The human activation inducer molecule (AIM/CD69) is a phosphorylated disulfide-linked 27/33-kDa transmembrane homodimeric glycoprotein (Sánchez-Mateos and Sánchez-Madrid, 1991). The CD69 molecule is rapidly expressed on the surface of T-lymphocytes upon in vitro activation with a wide variety of agents, including anti-CD3/T cell receptor and anti-CD2 mAbs, ()activators of protein kinase C, and phytohemagglutinin (Cebrián et al., 1988; Hara et al., 1986). Similarly, the expression of CD69 is inducible on the surface of NK cells, B-lymphocytes, and eosinophils (Hartnell et al., 1993; Lanier et al., 1988). In contrast, CD69 is constitutively expressed in vivo on platelets and on a small percentage of resident T- and B-cells of different lymphoid tissues (Sánchez-Mateos et al., 1989; Testi et al., 1990). Although a physiologic ligand for CD69 has not been identified so far, experiments with specific mAbs indicate that this antigen functions as a signal-transmitting receptor. Signals triggered by CD69 mAbs in T-lymphocytes include increase in intracellular calcium concentration and result in the synthesis of different cytokines and their receptors, enhancement of the expression of c-myc and c-fos protooncogenes, and cell proliferation (Cebrián et al., 1988; Nakamura et al., 1989; Santis et al., 1992; Testi et al., 1989; Tugores et al., 1992). In NK cells and platelets, CD69 also acts as a triggering molecule, being involved in the redirected target cell lysis by interleukin-2-activated NK cells (Moretta et al., 1991) and in the induction of platelet aggregation, Ca influx, and hydrolysis of arachidonic acid (Testi et al., 1990).

The molecular cloning of cDNAs encoding human and mouse CD69 revealed that this antigen is a member of the Cadependent (C-type) animal lectin superfamily of type II transmembrane receptors (Hamann et al., 1993; López-Cabrera et al., 1993; Ziegler et al., 1993). This superfamily includes the human NKG2, rat and mouse NKR-P1, and mouse Ly-49 families of NK cell-specific antigens as well as the low avidity IgE receptor (CD23), the Kupffer cell receptor, and the hepatic asialoglycoprotein receptor (Drickamer, 1993; Yokoyama, 1993). Characterization of the structure of genes encoding human and mouse CD69 further evidenced that CD69 is evolutionarily related to these C-type lectin receptors (Santís et al., 1994; Ziegler et al., 1994). In addition, the CD69 gene is clustered with other C-type lectin-encoding genes within a genetic region named the NK cell complex (Drickamer, 1993; López-Cabrera et al., 1993; Yokoyama, 1993; Ziegler et al., 1994).

The CD69 antigen is undetectable on peripheral blood lymphocytes; however, it is expressed at high levels by the majority of T-cells in the inflammatory cell infiltrates of several human diseases such as rheumatoid arthritis and chronic viral hepatitis (García-Monzón et al., 1990; Laffón et al., 1991), suggesting that inflammatory cytokines may be involved in CD69 expression. In this context, it has been reported that a large amount of TNF- is produced by human hepatocytes in chronic viral hepatitis (González-Amaro et al., 1994). Thus, TNF- and other cytokines may play a key regulatory role in the inducible expression of the CD69 antigen in vivo.

Northern blot analysis demonstrated that the constitutive and inducible expression of the CD69 molecule is regulated at the transcriptional level (Hamann et al., 1993, López-Cabrera et al., 1993; Ziegler et al., 1993, 1994). To determine the molecular basis for the pattern of CD69 expression, we have isolated the 5`-region of the CD69 gene and analyzed its inducible promoter activity. We describe herein that the upstream sequence of the CD69 gene functions as a PMA-inducible promoter element. In addition, we report that TNF- is capable of inducing both the surface expression of the CD69 antigen and the promoter activity of the 5`-region of the CD69 gene. The presence of NF-B motifs within the proximal promoter region of the CD69 gene may account for the TNF--inducible promoter activity.

EXPERIMENTAL PROCEDURES

Isolation of Genomic Clones Containing the 5`-Region of the CD69 Gene

Restriction analysis of the clones indicated that they possessed the same 3-kbp EcoRI insert, which was subcloned into the pBluescript plasmid (Stratagene, La Jolla, CA). DNA sequencing was performed by the dideoxy termination method either by subcloning restriction fragments into pBluescript vectors or by direct oligonucleotide-primed DNA sequencing with internal primers.

Plasmid Constructions, Transfections, and Luciferase Assays

K562 erythroleukemic cells were transiently transfected with 4 µg of each recombinant plasmid using 10 µg of Lipofectin reagent (Life Technologies, Inc.) according to the manufacturer's recommendations. To normalize transfection efficiency, 1 µg of pCMV-gal (CLONTECH, Palo Alto, CA), which contains the cytomegalovirus promoter ligated to the -galactosidase gene, was included in each transfection. After 48 h of transfection, luciferase activity was determined in cell extracts containing identical -galactosidase activity according to the instructions of the luciferase assay kit (Promega). Light emission was measured in a Lumat LB9501 luminometer (Berthold, Wildbad, Germany), and the results are expressed as relative light units. To analyze the effect of PMA and TNF- on the CD69 promoter activity, half of the transfected cells were treated for 12-16 h with either 20 ng/ml PMA or 50 ng/ml human recombinant TNF- (3.2 10 units/mg; Wichem, Vienna). As positive controls for PMA and TNF- stimulation, we used the plasmids pGL2P (Promega), in which the luciferase gene is under the control of the SV40 early promoter, and pKBF-Luc (a gift from Dr. A. Israël, Institut Pasteur, Paris), which contains a trimer of the NF-B motif of the H-2K gene (Yano et al., 1987) upstream of the herpes simplex virus thymidine kinase gene promoter.

To obtain K562 stable transfectants, cells were cotransfected by electroporation with 50 µg of pAIM1.4-Luc and 10 µg of pSV2-neo as described previously (Nueda et al., 1993), selected in the presence of G418 (1 mg/ml), and cloned by limiting dilution. To analyze the transactivation of the CD69 promoter by NF-B/Rel family members, K562 cells were cotransfected, in the presence of 20 µg of Lipofectin, with 5 µg of CD69 promoter-derived plasmids and 5-10 µg of the expression vector pRc/CMV-p50, pRc/CMV-c-Rel, or pRc/CMV-RelA (kindly provided by Dr. A. Israël), which contained the full-length cDNA encoding each protein, into the pRc/CMV plasmid (Invitrogen, San Diego, CA) (Le Bail et al., 1993).

Fluorescence-activated Cell Sorting Analysis

Nuclear Extracts and Electrophoretic Mobility Shift Assays

Binding reactions for gel retardation assays were performed at 0 °C in a volume of 20 µl containing 10 mM Hepes, pH 7.6, 10% glycerol, 50 mM KCl 6 mM MgCl, 0.1 mM EDTA, 1 mM dithiothreitol, 5 µg of poly(dI-dC), 0.5 ng of 3`-end labeled probe, and 2 µg of nuclear extract. After the binding reaction, the mixtures were electrophoretically separated on 4-5% nondenaturing polyacrylamide gels. When indicated, 0.5 µl of rabbit anti-p50 (Kieran et al., 1990), anti-RelA, anti-c-Rel, or anti-p52 specific polyclonal antibodies were added to the corresponding binding reaction prior to the addition of the radiolabeled probe. These antisera were kindly provided by Drs. A. Israël and N. R. Rice. For competition, a 50-fold molar excess of unlabeled oligonucleotide was added to the binding reaction prior to the addition of the probe. The sequences of the oligonucleotide probes (and their complementaries) used in this study were as follows: CD69-B-1, 5`-GATCAGACAACAGGGAAAACCCATACTTC-3` (nucleotides -170 to -144); and CD69-B-2, 5`-GATCAGAGTCTGGGAAAATCCCACTTTCC-3` (nucleotides -232 to -206). The synthetic oligonucleotides used for competition were as follows: VP9, 5`-CCCTGGGTTTCCCCTTGAAGGGATTTCCCTCCG-3` (a gift from Dr. J. M. Redondo, Hospital de la Princesa, Madrid), containing the two NF-B-binding sites from the vascular cell adhesion molecule-1 promoter; KBF, 5`-AGCTTGGGGATTCCCCAT-3`, containing the NF-B motif of the promoter of the H-2K gene (Yano et al., 1987); AP-1, 5`-CGCTTGATGAGTCAGCCGGAA-3`; and OCT-1, 5`-TGTCGAATGCAAATCACTAGA-3`.

RESULTS

Structure of the 5`-Region of the CD69 Gene

Figure 1: Structure of the 5`-region of the CD69 gene. A, restriction map of the cloned fragment encompassing the 5`-flanking region of the CD69 gene. The restriction sites are BglII (B), EcoRI (E), HindIII (H), PvuII (P), SacI (S), and XbaI (X). The position of the first exon is indicated by a filledbox. B, nucleotide sequence of the 5`-regulatory region of the CD69 gene. First exon nucleotides are underlined, and the major transcription initiation site is denoted by +1. The first intron sequence appears in lower-caseletters. Shadedareas correspond to consensus binding sequences for transcription factors. The TATA box is indicated by doubleunderlining. Prediction of putative transcription factor-binding sites was carried out by the C-coded program SITIOS (Dr. M. A. Vega, Instituto López Neyra, Consejo Superior de Investigaciones Científicas), which includes Release 5.0 of D. Ghosh's transcription factor data base (Ghosh, 1991).

The major transcription initiation site of the CD69 gene has been located 81 nucleotides upstream from the translation start codon (Santís et al., 1994). Nucleotide sequence analysis 1050 bp upstream from the CAP site revealed the presence of a canonical TATA box at position -30 and a GC-rich sequence at position -52. In addition, three potential NF-B/Rel-binding sequences were identified at positions -160, -223, and -373 (Fig. 1B). The two proximal NF-B-binding sites (kB-1 and kB-2 in Fig. 1B) were identical to those found in the gene promoters of c-myc and interleukin-6, respectively (Baeuerle, 1991), whereas the most upstream NF-B motif (kB-3 in Fig. 1B) was similar to that found in the major histocompatibility complex class I (H-2K) gene promoter (Yano et al., 1987).

Functional Analysis of the CD69 Gene Promoter

Figure 2: Functional analysis of the CD69 promoter. The schematic representation of the CD69 promoter-based reporter gene constructs is shown on the left, and the upstream region of the CD69 gene is represented at the top. The positions of the first exon and the first intron are indicated by filled and hatchedboxes, respectively. The nomenclature of the deletion plasmids is based on the most 5`-nucleotide of the CD69 gene sequence present, and its position is denoted relative to the transcription initiation site (position +1). The basal and PMA-induced promoter activity of the 5`-region of the CD69 gene was determined by transient expression of luciferase gene-based constructs in K562 cells. Each transfection was carried out at least four times, and the data from a representative experiment are shown on the right. INDUCT., induction.

TNF- Treatment Induces the Expression of the CD69 Antigen and the Activity of the CD69 Gene Promoter

Figure 3: TNF--induced expression of the CD69 antigen is mediated by an increase in the CD69 promoter activity. A, surface expression of the CD69 antigen was analyzed by flow cytometry on unstimulated K562 cells and on K562 cells treated for 16 h with PMA (20 µg/ml) or TNF- (50 ng/ml). Staining of cells with myeloma P3X63 antibody was included as a negative control. B, K562 cells were stably transfected with plasmid pAIM1.4-Luc, and the TNF--induced luciferase activity was analyzed in three independent clones. The luciferase activities are represented in light units/10 cells. C, activation of the CD69 gene promoter by the NF-B/Rel family members. Five µg of construct pAIM1.4-Luc were cotransfected into K562 cells with 5 or 10 µg of expression vector pRc/CMV-p50, pRc/CMV-RelA, or pRc/CMV-c-Rel. The luciferase activities are represented as -fold activation over the value obtained with construct pAIM 1.4-Luc cotransfected with 10 µg of empty vector pRc/CMV. Results shown are representative of four experiments.

It is known that the NF-B/Rel family of transcription factors plays an important role in the cytokine induction of many cellular and viral genes (Baeuerle, 1991). To characterize the functional role of NF-B-related proteins in the activation of the CD69 gene promoter, K562 cells were cotransfected with the construct pAIM1.4-Luc and expression vectors encoding the p50, RelA, and c-Rel members of the NF-B/Rel family. As shown in Fig. 3C, p50 was unable to transactivate the promoter, whereas c-Rel and RelA efficiently induced the promoter activity (4-5- and 12-18-fold, respectively). Interestingly, cotransfection with a combination of p50- and RelA-encoding plasmids did not result in activation of the CD69 promoter (data not shown).

The B-2 Site Is Responsible for TNF- Inducibility of the CD69 Gene Promoter

Figure 4: Activation of the CD69 promoter by TNF- is achieved through the sequence spanning positions -255 to -170, which contains the NF-B-2 motif. A, CD69 promoter-based luciferase plasmids were transfected into K562 cells, and half of the transfected cells were treated with TNF- for 16 h. Each transfection was carried out five times, and a representative experiment is shown. B, 5 µg of each of the CD69 promoter-derived constructs were cotransfected into K562 cells with 10 µg of expression vector pRc/CMV-RelA. Numbers above the bars indicate -fold transactivation over the values obtained by cotransfection with the empty vector pRc/CMV.

To confirm this point, K562 cells were cotransfected with the CD69 promoter constructs and the expression vector pRc/CMV-RelA. As shown in Fig. 4B, the pattern of transactivation of the different CD69 promoter fragments by RelA correlated with the response of these constructs to TNF- (Fig. 4A). Therefore, deletion of the sequences containing the B-2 site greatly diminishes the transactivation of the CD69 promoter by RelA.

Members of the NF-B/Rel Family Bind to the B-2 and B-1 Motifs of the CD69 Gene

Figure 5: The B-2 and B-1 motifs of the proximal CD69 promoter region are recognized by NF-B/Rel family members. A, mobility band shift assays were performed with oligonucleotide probes CD69-B-1 (lanes 1-5) and CD69-B-2 (lanes 6-9), which contained the putative NF-B motifs located at positions -160 and -223, respectively. Nuclear extracts from COS-7 cells (lane1) and from COS-7 cells transfected with pRc/CMV-p50 and pRc/CMV-RelA (lanes 2-9) were incubated with 0.5 ng of each double-stranded oligonucleotide probe. Competitor oligonucleotides were added at 50-fold molar excess and included oligonucleotide KBF, which contains the NF-B motif of the promoter of the H-2K gene (lanes3 and 7) and the unrelated oligonucleotides OCT-1 (lanes4 and 8) and AP-1 (lanes5 and 9). B, oligonucleotide probes CD69-B-2 (lanes 1-6) and CD69-B-1 (lanes 7-12) were incubated with nuclear extracts from uninduced K562 cells (lanes1 and 7) and from K562 cells stimulated with PMA (P; lanes2 and 8) or TNF- (lanes 3-6 and 9-12). Competitor oligonucleotides were added at 50-fold molar excess and included the specific competitors (lanes5 and 11), oligonucleotide VP9 (which contains two NF-B-binding sites from the vascular cell adhesion molecule-1 gene promoter) (lanes6 and 12), and the unrelated oligonucleotide OCT-1 (lanes4 and 10). Specific DNA-protein complexes (a-d) are indicated. C, nuclear extracts from uninduced K562 cells (lanes1 and 7) or from K562 cells stimulated with TNF- (lanes 2-6 and 8-12) were preincubated either with preimmune serum (PRE; lanes1, 2, 7, and 8) or with antiserum specific to p50 (lanes3 and 9), RelA (lanes4 and 10), c-Rel (lanes5 and 11), or p52 (lanes6 and 12) prior to the addition of probe CD69-B-2 (lanes 1-6) or CD69-B-1 (lanes 7-12). The DNA-protein complexes obtained with probe CD69-B-1 were exposed three times longer than those obtained with CD69-B-2 to get a similar intensity of the bands.

To characterize the NF-B/Rel-related proteins that bind to the NF-B sites of the CD69 promoter in CD69-expressing cells, the two oligonucleotide probes were incubated with nuclear extracts prepared from untreated K562 cells and from K562 cells treated with either PMA or TNF-. Four inducible DNA-protein complexes (a-d) were observed with both oligonucleotide probes when they were incubated with extracts from PMA- and TNF--treated K562 cells (Fig. 5B). The specific DNA-protein complexes observed with both probes displayed identical electrophoretic mobility, suggesting that they bind the same nuclear factors. It is interesting to note that oligonucleotide CD69-B-1, which bound a lesser amount of nuclear proteins, contained one mismatch with respect to the consensus NF-B-binding site (5`-GGGRNTYYC-3`), whereas oligonucleotide CD69-B-2 perfectly matched the consensus sequence (Baeuerle, 1991). In both cases, the addition of an excess of unlabeled specific oligonucleotide to the binding reaction completely abolished the formation of the inducible DNA-protein complexes (Fig. 5B). Similarly, an equal amount of the heterologous oligonucleotide VP9, which contained two NF-B-binding sites from the vascular cell adhesion molecule-1 gene promoter, efficiently competed the specific complexes (Fig. 5B). In contrast, the formation of these DNA-protein complexes was not blocked by the addition of a heterologous competitor that lacked NF-B binding sequences (Fig. 5B).

To identify the nature of the NF-B/Rel family members that bind to the NF-B motifs of the CD69 gene promoter, the binding reactions were preincubated with antiserum specific to p50, RelA, c-Rel, or p52 (Fig. 5C). The anti-p50 antiserum induced the disappearance or reduced the intensity of complexes b-d. The anti-RelA antiserum blocked the formation of complexes a and b, whereas the anti-c-Rel antiserum inhibited only complex c. In contrast, the anti-p52 antiserum did not interfere with the formation of any of these complexes. These results indicate that the slower migrating complex (complex a) corresponds to a homodimer of RelA, which is more easily detected when using oligonucleotide CD69-B-2. Then, from top to bottom, the complexes correspond to p50/RelA (complex b) and p50/c-Rel (complex c) heterodimers and to a p50 homodimer (complex d). Taken together, these results confirm that the NF-B motifs of the proximal CD69 promoter are capable of binding to inducible NF-B/Rel factors, and therefore, they may be involved in the TNF--mediated induction of CD69 gene expression.

DISCUSSION

The transcription of the CD69 gene appears to be tightly regulated in vivo, as it is almost exclusively expressed at sites were inflammation takes place, suggesting that inflammatory cytokines may participate in the control of the expression of this gene. In this report, we describe the isolation and functional characterization of the human CD69 gene promoter region. We have focused mainly on the identification of the cis-acting sequences and the nuclear factors involved in the inducible expression of the CD69-encoding gene.

Deletion analysis of the 5`-flanking region of the CD69 gene has allowed the identification of a proximal fragment of 94 bp (nucleotides -78 to +16) that is sufficient to govern basal and PMA-induced promoter activity. This proximal promoter domain contains a canonical TATA box and a GC-rich sequence that could be a target for Sp1, a ubiquitous and constitutive transcription factor that binds to the promoter of many genes, and Egr-1/Krox-24, a zinc-finger transcription factor whose expression can be induced by various agents, including phorbol esters, and that has been implicated in the activation of T- and B-lymphocytes (McMahon and Monroe, 1995; Krämer et al., 1994; Pérez-Castillo et al., 1993). Since the synthesis of CD69-encoding mRNA is rapidly induced upon PMA stimulation of the cells (López-Cabrera et al., 1993), additional pre-existing transcription factors should mediate the PMA-induced expression of the CD69 gene to ensure a rapid transcriptional response. In this context, computer-aided analysis of the 5`-flanking region of the CD69 gene revealed the presence of three NF-B motifs (positions -160, -223, and -374) and at least one putative AP-1-binding site (position -956), which may cooperate with the proximal cis-acting elements in the response to phorbol esters.

We demonstrate herewith that TNF- is capable of inducing the expression of the CD69 antigen and that this induction is mediated by an increase in the CD69 promoter activity. Cotransfection experiments with CD69 promoter-derived reporter constructs and NF-B-encoding vectors show that this promoter is transactivated by members of the NF-B/Rel family, specially by RelA (formerly p65). Thus, the TNF--induced expression of the CD69 gene may be mediated by the binding of NF-B/Rel proteins to one or more NF-B motifs of the CD69 promoter. This point is further supported by the fact that deletion of the sequence that contains the B-2 motif (nucleotides -255 to -170) completely abolishes the response to TNF- and markedly reduces RelA-mediated transactivation. Since no additional TNF--responsive elements are present in this sequence, our results strongly indicate that the B-2 motif plays a key role in the TNF--induced promoter activity. However, to define unambiguously the contribution of B-2 and the other NF-B motifs to the response to TNF-, we are currently performing site-directed mutagenesis of these motifs in the context of the intact promoter.

Our results also demonstrate that the two most proximal NF-B motifs of the CD69 promoter (B-2 and B-1) bind four PMA- and TNF--inducible NF-B/Rel-related complexes. The higher affinity of these proteins for the B-2 sequence further emphasizes the functional role of this motif and may explain why constructs containing only the B-1 site are unresponsive to TNF-. Antisera directed against the different NF-B/Rel proteins were used to identify the family members present in the DNA-protein complexes detected with the NF-B motif-derived probes. These experiments revealed that the DNA-binding activities consisted of a RelA homodimer, p50/RelA and p50/c-Rel heterodimers, and a p50 homodimer. The most prominent complexes were composed of p50/RelA and p50/c-Rel heterodimers. The detection of RelA homodimer binding to the B-2 motif, a complex that is not easily detected with the NF-B sites of other promoters (Ganchi et al., 1993), is consistent with the observation that the sequence of this motif matches perfectly with the consensus binding sequence for RelA, 5`-GGGRNTTTCC-3` (Kunsch et al., 1992).

We have recently demonstrated that the expression of the CD69 gene is regulated at the post-transcriptional level by a rapid degradation pathway associated with AU-rich sequence motifs (Santís et al., 1995). This mechanism of regulation has been found in many genes involved in inflammatory and activation responses. Most of these genes code for cytokines and oncoproteins, which are implicated in the initial events leading to activation and proliferation of the cells. In addition, the transcription of these genes appears to be highly regulated by the NF-B motifs of their promoters (Baeuerle, 1991; Baeuerle and Henkel, 1994). Therefore, our findings, which highlight the functional relevance of the NF-B motifs of the CD69 gene, further support the idea regarding a general mechanism involved in the control of expression of activation-associated genes during the early phase of the immune response.

The expression of CD69-encoding mRNA can be easily induced in most leukocytes by treatment ``in vitro'' with a wide range of stimuli such as PMA or anti-CD3/T cell receptor mAb (López-Cabrera et al., 1993; Ziegler et al., 1994). In addition, the expression of CD69 on activated T-lymphocytes from cell infiltrates of various chronic inflammatory diseases such as rheumatoid arthritis and chronic viral hepatitis has been documented (García-Monzón et al., 1990; Laffón et al., 1991). It is also known that TNF- is an important inflammatory mediator that is actively secreted by several cell types at inflammatory sites (González-Amaro et al., 1994; Vassalli, 1992). Our findings strongly suggest that the pro-inflammatory cytokine TNF- may play a key role ``in vivo'' in the expression of CD69 by inflammatory cells. Interestingly enough, we have previously described that CD69 antigen is in turn capable of generating signals that induce the synthesis of TNF- by lymphoid cells (Santís et al., 1992). Thus, it is feasible that, at sites of inflammation, a positive feed-back loop is established between the expression of CD69 and the production of TNF-. The putative ligand of CD69 should be clearly involved in this condition. The above phenomenon could have an important role in the perpetuation of several inflammatory diseases and could be related to the resistance of some patients with these conditions toward the current anti-inflammatory therapy.

FOOTNOTES

*

This work was supported by Grant PB92-0318 (to F. S-M.), Grant FIS 95/0208 (to M. L.-C.), and Grant SAF 95/0474 (to E. M.) from the Ministries of Education and Health. 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 nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z38109[GenBank].

§

To whom correspondence should be addressed: Servicio de Inmunología, Hospital de la Princesa, Diego de León, E-28006 Madrid, Spain. Fax: 34-1-309-24-96.

()

The abbreviations used are: mAbs, monoclonal antibodies; NK, natural killer; TNF-, tumor necrosis factor-; PMA, phorbol 12-myristate 13-acetate; bp, base pair(s); kbp, kilobase pair(s).

ACKNOWLEDGEMENTS

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