competing antibodies are indicated on the left side of each panel. Panel C, enlarged view of the kappa B-binding complexes from control cells (C) and the A3 cells after 7 h of PMA plus PHA activation.


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Fig. 3.   Increase of RelB protein in nuclei of A3 and HPB-ALL cells upon cell stimulation. Nuclear extracts from A3 (lower panel) and HPB-ALL cells (upper panel) (70 µg/lane) were obtained from unstimulated (0) and PMA plus PHA-treated cells for 7 h (7) and 24 h (24). Proteins were fractionated by 10% polyacrylamide gel electrophoresis and analyzed for RelB protein with RelB-specific antiserum (1/500) as described under "Materials and Methods." The position of RelB is indicated by the arrow.

Ikappa Balpha 32/36A Expression Is Increased in PMA-stimulated Cells-- In the absence of stimulation, wild type Ikappa Balpha has a rapid turnover that is independent of serines 32 and 36 phosphorylation and of ubiquitination (46). After stimulation with PMA or TNF-alpha , Ikappa Balpha is modified by phosphorylation and ubiquitination, and the balance between degraded and newly synthesized Ikappa Balpha turns transiently in favor of the degradation (47). As a result, Ikappa Balpha is detected in lower amounts in cytosol from short term activated cells. However, the resulting activation of NF-kappa B induces newly synthesized Ikappa Balpha that is detectable within 1-2 h after activation. This neosynthesized Ikappa Balpha is, in turn, probably responsible for the inhibition of the RelA-containing NF-kappa B at later time points of PMA plus PHA treatment (see Fig. 2B). This cycle of activation-induced proteolysis/resynthesis of Ikappa Balpha is initiated by the phosphorylation of serines 32 and 36. To examine the fate of the Ikappa Balpha 32/36A versus the wild type Ikappa Balpha in activated cells, we performed kinetic experiments in which the A3 and the control clones were treated with PMA plus PHA for increasing lengths of time. Western blot analysis of Ikappa Balpha after up to 2 h (Fig. 4A) and 24 h (Fig. 4B) of activation by PMA plus PHA showed that the wild type Ikappa Balpha was degraded almost completely within 30 min in control cells. After 1 h of PMA plus PHA treatment it was resynthesized progressively, reaching initial levels after as soon as 3 h of activation (Fig. 4B). In the A3 clone, the wild type Ikappa Balpha was also degraded rapidly in response to cell activation, but neosynthesized wild type Ikappa Balpha was detectable only after 7 h of PMA plus PHA treatment, reaching initial levels at the 9 h time point (Fig. 4, A and B). In contrast to the wild type Ikappa Balpha , the Ikappa Balpha 32/36A was not degraded in response to cell activation. In fact, levels of Ikappa Balpha 32/36A increased from the 30 min time point to reach a steady maximum at 2 h (Fig. 4A) probably because the CMV promoter is activated independently of NF-kappa B activation. These experiments clearly demonstrated the stability of the mutant Ikappa Balpha in activation conditions that lead to wild type Ikappa Balpha proteolysis.


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Fig. 4.   Stability of the wild type Ikappa Balpha and the Ikappa Balpha 32/36A during cell activation. Panel A, Western blotting of cytoplasmic proteins extracted from control cells (pcDNA3-tranfected) (C) and the A3 clone (A3) was performed with the MAD 10B monoclonal antibody. Cells were activated for 0-2 h with PMA plus PHA. Positions of the wild type Ikappa Balpha and the Ikappa Balpha 32/36A are indicated by arrows. Panel B, as in panel A except for the time length of PMA plus PHA activation.

Differential Effect of Ikappa Balpha 32/36A on Several kappa B-dependent Promoters-- To test the functional consequences of the selective inhibition of RelA containing NF-kappa B, we performed transient transfections of A3, D7, F10, and control cells, with a series of reporter gene constructs (CAT or Luc) linked to promoter regulatory regions of seven genes suspected to constitute targets for NF-kappa B. In each of these promoters, at least one kappa B consensus sequence was identified in addition to sites specific for other regulatory transcription factors. The specificity of the Ikappa Balpha 32/36A inhibition on NF-kappa B-driven transcription was assessed with a reporter plasmid dependent on serum response element (c-fos CAT) (41). The results are summarized in Tables I and II. All of the promoters used in this study were activated by PMA plus PHA in control cells. The inductions of CAT and Luc constructs by PMA plus PHA ranged from 2.2-fold (c-myc) to 189-fold (p105) in control cells (Table I). The effect of the Ikappa Balpha 32/36A transgene on activation of CAT and Luc transcription depended on the promoter used. The transcription of the reporter genes driven by HIV LTR, MAD3, IL-6, IL-2, and p105 promoters was strongly inhibited in the three clones used (Table II). For example, in clone A3, activation of the IL-2 promoter reached only 3% of that obtained in control cells. In contrast, only 50% inhibition was observed with the ICAM-1 promoter. Finally, c-myc promoter driven transcription of CAT was not inhibited at all in the Ikappa Balpha 32/36A-transfected clones. Transient transfections performed with the control fos-CAT showed no difference in transcriptional activation between control cells and the A3 clone and a small inhibition in the D7 and F10 clones. Together these results indicated a selective effect of the Ikappa B transgene on NF-kappa B-driven transcription. Three sets of promoters could be distinguished on the basis of their responsiveness to NF-kappa B inhibition: promoters that were strongly inhibited by the transgene (IL-6 and IL-2, for example), promoters that were partially inhibited (ICAM-1), and promoters that were not affected by the lack of NF-kappa B translocation (c-myc). Activation of the Ikappa Balpha (0.2SK) promoter, which was shown to contain the kappa B1 sequence responsible for the transcriptional induction by RelA-p50 in Jurkat cells (39), was abolished totally in two of the three clones (A3 and D7). To estimate the potential contribution to Ikappa Balpha -promoter activation by RelB through binding to the two upstream kappa B sites, we performed Luc assays with two additional Ikappa Balpha -promoter constructs, the 0.4SK-Luc, containing all three kappa B sites, and the 0.4SKDelta kappa B, which contains only the kappa B2 and kappa B3 upstream sites. With the 0.4SK-Luc construct, only a little activation was obtained in A3 clone (2.1-fold) after PMA plus PHA treatment compared with control cells (23.6-fold) (Fig. 5). The 0.4SKDelta kappa B construct was not activated by PMA plus PHA in control and A3 clones (Fig. 5), indicating that kappa B2 and kappa B3 sites are not capable of enhancing Ikappa Balpha transcription in the absence of the kappa B1 site. Together, these results suggested that if RelB is implicated in Ikappa Balpha gene up-regulation, it does so through the involvement of a site(s) distinct from those contained in the promoter region reported up to now. Thus, to determine the impact of NF-kappa B inhibition on gene expression in the context of the genome, we analyzed the mRNA and/or protein production of several NF-kappa B target genes.

                              
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Table I
Effect of NF-kappa B inhibition on PMA plus PHA activation of various promoters
The results are expressed as the fold increase of CAT or Luc relative units in PMA plus PHA-treated cells relative to control level activities.

                              
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Table II
Percentage of residual Luc and CAT activities in Ikappa Balpha 32/36A-transfected cell clones
The effect of the transgenic Ikappa Balpha on promoter activation was calculated by comparing the fold increases in transgenic clones and in the control clone, taking the fold increase in control cells as 100%.


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Fig. 5.   Luciferase assays in control (C) and A3 clones. Cells were transfected with the MAD3 promoter-luciferase construct (0.4SK, O.2SK, and 0.4SKDelta kappa B; see "Materials and Methods"). The luciferase activity was determined 24 h after PMA plus PHA treatment in A3 clone extracts (white bars) and control cell extracts (black bars). The numbers above the black bars represent the fold increase of relative luciferase activities in PMA plus PHA-activated cells relative to control levels. The relative Luc activity measurements were performed in quadruplicate in at least three independent experiments. The deviations represented less than 10% of the mean value.

Effect of Ikappa Balpha 32/36A on mRNA Production-- We compared the induction of five kappa B target genes by a time course analysis of mRNA production by Northern blotting. Hybridization of the beta -actin probe was used as control (Fig. 6). In the empty vector-transfected cell clone (control), p105 (NFKB1), IL-2, wild type Ikappa Balpha mRNA, and IL-6 (data not shown) were induced in a time course-dependent manner. In contrast, no transcripts for IL-2 and IL-6 (data not shown) were detected in the A3 clone. The NFKB1 (p105) transcript was produced constitutively in A3 cells, but no induction by PMA plus PHA was observed. In the control cells, MAD3 (Ikappa Balpha ) mRNA reached a steady state after as little as 30 min of PMA plus PHA treatment. In A3 cells, two transcripts were detected with the MAD3 probe. The higher mobility transcript, corresponding to the mutant Ikappa Balpha , was strongly augmented in the early time points of activation, peaking at 1 h, whereas the slower migrating wild type messenger was detected at later time points (6-24 h). Thus, whereas transcription of IL-2 and IL-6 genes was inhibited completely in the A3 clone, induction of the wild type Ikappa Balpha messenger RNA was delayed in the A3 clone compared with the control cells. Finally, the c-myc messenger was produced constitutively in these cells, and no induction by PMA plus PHA was detected in either control or A3 cells.


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Fig. 6.   Effect of Ikappa Balpha 32/36A on target gene expression. Gene transcription of IL-2, NFKB1 (p105), MAD3 (Ikappa Balpha ), and c-myc genes was examined by Northern blotting. The experimental procedure is described under "Materials and Methods." Total RNA was extracted from A3 cells (left panels) and control pcDNA3-transfected HPB-ALL cells (right panel) treated with medium (lane 1) or with PMA plus PHA for 30 min (lane 2), 1 h (lane 3), 4 h (lane 4), 6 h (lane 5), 8 h (lane 6), or 12 h (lane 7). The Hybond membranes were reblotted with the beta -actin cDNA probe as control. The migration of the wild type Ikappa Balpha RNA is shown by the star.

Both IL-2 and IL-2Ralpha Productions Are Inhibited by the Ikappa Balpha 32/36A Mutant-- We further investigated the regulation of IL-2, an important T cell proliferation regulator, by measuring its production at the protein level in two of the stable clones (A3 and D7) by ELISA (Fig. 7). In both transgenic clones, IL-2 production was 10% of the control after 12 h of PMA and PHA activation. Thus, the result obtained with the Northern blot analysis of IL-2 induction was confirmed at the protein level.


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Fig. 7.   Dosage of IL-2 secretion by ELISA. IL-2 was measured in A3, D7, and control cell supernatants by ELISA after 12 h of PMA plus PHA activation as described under "Materials and Methods." The experiments were performed twice in quadruplicate. Standard deviations are shown with vertical bars and represent less than 10% of the mean value.

The alpha  subunit of the IL-2 receptor (CD25) is another potential target for NF-kappa B regulation (48, 49). To analyze further the effect of Ikappa Balpha 32/36A on the IL-2-regulated growth control we measured the expression of the CD25 by flow cytometry. In the absence of activation, no CD25 was detected on the surface of the clones. After 24 h of PMA plus PHA treatment, 68% of the control cells expressed CD25. In the A3 clone, a strong inhibition was observed because only 7% of cells were labeled with the CD25-specific antibody. In the F10 and D7 clones, the inhibition was less potent; 40% and 28%, respectively, of these cells were found to be CD25 positive (summarized in Table III). Despite the variability among the three clones, together these results indicated that the transcription of IL-2Ralpha requires RelA-NF-kappa B activation.

                              
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Table III
Induction of CD25 (IL-2Ralpha ) by PMA plus PHA in control and Ikappa Balpha 32/36A-clones
The data were obtained by flow cytometry using fluorescein isothiocyanate-conjugated antibodies to CD25.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present paper, we have reported the effect of a selective inhibition of the RelA-containing NF-kappa B on gene expression in T cell clones. We have generated this cell system by stable transfection of a mutant form of Ikappa Balpha which has been shown previously to block inducible RelA and c-Rel nuclear translocation (32). The particular T cell line that we used also naturally expresses RelB. Three independent cell clones, A3, D7, and F10, were selected by limiting dilution. In agreement with previous reports (30-32), no proteolysis or ubiquitination of the mutant Ikappa Balpha was detected under conditions in which signal-induced degradation of the wild type Ikappa Balpha occurred. In all three of the clones, the stability of the transgene led to an efficient inhibition of the RelA-containing NF-kappa B DNA binding, whereas RelB-p50 DNA binding capacity was unchanged, as expected. Therefore the signal-activable NF-kappa B was inhibited selectively, whereas the inducible Ikappa Balpha -independent RelB-p50 complex remained potentially active.

Expression of RelB is unusual in T cells. RelB is dominantly expressed in dendritic cells from both primary and secondary lymphoid organs (3, 5, 6) and in B cells at later stages of development. Its involvement in dendritic cells development was demonstrated clearly in RelB knockout mice (4, 50), but the role of RelB in gene expression remains obscure. Thus, our cell system is a convenient model for discriminating between gene transcription regulated selectively by the RelA-NF-kappa B and genes that may be regulated by RelB. In this respect, among the genes that we have studied the regulation of Ikappa Balpha expression is of particular interest. It has been shown, indeed, that among the three kappa B consensus sites found in the human MAD3 promoter region, it is the most proximal site, kappa B1, that mediates PMA and TNF activation through binding of RelA complexes (39). The kappa B2 site is recognized by RelA complexes, but it is not able to mediate efficient activation of the MAD3 promoter in cells producing only RelA and c-Rel complexes. The kappa B3 site was unable to bind NF-kappa B proteins extracted from myeloid cells (39). In HeLa cells all three kappa B sites were reported to contribute efficiently to TNF-alpha activation of Ikappa Balpha promoter (51). In addition, transfection of Jurkat cells with RelB vectors led to increased levels of Ikappa Balpha , suggesting that RelB is capable of enhancing Ikappa Balpha expression (13). We used three-luciferase reporter constructs that contain, respectively, all three (0.4SK), the two upstream (0.4SKDelta kappa B), or only the kappa B1 (0.2SK) sites of the Ikappa Balpha promoter. With these constructs the induced transcription of Luc was inhibited potently in all of the Ikappa Balpha 32/36A transgenic clones. This result demonstrates that in the MAD3 promoter, the three kappa B sites mediate Ikappa Balpha transcription by selectively binding forms of NF-kappa B which are themselves regulated by Ikappa Balpha . RelB was unable to compensate for the lack of NF-kappa B activation in this assay. However, expression of Ikappa Balpha did occur but at later time points of PMA stimulation than in control cells. The expression of the wild type Ikappa Balpha mirrored the increase of RelB DNA binding activity and protein. In addition, resynthesis of Ikappa Balpha did not occur in the MCF7 cells stably transfected with Ikappa Balpha 32/36A, cells that do not express RelB (52). These results strongly suggest that not only does RelB regulate Ikappa Balpha transcription through a site different from that which binds the prototypical NF-kappa B, but that NF-kappa B is not required for full Ikappa Balpha expression when RelB is produced in sufficient amounts. This effect might be specific for human Ikappa Balpha since the porcine Ikappa Balpha promoter domain, which contains six kappa B consensus sites, was not activated by overexpression of RelB (53). The alternative RelB-specific regulation of Ikappa Balpha could have functional consequences in cells that produce high levels of RelB such as dendritic cells. In such cells, NF-kappa B activity could be regulated negatively by Ikappa Balpha overexpression due to to RelB. However, the correlation between MAD3 expression and RelB activation remains to be established.

The potential compensatory effect of RelB was not observed with all of the genes that we examined. For example, expression of IL-2 was inhibited dramatically at both RNA and protein levels. IL-2 promoters possess multiple regulatory sequences among which are a single kappa B consensus site and multiple sites capable of interacting with c-jun protein complexes. Expression of a dominant negative mutant of c-jun abolishes IL-2 expression (54) probably because it coordinately blocks the IL-2 transcriptional regulation at multiple sites. In activated T cells, the major forms of NF-kappa B which bind to the kappa B site in the IL-2 promoter were shown to be p50 homodimers (55) and RelA homo- and heterodimers (56). Paradoxically, disruption of the c-rel gene in mice also inhibited induced IL-2 production despite the presence of RelA and p50 (57). A possible explanation of this effect was reported recently by Smith Shapiro et al. (58), who show that c-Rel regulation of the IL-2 promoter might be mediated by AP1 rather than directly through binding to kappa B sites. In contrast to c-Rel, RelA was not able to activate AP1-dependent luciferase expression (58). Here we show that in the absence of RelA dimers, RelB-p50 cannot rescue IL-2 expression. Further, the degree of IL-2 inhibition by Ikappa Balpha 32/36A transfection brings additional strong evidence that activation of RelA dimers is a limiting step for IL-2 transcriptional initiation.

Contrary to c-Rel disruption, inhibition of RelA dimers also diminished expression of the IL-2 receptor (CD25). Therefore, the classical NF-kappa B dimers seem to be involved in regulating the whole IL-2 growth control system.

In contrast to MAD3, RelB was not able to enhance the signal induced expression of NFKB1 (p105), indicating that selective activation of RelA dimers is required for the signal-induced expression of p105. However, the p50 protein (the processed, functional product of the NFKB1 gene) and the p105 mRNA were produced in both parental and the Ikappa Balpha transgenic cells, independent of cell activation. This suggests that the constitutive expression of NFKB1 is independent of kappa B enhancers.

Interestingly, c-myc expression was not inhibited by the inhibition of NF-kappa B. The c-myc promoter upstream kappa B site was shown to bind to RelA and c-Rel dimers and to be a positive regulator of the c-myc promoter in CAT assays in B lymphoma cells (59). Here we show that c-myc is expressed constitutively, not only in the parental HPB-ALL cells, but also in the Ikappa Balpha 32/36A transgenic cell clones. The c-myc promoter activity was only feebly enhanced by PMA plus PHA, and it was not decreased by the inhibition of RelA-NF-kappa B. It is therefore possible that RelB is able to activate c-myc expression constitutively. Alternatively, c-myc expression could be independent of the kappa B sites in HPB-ALL cells.

HIV LTR contains two direct repeats of the kappa B site in tandem. These kappa B sites are critical for the initial steps of HIV replication (24, 60, 61). It has been shown that although RelA-p50 up-regulates the HIV promoter through binding to the kappa B tandem sequence, c-rel behaves as a repressor of the RelA-p50 in the context of HIV LTR and the CD25 promoter (62). In control HPB-ALL cells, PMA plus PHA activated the transcription from the HIV LTR by 42-fold over the basal level. This activation was inhibited by 90% in the Ikappa Balpha 32/36A transgenic cell clones. Thus RelB was unable to substitute for RelA dimers. Ikappa Balpha 32/36A could be a powerful tool for repressing HIV replication in infected cells. However, since HIV replication is independent of NF-kappa B in the presence of the HIV Tat regulatory factor (61), we are currently investigating by infection experiments whether the effect seen in the CAT assay can be extrapolated to the viral replicative cycle.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Alain Israël and Douglas Ferris for reading the manuscript and for helpful discussion and to Dr. Catherine Dargemont and Catherine Amarger for helping with the graphical processing of the data. We are indebted to Dr. Fernando Arenzana-Seisdedos for the Ikappa Balpha -specific monoclonal antibody and to Dr. Patrick Baeuerle for providing the Ikappa Balpha 32/36A expression vector. We thank Drs. A. Israël, R. B. Gaynor, C. G. Crabtree, K. Roebuck, A. Harel-Bellan, J. Wietzerbin, and G. Haegeman for providing the various CAT and Luc constructs as well as some of the cDNA constructs used in the Northern blot assays, and we especially thank Prof. Patrice Debré for constant encouragement.

    FOOTNOTES

* This work was supported by the European Community Grant BiotechII BIO2-CT92-0130 and by a grant from the Agence Nationale de Recherche sur le Sida.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 33-1-4217-7513; Fax: 33-1-4217-7491; E-mail: KORNER{at}ccr.jussieu.fr.

1 The abbreviations used are: NF-kappa B, nuclear factor kappa B; IL, interleukin; TNF, tumor necrosis factor; ICAM-1, intercellular adhesion molecule-1; HIV, human immunodeficiency virus; CMV, cytomegalovirus; EMSA, electrophoretic shift assay; LTR, long terminal repeat; CAT, chloramphenicol acetyltransferase; Luc, luciferase; PMA, phorbol 12-myristate 13-acetate; ELISA, enzyme-linked immunosorbent assay; PHA, phytohemagglutinin.

2 J. Feuillard and M. Körner, unpublished observation.

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Abstract
Introduction
Materials & Methods
Results
Discussion
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