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The Journal of Immunology, 2001, 167: 6412-6420.
Copyright © 2001 by The American Association of Immunologists

A Novel NF-{kappa}B/Rel Site in Intron 1 Cooperates with Proximal Promoter Elements to Mediate TNF-{alpha}-Induced Transcription of the Human Polymeric Ig Receptor1

Hilde Schjerven2, Per Brandtzaeg and Finn-Eirik Johansen

Laboratory for Immunohistochemistry and Immunopathology, Institute of Pathology, University of Oslo, Rikshospitalet, Oslo, Norway


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Secretory Abs constitute the first line of specific immune defense at mucosal surfaces. Such Abs are generated by the active transport of polymeric Ig (pIg) across secretory epithelia mediated by the pIgR, also known as transmembrane secretory component (SC). The proinflammatory cytokine TNF-{alpha} is a key mediator of host responses to infections, and it can stimulate protein synthesis-dependent transcriptional up-regulation of pIgR/SC in the HT-29 intestinal adenocarcinoma cell line. By reporter gene assay we identified a novel TNF-{alpha}-responsive region located within a 748-bp fragment in intron 1 of the human pIgR/SC gene which depended on an NF-{kappa}B/Rel site for full responsiveness. EMSAs demonstrated preferential binding of the NF-{kappa}B/Rel family member p65 (RelA) to this DNA element after TNF-{alpha} stimulation, with weaker and more delayed binding of p50. Furthermore, the TNF-{alpha}-responsive region in intron 1 required cooperation with DNA elements located in the proximal promoter region of the gene. Mutational analysis demonstrated that an IFN-stimulated response element near the transcriptional start site in exon 1 was involved in the TNF-{alpha} responsiveness. Thus, DNA elements located >4 kb apart were found to cooperate in TNF-{alpha}-induced pIgR/SC up-regulation. The intronic TNF-{alpha}-responsive enhancer overlapped with a recently identified IL-4-responsive enhancer. Several intronic DNA elements found to be functionally important in the human gene are highly conserved between the human and mouse pIgR/SC genes, suggesting the presence of a conserved cytokine-responsive enhancer region.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The first line of adaptive immune defense at mucosal surfaces depends on the production of secretory Igs (SIgs)3 to exclude ingested or inhaled potentially harmful agents. SIgs are produced by a cooperation between local J chain-expressing plasma cells that produce polymeric Igs (pIgs; mainly dimeric IgA, but also larger polymers of IgA and pentameric IgM) (reviewed in Ref. 1) and secretory epithelial cells that express the pIgR. Such a common receptor-mediated transport mechanism for pIgs was first proposed by Brandtzaeg (2) in 1974. The receptor, also known as the transmembrane secretory component (SC), binds the pIgs at the basolateral side of the epithelial cells and translocates the receptor-pIg complexes to the mucosal surface. At the apical epithelial domain pIgR/SC is cleaved, allowing release of SIgA and SIgM with bound SC as well as free SC derived from unoccupied receptor (reviewed in Refs. 3 and 4). Cleavage of pIgR/SC involves a sacrificial pathway, which requires a high level of constitutive receptor expression as well as receptor up-regulation when it is of advantage to increase external SIg transport. This receptor is expressed by all kinds of secretory epithelia, with the highest levels seen in the intestine (3). Several clinical and experimental studies have provided evidence for the importance of SIgA Abs in the protection of mucosal surfaces (5, 6). Recently, pIgR/SC knockout mice have confirmed the essential role of this receptor in the transport of pIgs to external secretions (7, 8). Analysis of such mice demonstrated the importance of secretory Abs in maintaining the integrity of surface epithelium (7). Together, these data suggest that pIgR/SC is critical for the mucosal barrier function and that regulation of the expression of this receptor may be an important means to reinforce secretory immunity.

TNF-{alpha} was initially discovered for its ability to kill tumor cells, and as a mediator of acute and chronic inflammation (9). Although this cytokine is produced mainly by macrophages, it can be derived from several other cell types and is a key mediator of the host response to many infectious agents (reviewed in Ref. 9). Signaling through the TNF-{alpha} receptor initiates a cascade of phosphorylation events, which eventually lead to the release of prestored NF-{kappa}B/Rel from its inhibitor in the cytoplasm, thereby allowing translocation to the nucleus (reviewed in Refs. 10, 11, 12, 13). This relatively simple activation scheme of NF-{kappa}B/Rel contrasts with the enormous complexity of the converging activation pathways and the diverging downstream effects. NF-{kappa}B/Rel activation is important both for innate and adaptive immune responses, but also in biological processes such as apoptosis, cell proliferation, stress responses, and carcinogenesis (reviewed in Refs. 12, 13, 14). Importantly, NF-{kappa}B/Rel is believed to play a central role in both the initiation and perpetuation of relapsing inflammatory processes (10), including inflammatory bowel disease, and is thus a potential target of therapeutic agents (10, 12, 15).

In situ studies have shown up-regulation of pIgR/SC in several chronic mucosal disorders such as celiac disease, Helicobacter pylori gastritis, and Sjögren’s syndrome—most likely as a result of locally produced cytokines (reviewed in Ref. 16). Consistent with these data, TNF-{alpha} (17), IFN-{gamma}, IL-1, and IL-4 enhance pIgR/SC expression in culture models of secretory epithelial cells (reviewed in Ref. 3). This cytokine-mediated transcriptional gene activation has been shown to depend on de novo protein synthesis (18, 19, 20). However, in inflammatory bowel disease the pattern of pIgR/SC expression is quite variable, but an overall down-regulation is seen in lesions with dysplastic epithelium (21, 22, 23).

The central role that the pIgR/SC plays in the protection of mucosal surfaces, and its observed deregulation in dysplastic epithelium, have led to an increased interest in the mechanisms that control pIgR/SC expression. Thus, such mechanisms have been extensively studied with regard to both the constitutive and hormone- or cytokine-mediated receptor expression (reviewed in Refs. 3 and 24). TNF-{alpha}-mediated transcriptional up-regulation of pIgR/SC was shown to depend on NF-{kappa}B/Rel activation (25) and an IFN-stimulated response element (ISRE) located in exon 1 (26, 27). However, the effect mediated by described DNA elements could not account for the degree of up-regulation indicated by the increased level of mRNA and by nuclear run-on experiments (18). In this report, we describe a novel 748-bp TNF-{alpha}-responsive region in the 5.7-kb intron 1, located ~4.1 kb downstream of the transcriptional start site. We further show that a consensus NF-{kappa}B/Rel site within this intronic enhancer, which preferentially binds p65/RelA, is required for full TNF-{alpha} responsiveness. Finally, we demonstrate that the intronic TNF-{alpha}-responsive region requires cooperation with quite distant DNA elements in the pIgR/SC promoter.


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

Plasmids denoted pSC1 through pSC9 and pSC16 (described in Ref. 24) contained different lengths of regulatory sequences (including the promoter) from the human pIgR/SC gene subcloned into XhoI/NcoI-digested pGL3 enhancer vector (Promega, Madison, WI). pSC1 extended from -2684 (relative to the transcriptional start site; Ref. 28) to the ATG start codon in exon 2 of the human pIgR/SC gene. pSC2 through pSC9 and pSC16 contained internal deletions in intron 1.

Plasmids denoted p1 and p2 contained a 1.3-kb SacI-HindIII fragment from intron 1 subcloned upstream of the SV40 promoter of the pGL3 promoter vector (designated p0; Promega) in either orientation. The plasmid pSC15 was constructed from pSC1 by deletion of upstream promoter sequences down to a SacI restriction enzyme site at -537. Plasmids denoted pSC11, pSC27, and pSC28 were derived from pSC2 and contained different lengths of the pIgR/SC promoter, extending from -537, -177, and -81, respectively. pSC12, pSC29, and pSC30 contained the 1.3-kb SacI-HindIII fragment from intron 1, subcloned upstream of the pIgR/SC promoter in pSC11, pSC27, and pSC28, respectively. The point mutations in pSC45–pSC47 and pSC51–pSC58 were introduced with the QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). For mutation of the exon 1 ISRE and the NF-{kappa}B/Rel site in intron 1 (pSC45–pSC47, pSC51–pSC54, pSC56–pSC58), point mutations were designed that changed the nucleotide from a purine to the noncomplementary pyrimidine and vice versa (i.e., A{leftrightarrow}C, T{leftrightarrow}G). For mutation of the NF-{kappa}B/Rel site in the pIgR/SC promoter (pSC55–pSC58), the same mutation was introduced (GGG->CCC) as used for EMSA by Nilsen et al. (18). The integrity of the vector-insert boundary of all subcloned DNA fragments, as well as all mutations, was confirmed by sequencing with the cycle sequencing kit (Amersham International, Slough, U.K.), or by the sequencing service offered by MediGenomix (Martinsried, Germany). The Renilla luciferase control vector, pRL-PGK, has been described previously (24).

Cell culture and transfections

The human colonic adenocarcinoma cell line, HT-29.m3, previously selected for high expression of pIgR/SC (29), was maintained in RPMI 1640 medium supplemented with 50 µg/ml gentamicin, 2 mM L-glutamine, and 10% FCS. Transient transfections were performed with FuGENE 6 reagent (Roche Diagnostic, Indianapolis, IN) as previously described (24). Transfected cells were either left untreated or stimulated with 10 ng/ml recombinant human TNF-{alpha} for 12 or 24 h (as indicated in the figures). A time point of 24 h was initially chosen based on earlier experiments with the endogenous pIgR/SC gene (18). However, to minimize the effect of TNF-{alpha} on the internal control plasmid (pRL-PGK), we later chose 12 h for cytokine incubation, which yielded similar levels of pIgR/SC gene induction as 24 h. The luciferase activity of both the reporter gene (Firefly luciferase) and the internal control plasmid pRL-PGK (Renilla luciferase) was measured in a luminometer (Victor; Wallace, Turku, Finland) with the Dual Luciferase Reporter Assay System (Promega). We tested several Renilla luciferase plasmids and found that all were reduced upon TNF-{alpha} treatment, but pRL-PGK was the least affected (data not shown). To minimize this problem, fold induction of two replicate wells per treatment was calculated directly from the Firefly luciferase signal, which was normalized in the following way: ((lcf-Firefly-A/lcf-Renilla-A) + (lcf-Firefly-B/lcf-Renilla-B))/2 x average of lcf-Renilla (A and B). Data in Figs. 1GoB, 2, and 4, A and B show the mean + SEM of three or more independent experiments. Experimental values with SEMs that did not overlap were regarded to be significantly different.



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  		FIGURE 1. Intron 1 of the human pIgR/SC gene contains a promoter-dependent TNF-{alpha}-responsive region. HT-29.m3 cells were transiently transfected with the indicated luciferase reporter constructs and either left untreated or treated with TNF-{alpha} (10 ng/ml) for 24 h before harvesting and measurement of luciferase activity. Designations and diagrams of the reporter constructs are given on the left, and fold induction after TNF-{alpha} stimulation is on the right. A, Mapping of the TNF-{alpha}-responsive region in intron 1. Indicated on the diagram are the complete intron 1 (5.7 kb), the positions of exon 1 and exon 2, and the restriction enzyme sites used for internal deletions (N, NcoI; X, XhoI; S, SacI; H, HindIII; and A, ApaI). The results show the mean of two independent experiments; similar results were obtained with different incubation periods and concentrations of TNF-{alpha}. B, The 1.3-kb SacI/HindIII fragment from intron 1 (A) was inserted in the forward or reverse orientation (indicated by arrows) upstream of an unrelated basal promoter. p0 is the pGL3-promoter vector containing the minimal SV40 promoter (Promega).

 
Nuclear extract preparation and EMSA

Preparation of nuclear extracts from HT-29.m3 cells was performed essentially as described (30), with the modifications introduced by Schjerven et al. (24). Approximately 5 µg of nuclear proteins was incubated with 32P-end-labeled double-stranded oligonucleotide probe (0.5 pmol/reaction). For the NF-{kappa}B/Rel site in intron 1, the EMSA reactions were performed in buffer containing 0.8 mM EDTA, 70 mM KCl, 0.8 mM DTT, 0.1 µg/µl dI/dC, 0.05% Nonidet P-40, 10 mM Tris, 0.2 mM MgCl2, and 4% glycerol for 30 min at room temperature. For the NF-{kappa}B/Rel site in the upstream promoter region, the EMSA reactions were performed with the NF-{kappa}B/Rel Family Nushift kit (Geneka, Montreal, Canada) according to the manufacturer’s protocol. Bound and free probes were separated by electrophoresis in a 5% polyacrylamide gel (0.25 x Tris/borate/EDTA) at 150 volts for ~1.5 h at room temperature, dried, and visualized on x-ray film overnight. Cold competitors were added in 100-fold excess before addition of the labeled probe when indicated. For supershift experiments, 2 µl of polyclonal Ab (Geneka) to either p65 (RelA), p50, or c-Rel was added to the reaction mixture and incubated at room temperature for 20 min. The labeled probe was added to the reactions and incubated for another 30 min before electrophoresis. The mutated oligonucleotide probes contained the same mutations as used in the plasmid constructions (pSC45–pSC58). The top strands of the oligonucleotide probes used were: NF-{kappa}B/Rel(intron 1), 5'-CTTGCTGGGAAATTCCCCTGCAAC-3'; mutNF-{kappa}B/Rel(intron 1), 5'-CTTGCTGTTCCATTCCCCTGCAAC-3'; NF-{kappa}B/Rel(-450), 5'-gatccGAGGGGATTCCAGAGtcga-3' (18); and mutNF-{kappa}B/Rel(-450), 5'-gatccGAGCCCATTCCAGAGtcga-3' (18).

Computer-assisted analysis of DNA sequences

Analysis of DNA sequences was performed with the Genetics Computer Group package (Genetics Computer Group, Madison, WI) and MatInspector (http://www.gsf.de/cgi-bin/matsearch.pl) (31).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A 748-bp region in intron 1 is required for maximal TNF-{alpha}-mediated pIgR/SC gene induction

To identify DNA elements that mediate TNF-{alpha}-induced transcriptional up-regulation of pIgR/SC, we tested luciferase reporter constructs containing putative regulatory sequences of the human pIgR/SC gene. The largest construct, pSC1, contained ~2.7 kb of upstream promoter sequences, exon 1, and the complete 5.7-kb intron 1; it was fused in frame with the luciferase gene at the ATG start codon in exon 2. A second reporter construct, pSC2, contained the same 2.7-kb upstream sequences, exon 1, and exon 2, but lacked the entire intron 1. Transient transfections of these constructs were performed into HT-29.m3 cells, which were either left untreated or treated with TNF-{alpha} for 24 h. The activity of the intron-containing reporter gene was enhanced ~4-fold after TNF-{alpha} treatment (Fig. 1GoA, pSC1), whereas that of the intron-less construct was enhanced only ~2-fold (Fig. 1GoA, pSC2). Thus, although the pIgR/SC promoter displayed some TNF-{alpha} responsiveness, maximal TNF-{alpha}-induced up-regulation of the reporter construct depended on DNA elements located in intron 1.

To map the TNF-{alpha}-responsive element(s) in intron 1 more closely, we made several constructs with sequential internal deletions. When transiently transfected into HT-29.m3 cells, deletion of bases 847-1855, 1855–4118, 847-4118, or 4866–5163 in the reporter constructs pSC3, pSC4, pSC5, and pSC9, respectively, did not significantly reduce their TNF-{alpha}-mediated induction (Fig. 1GoA). In contrast, deletion from position 3464 to 4866 in intron 1 (pSC8), or deletion of bases 4118–4866 (pSC16), greatly reduced the TNF-{alpha} responsiveness to a level comparable to that of the intron-less reporter construct (pSC2) (Fig. 1GoA). Therefore, the 748-bp fragment between position 4118 and 4866 (present in pSC3–5 and pSC9, but absent in pSC8 and pSC16), contained DNA elements required for full TNF-{alpha} responsiveness.

Full TNF-{alpha} responsiveness requires cooperation between DNA elements in the pIgR/SC promoter and intron 1

To determine whether the identified TNF-{alpha}-responsive region in intron 1 behaved like a general enhancer, we subcloned the 1.3-kb fragment corresponding to the deleted region in pSC8 (Fig. 1GoA) upstream of the minimal SV40 promoter in both orientations. The luciferase activity of these reporter constructs (designated p1 and p2, respectively) was tested as above (Fig. 1GoB). The basal viral promoter (designated p0) was not activated by TNF-{alpha} treatment. Introduction of the 1.3-kb intronic fragment in p1 and p2 resulted in slight activation with <1.5-fold induction after TNF-{alpha} treatment (Fig. 1GoB). Thus, the 1.3-kb intronic fragment only conferred marginal TNF-{alpha} inducibility to a heterologous promoter.

To map putative TNF-{alpha} responsive elements in the pIgR/SC promoter, we deleted the promoter to -537 in the presence or absence of intron 1 (Fig. 2Go, pSC15 and pSC11, respectively). This deletion reduced TNF-{alpha} responsiveness to ~1.2-fold in the absence of intron 1, suggesting the presence of positive regulatory DNA elements in the far upstream promoter. However, we did not observe a decrease in TNF-{alpha} responsiveness when the bases from -2.7 kb to -537 were deleted in the context of the intact intron (Fig. 2Go). Further deletion (in the absence of intron 1) to -177 (pSC27) produced slightly increased responsiveness (from 1.2-fold to 1.6-fold), while deletion to -83 (pSC28) abolished all TNF-{alpha} responsiveness (Fig. 2Go).



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  		FIGURE 2. DNA elements in the promoter region and far downstream in intron 1 of the pIgR/SC gene cooperate to mediate TNF-{alpha} responsiveness. HT-29.m3 cells were transiently transfected with the indicated reporter constructs and treated as described in Fig. 1Go. Indicated on the left diagram are (see key at the bottom) the 1.3-kb SacI/HindIII fragment from intron 1, a consensus NF-{kappa}B/Rel element within this 1.3-kb fragment, the ISRE in exon 1, the putative NF-{kappa}B/Rel element in the upstream promoter, and the length of upstream promoter sequence of the different reporter constructs.

 
To study cooperation between DNA elements in the 1.3-kb intronic fragment and the pIgR/SC promoter, we inserted that fragment upstream of the above-described different lengths of the pIgR/SC promoter (Fig. 2Go). Deleting the entire intron 1 in the context of the -537 pIgR/SC promoter construct reduced TNF-{alpha} responsiveness from 4-fold to 1.2-fold (compare pSC15 with pSC11). However, insertion of the 1.3-kb intronic fragment upstream of the intron-less pIgR/SC promoter restored part of the TNF-{alpha} responsiveness (Fig. 2Go; compare pSC12 with pSC11 and pSC15). Furthermore, insertion of the 1.3-kb intronic fragment upstream of pSC27 and pSC28 similarly partly restored TNF-{alpha} responsiveness (Fig. 2Go, pSC29 and pSC30). The best response was seen in the -177 promoter context, in which upstream insertion of the 1.3-kb intronic fragment resulted in a 2.5- (Fig. 2Go, pSC29) to 3-fold (Fig. 4GoA, pSC29) induction after TNF-{alpha} stimulation.



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  		FIGURE 4. A consensus NF-{kappa}B/Rel site in intron 1 and an ISRE in exon 1 of the human pIgR/SC gene are required for full TNF-{alpha} responsiveness. HT-29.m3 cells were transiently transfected with the indicated reporter constructs (left), and treated and examined (right) as described in Fig. 1Go, except that the cells were stimulated with TNF-{alpha} for 12 h. The consensus NF-{kappa}B/Rel site in intron 1, the ISRE in exon 1, and the putative NF-{kappa}B/Rel site in the upstream promoter are indicated in the left diagrams of the reporter constructs (see key at the bottom). A, The reporter constructs contain human pIgR/SC promoter sequence from -177 with the 1.3-kb intronic fragment inserted upstream of the promoter (where indicated). B, Reporter constructs containing the human pIgR/SC promoter sequence from -2.7 kb to +5.9 kb in exon 2, with the indicated mutations of specific putative regulatory DNA elements.

 
A consensus NF-{kappa}B/Rel element in intron 1 is required for full TNF-{alpha} responsiveness of the pIgR/SC gene

Within the 1.3-kb intronic fragment, we identified a consensus NF-{kappa}B/Rel site (31). This was also found to be conserved in intron 1 of the mouse pIgR/SC gene (Fig. 3GoA). To investigate its putative functional role, we made a 4-bp point mutation that destroyed this site in the context of both pSC29 and pSC1 (Fig. 3GoA; mutated nucleotides are underlined). These two new reporter constructs (pSC51 and pSC53) were transiently transfected into HT-29.m3 cells, stimulated with TNF-{alpha} for 12 h, and analyzed as above. Mutation of the NF-{kappa}B/Rel site resulted in a distinct reduction of TNF-{alpha} responsiveness, i.e., from 3- to 1.7-fold in the context of pSC29 (Fig. 4GoA, pSC51), and from 4- to <2-fold in the context of pSC1 (Fig. 4GoB, pSC53). Thus, this novel NF-{kappa}B/Rel site in intron 1 was critical for the TNF-{alpha} responsiveness of the pIgR/SC gene.



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  		FIGURE 3. Sequence homology for two putative TNF-{alpha}-responsive regulatory DNA elements in the pIgR/SC gene. The consensus DNA elements are boxed and conserved nucleotides are indicated with dashes. The mutated nucleotides in the reporter constructs used in Fig. 4Go are underlined. A, NF-{kappa}B/Rel element in intron 1. The human sequence is from nucleotides 4391 to 4411 and the murine sequence is from nucleotides 5923 to 5942 (relative to start of exon 1). GenBank accession numbers: human, AJ276452 and murine, AB001489. B, ISRE in exon 1. All sequences start from position +1. Two transcriptional start sites have been suggested both for the human (28 32 ) and the murine (33 34 ) sequences (indicated with the first one in parentheses). GenBank accession numbers: human, X95880 (35 ) and Y08254 (32 ); murine, U83426 (33 ) and AB001489 (34 ); and rat, AF039920 (36 ).

 
An ISRE in exon 1 is required for full TNF-{alpha} responsiveness of the pIgR/SC gene

Next, we investigated the role of two previously reported putative TNF-{alpha}-responsive elements: an ISRE in exon 1 (26, 27) and an NF-{kappa}B/Rel site centered around position -450 in the upstream promoter (18, 25). The former is 100% conserved between human, rat, and murine sequence, although the position relative to the transcriptional start site differs somewhat (Fig. 3GoB and Ref. 27). In contrast, the putative NF-{kappa}B/Rel site in the upstream promoter of the human pIgR/SC gene was not found in the murine or rat upstream promoter. Computer-assisted methods revealed high homology between the murine and rat pIgR/SC promoter sequences. Comparing the murine and rat sequences with the human one, we found that they were homologous to nucleotide position -206/207 in the human promoter (except for one 18-bp gap centered around position -96). However, upstream of this position the rat and murine sequences diverged from the human one, and an NF-{kappa}B/Rel site homologous to the human site around nucleotide -450 could not be found.

To investigate the functional importance of the exon 1 ISRE and the promoter NF-{kappa}B/Rel element, we introduced specific point mutations destroying these two sites. The mutations were introduced in the context of pSC27 and pSC29 (for mutation of ISRE) and in pSC1 (for mutations of both ISRE and the promoter NF-{kappa}B/Rel site), either alone or in combination with each other and the above-described mutation of the intronic NF-{kappa}B/Rel site. These new reporter constructs were then transiently transfected into HT-29.m3 cells, stimulated with TNF-{alpha} for 12 h, and analyzed as above. Mutation of the ISRE in pSC27 and pSC29 (Fig. 3GoB; mutated nucleotides are underlined) both reduced TNF-{alpha} responsiveness significantly, from ~1.5-fold induction to nearly none (1.2-fold) in the context of pSC27, and from 3- to 2-fold in the context of pSC29 (Fig. 4GoA, pSC45 and pSC46). By contrast, mutation of the exon 1 ISRE in the context of the full-length wild-type construct (pSC1) did not significantly reduce the TNF-{alpha} responsiveness (Fig. 4GoB, pSC47). However, in the context of a mutated intronic NF-{kappa}B/Rel site, mutation of the exon 1 ISRE further reduced the TNF-{alpha} responsiveness of the reporter gene significantly (Fig. 4GoB, pSC54-pSC58). Mutation of the NF-{kappa}B/Rel site in the promoter did not reduce TNF-{alpha} responsiveness in any context (Fig. 4GoB, pSC55). By contrast, this mutation alone in the pSC1 context actually increased the TNF-{alpha} responsiveness, from ~4-fold for pSC1 to >5-fold for pSC55 (Fig. 4GoB). This accorded with the fact that deleting promoter sequences from bases -537 to -178 (including the NF-{kappa}B/Rel site at -450) did not reduce TNF-{alpha} responsiveness but rather enhanced it (Fig. 2Go; compare pSC11 with pSC27 and pSC12 with pSC29).

TNF-{alpha} stimulation induces preferential binding of NF-{kappa}B-p65 (RelA) to the intronic NF-{kappa}B/Rel site

To determine whether TNF-{alpha} stimulation induced binding of nuclear factors to the identified NF-{kappa}B/Rel site in intron 1, we isolated nuclear extracts from HT-29.m3 cells treated with TNF-{alpha} for various time periods and performed in vitro EMSA experiments with a probe spanning the intronic NF-{kappa}B/Rel site (Fig. 5Go). We found that TNF-{alpha} induced the formation of three protein-DNA complexes with markedly different kinetics. The fastest migrating complex, designated complex I, was barely detectable after 30 min of TNF-{alpha} stimulation but slowly increased its binding intensity throughout the test period of 24 h (Fig. 5GoA). Complex II was induced within 10 min of TNF-{alpha} stimulation and showed relatively constant intensity for the time points investigated. The lowest mobility complex (complex III) was induced early, appearing after as little as 10 min and remaining up to 2 h, but then disappearing after 6 h of TNF-{alpha} stimulation (Fig. 5GoA). Competition experiments demonstrated that all three complexes were specific, as they were competed by an excess of wild-type oligonucleotide, while an oligonucleotide with a 4-bp mutation of the NF-{kappa}B/Rel site (identical with the mutation introduced in the reporter constructs) could not compete for binding to any of these complexes (Fig. 5GoA). To identify the NF-{kappa}B/Rel subunits in the three complexes, we incubated the nuclear extracts from 30 min (Fig. 5GoB) and 24 h of TNF-{alpha} stimulation (Fig. 5GoC) with polyclonal Ab against the NF-{kappa}B/Rel subunits p65 (RelA), c-Rel, and p50. Polyclonal Ab against p65 further shifted complexes II and III, causing the appearance of a supershifted complex (Fig. 5Go, B and C, complex V, lane 4). Ab against c-Rel did not react with any of the three induced complexes, while Ab against p50 led to a shift of complex I and the appearance of the supershifted complex IV (Fig. 5Go, B and C, lane 9). Specificity of the latter supershifted complex (IV) was demonstrated by incubating with a p50 peptide epitope that competed effectively (Fig. 5Go, B and C, lane 10).



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  		FIGURE 5. TNF-{alpha} treatment induces binding of both NF-{kappa}B-p65 (RelA) and NF-{kappa}B-p50 to the TNF-{alpha}-responsive DNA element in intron 1 of the human pIgR/SC gene. EMSA experiments were performed with nuclear extracts from HT-29.m3 cells stimulated with TNF-{alpha} for the indicated time periods. A 24-bp fragment, spanning the NF-{kappa}B/Rel site from pIgR/SC intron 1, was used as a labeled probe, and a 100-fold molar excess of unlabeled wild-type (+) or mutated (-) oligonucleotide (cc) or epitope-specific peptides (pept) were added as indicated. A, Nuclear extracts from HT-29.m3 cells treated with TNF-{alpha} for different time periods (10 min to 24 h) induced three complexes (I, II, and III) indicated with arrows. B, Supershift experiments with Ab against p65, cRel, or p50, and nuclear extracts from 30 min of TNF-{alpha} stimulation, revealed that complexes II and III contained the p65 subunit (see complex V, indicated with an arrow) and that complex I (lane 2) contained the p50 subunit (see complex IV, lane 9, indicated with an arrow). C, Supershift experiments with nuclear extracts after 24-h TNF-{alpha} stimulation confirmed that complex II contained the p65 subunit (see complex V, indicated with arrow) and that complex I contained the p50 subunit (see complex IV, indicated with arrow).

 
TNF-{alpha} stimulation induces strong binding of both p50- and p65-containing complexes to the promoter NF-{kappa}B/Rel site

Previous studies have demonstrated binding of nuclear factors to the NF-{kappa}B/Rel site in the pIgR/SC promoter (18, 25). Therefore, we next investigated the nature of factors binding to this site. Similar to the intronic NF-{kappa}B/Rel element, we found that TNF-{alpha} specifically induced (with different kinetics) the formation of three protein-DNA complexes (Fig. 6GoA). However, the preference for binding to this site differed from the intronic NF-{kappa}B/Rel site. The highest mobility complex (complex I) was already significantly induced after 30 min and demonstrated increased binding intensity up to 24 h, reaching intensity comparable to that of complex II after as little as 6 h. Supershift experiments demonstrated that this complex (I) contained p50 but not p65 or c-Rel (Fig. 6GoB). As for the intronic element, complex II was rapidly induced and showed a relatively constant binding intensity throughout the time points investigated. Finally, complex III produced a weak band visible only up to 2 h (Fig. 6GoA). As for the intronic site, incubation with Ab to p65, but not the other Abs, resulted in a supershift of complexes II and III (Fig. 6GoB).



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  		FIGURE 6. The NF-{kappa}B/Rel element in the upstream promoter of the pIgR/SC gene binds both NF-{kappa}B-p65 (RelA) and NF-{kappa}B-p50 after TNF-{alpha} stimulation, but displays earlier and stronger p50-containing complexes than the intronic NF-{kappa}B/Rel element (compare Fig. 5Go). EMSA experiments were performed as described in Fig. 5Go, except that a 24-bp oligonucleotide containing the putative NF-{kappa}B/Rel site from position -450 in the pIgR/SC promoter was used as a labeled probe. A, TNF-{alpha} treatment for different time periods (10 min to 24 h) induced three complexes (I, II, and III), indicated with arrows. B, Supershift experiments with nuclear extracts after 30-min TNF-{alpha} stimulation and Ab against p65 led to the disappearance of both complexes II and III and to the appearance of complex V (indicated with arrow). Adding Ab against p50 revealed that complex I contained the p50 subunit (see complex IV, indicated with arrow).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study identified an NF-{kappa}B/Rel site in intron 1 of the human pIgR/SC gene, with a key role in its TNF-{alpha} responsiveness. This site preferentially bound p65/RelA-containing complexes after TNF-{alpha} stimulation, was located within a 748-bp TNF-{alpha}-responsive enhancer in intron 1, and required cooperation with DNA elements in the pIgR/SC promoter to confer TNF-{alpha} responsiveness. We further investigated the role of an ISRE in exon 1 and an NF-{kappa}B/Rel site in the upstream promoter; the former was shown to be involved in the TNF-{alpha}-mediated up-regulation of pIgR/SC, while the latter did not appear to contribute positively to the response.

Mapping of the TNF-{alpha} responsiveness of the human pIgR/SC gene: cooperation between intronic DNA elements and the promoter

We have identified a TNF-{alpha}-responsive enhancer within a 748-bp fragment in intron 1, containing several stretches of high homology between the murine and the human pIgR/SC gene sequences. Within this region we identified a 100% conserved consensus NF-{kappa}B/Rel site, and mutation of this element led to a dramatic reduction of TNF-{alpha} responsiveness. The intronic enhancer was shown to be promoter dependent but not position dependent, suggesting that intronic DNA elements cooperate with pIgR/SC promoter elements. Also, the promoter itself showed some degree of TNF-{alpha} responsiveness when containing a sufficient stretch of the upstream sequence (from -177 or longer). It is possible that SV40-derived DNA elements in our reporter gene constructs could support the weak induction seen by the pIgR/SC promoter or intronic enhancer when either one is operating on its own. However, these elements did not by themselves confer any TNF-{alpha} responsiveness, as demonstrated by the unresponsiveness of the SV40 promoter alone (Fig. 1GoB, p0) or the SV40 enhancer in the context of the -83 pIgR/SC promoter (Fig. 2Go, pSC28). Exon 1 of the pIgR/SC gene contains an ISRE involved in its IFN-{gamma} responsiveness (32), which has also been implicated in its TNF-{alpha} responsiveness (26, 27). This exon 1 ISRE is 100% conserved among human, rat, and mouse (Fig. 3GoB), and we investigated its role by mutational analysis. Mutation of this site in different reporter gene constructs reduced the response to TNF-{alpha} by 14–35%, clearly demonstrating its functional role.

Piskurich et al. (32) reported that treatment with IFN-{gamma} induced binding of IFN regulatory factor (IRF)-1 to the exon 1 ISRE, and we have previously shown that TNF-{alpha} was able to induce the same set of complexes as IFN-{gamma} (although somewhat weaker) with this probe (18). Both IFN-{gamma} and TNF-{alpha} have been demonstrated to induce production of IRF-1 in HT-29 cells (26, 27, 32), suggesting that this transcription factor participates in TNF-{alpha}-mediated transcriptional activation of the pIgR/SC gene via binding to the exon 1 ISRE. The idea that NF-{kappa}B might synergize with a member of the IRF family to regulate pIgR/SC expression is supported by the documented cooperation between these two transcription factor families in the regulation of the IFN-{beta} gene (37, 38).

Possible role of other transcription factor binding sites

Several lines of evidence have suggested that there are other DNA elements in the pIgR/SC gene that contribute to the TNF-{alpha}-mediated induction. Deletion of upstream promoter sequences from ~-2.7 kb down to -537 reduced TNF-{alpha} responsiveness of the pIgR/SC promoter (without intron 1) from 2- to 1.2-fold, indicating the presence of positive regulatory elements in the far upstream promoter. However, the significance of these sequences appeared to be context dependent, because their deletion in the construct containing intron 1 did not reduce its TNF-{alpha} responsiveness. This disparity suggested redundancy among DNA elements in the far upstream promoter and intron 1. However, it is known that transcription of a gene from a plasmid can be more permissive than for the same gene packed in the genome (39). Therefore, an apparent redundancy of DNA elements observed in a transient reporter gene system should be interpreted with caution; all the actual DNA elements might be required for the regulation of the endogenous gene (39). Also, deletion of nucleotides from -537 to -177 in the promoter led to increased TNF-{alpha} responsiveness, which suggested the presence of negative regulatory DNA elements in this region.

Interestingly, deletion of the pIgR/SC promoter sequence from position -177 down to -83 abolished all TNF-{alpha} responsiveness of the promoter alone and strongly reduced the responsiveness of the promoter together with the intronic 1.3-kb fragment. This finding strongly suggested that the exon 1 ISRE required cooperation with DNA elements within this promoter region. Piskurich et al. (32) reported the presence of two other putative ISREs in the promoter region, centered around position -132 and -99, respectively, which bound constitutively expressed nuclear factors. Based on deletional analysis, they suggested the involvement of these two ISREs both in the IFN-{gamma}- (32) and TNF-{alpha}-mediated (26) up-regulation of human pIgR/SC. However, the direct involvement of either of these two ISREs has not yet been demonstrated by mutational analysis.

Finally, by comparing the TNF-{alpha} responsiveness of the two reporter constructs pSC15 (containing the entire intron 1) and pSC12 (containing the 1.3-kb intronic fragment inserted upstream of the promoter), we demonstrated that the 1.3-kb intronic fragment could not completely compensate for the deletion of intron 1. The reason might be additional regulatory DNA elements elsewhere in intron 1, although we could not exclude the possibility that the reduced TNF-{alpha} responsiveness was due to positional effects of the intronic enhancer relative to the promoter. Taken together, as-yet-unidentified DNA elements are probably involved in TNF-{alpha}-mediated up-regulation of human pIgR/SC expression.

Role of NF-{kappa}B/Rel in TNF-{alpha}-mediated induction of the human pIgR/SC gene

The transcription factor family NF-{kappa}B/Rel is known to be activated by several extracellular signals, including TNF-{alpha} (10, 11, 12). This family consists of five transcription factors: p65 (RelA), RelB, c-Rel, p50, and p52. The first three contain a trans-activating domain and can thus activate transcription, whereas the latter two (derived from the precursors p105 and p100, respectively) lack this domain and can (as homodimers) act as repressors (10, 12). The NF-{kappa}B/Rel proteins recognize a common 10-bp consensus DNA element, but the different homo- and heterodimeric complexes display some degree of preference in their binding specificities to different variations of the NF-{kappa}B/Rel-site (12, 31).

In this work, we studied the time-dependent activation of different family members of NF-{kappa}B/Rel in HT-29 cells and their binding to the intronic NF-{kappa}B/Rel site and a putative comparable site in the promoter. We found that TNF-{alpha} produced immediate activation of p65 (RelA), persisting up to 24 h (the longest time point investigated). This accorded with a previous study in which activated p65 was found in the epithelium from inflamed but not uninflamed human gut mucosa (15). In addition, we observed a more delayed and gradually increasing activation of p50, which could reflect an overall down-regulation of TNF-{alpha} responsiveness by the inhibitory subunit p50. Interestingly, there seemed to be no binding of the classical p65/p50 heterodimer to either of the two probes, because the p65-containing complexes exclusively bound Ab to p65, while the p50-containing complex exclusively bound the Ab to p50. This suggested that the shifted bands predominantly consisted of either p65 or p50 homodimers, but we could not exclude the possibility of heterodimerization with other partners (i.e., p52 or RelB). Indeed, TNF-{alpha} induced two differently migrating p65-containing complexes with both NF-{kappa}B/Rel probes, suggesting that p65 could interact with other proteins.

Despite similarities in protein binding to the two investigated NF-{kappa}B/Rel sites, we observed one major interesting difference: the novel functional NF-{kappa}B/Rel site in intron 1 bound preferentially p65, which accorded with the activating function of this DNA element. However, the same site could also bind p50-containing complexes with increasing (although weak) intensity over time, suggesting a requirement for higher concentrations of activated p50 to compete with p65 for binding to this DNA element. In the endogenous gene, factors binding to the surrounding sequences might stabilize the p65-containing complexes and thus prevent access of p50-containing complexes to this intronic NF-{kappa}B/Rel site, even when active p50 becomes abundant.

Previous studies have demonstrated TNF-{alpha}-induced binding of nuclear factors to an NF-{kappa}B/Rel site in the upstream promoter (18, 25). We found that this DNA element had no positive, but rather a possible negative, regulatory effect in the context of our reporter genes. A similar NF-{kappa}B/Rel site is not conserved in the rat and mouse promoter sequences, because only some 200 initial nucleotides of the pIgR/SC promoter show homology among all three species (data not shown). This element appeared to have a higher affinity for p50 than the intronic NF-{kappa}B/Rel site, judged from the ratio of the EMSA band intensities between the p50- and p65-containing complexes. This apparent preference for p50 homodimers supported the possibility of a negative role of this DNA element in TNF-{alpha}-mediated induction of the pIgR/SC gene.

Cytokine-mediated up-regulation of pIgR/SC expression

We identified a novel regulatory 748-bp fragment in intron 1, located 4 kb downstream of the transcriptional start site in the human pIgR/SC gene. Within this region, which contains a cluster of DNA elements important for both TNF-{alpha} and IL-4 (24) responsiveness, there is high homology between the murine and the human sequence (Fig. 3GoA, Ref. 24 , and data not shown), including 100% conservation of several functional cytokine-responsive regulatory DNA elements demonstrated in the human gene. Two of these elements have been identified as an NF-{kappa}B/Rel site (Figs. 3GoA and 5) and a STAT6 site (24), respectively, while the identity of the factors binding to the other DNA elements remains unknown (data not shown). NF-{kappa}B/Rel is a key mediator of many different extracellular stimuli such as IL-1, TNF-{alpha}, LPS, Toll-like receptor ligands, and oxidative stress (10, 11, 12). Likewise, the transcription factor STAT6 can be activated both by IL-4 and IL-13. In addition, the presence of other homologous sequences between the two species, to which we as yet have not assigned any functional importance, allows for the possibility that this enhancer region within intron 1 of the pIgR/SC gene contains regulatory DNA elements responsive also to other stimuli, e.g., other cytokines, hormones, or bacterial products. Interestingly, it was recently demonstrated that intestinal pIgR/SC mRNA was up-regulated when germ-free mice were colonized with Bacteroides thetaiotaomicron, a prominent component of the normal intestinal microflora (40).

Transcriptional activation of the human pIgR/SC gene in response to both TNF-{alpha} and IL-4 is slow and depends on novel protein synthesis. Yet the transcriptional mechanisms for both of these two cytokine responses use direct binding of rapidly activated latent transcription factors, NF-{kappa}B/Rel for TNF-{alpha} (this work) and STAT6 for IL-4 (24). However, it was recently shown that NF-{kappa}B-p65 bound with different kinetics to distinct promoters in LPS-stimulated macrophages; promoters with hypoacetylated histone H4 required histone acetylation before NF-{kappa}B binding (41). It is possible that a similar mechanism delays the binding of STAT6 and NF-{kappa}B to their respective sites in intron 1 of the pIgR/SC gene. Furthermore, incomplete and delayed degradation of the NF-{kappa}B inhibitor, I{kappa}B, in intestinal epithelial cells might also contribute to the delayed transcriptional response to TNF-{alpha} stimulation (11). The requirement for de novo protein synthesis for the TNF-{alpha} responsiveness might be accounted for by the transcription factor IRF-1, because it is synthesized via the NF-{kappa}B/Rel-pathway (12). The de novo-synthesized factor required for the effect of IL-4 remains unknown (24).

Despite the similarities between the TNF-{alpha}- and the IL-4-responsive enhancers, there is one important difference. For the latter, all the required DNA elements seemed to be contained within a 300-bp fragment in this common enhancer region, thus constituting a promoter-independent general IL-4-responsive enhancer (24). Conversely, the DNA element(s) used for TNF-{alpha} response within the intron 1 enhancer region required cooperation with promoter elements located >4 kb upstream. Nevertheless, such spatial separation was not essential for this cooperation, because the 1.3-kb intronic fragment also conferred TNF-{alpha} responsiveness when located upstream of different lengths of the pIgR/SC promoter.

In this study, we demonstrated that a consensus NF-{kappa}B/Rel binding site within intron 1 of the human pIgR/SC gene is necessary for its full TNF-{alpha}-mediated induction. Interestingly, this DNA element depended on cooperation with promoter-specific DNA elements, including an ISRE in exon 1 and possibly other DNA elements located both elsewhere in intron 1 and upstream in the promoter, thus suggesting a complex cooperation among various transcription factors. The proximity of this novel NF-{kappa}B/Rel binding site to a STAT6 binding site essential for IL-4 induction of pIgR/SC transcription (24) suggests the presence of an enhancer region in intron 1 important for interpretation of signals from distinct signal transduction pathways. Further studies are needed to delineate other functional elements of this regulatory region and to determine whether input from distinct signaling pathways might be integrated at the DNA level at this site.


   Acknowledgments
 
We thank the technical staff at the Laboratory for Immunohistochemistry and Immunopathology, Institute of Pathology (University of Oslo, Oslo, Norway), for excellent laboratory assistance.


   Footnotes
 
1 This work was supported by the Norwegian Cancer Society, the Research Council of Norway, and Anders Jahre’s Fund. Back

2 Address correspondence and reprint requests to Hilde Schjerven, Laboratory for Immunohistochemistry and Immunopathology, Institute of Pathology, Rikshospitalet, N-0027 Oslo, Norway. E-mail address: hilde.schjerven{at}labmed.uio.no Back

3 Abbreviations used in this paper: SIg, secretory Ig; IRF, IFN regulatory factor; ISRE, IFN-stimulated response element; pIg, polymeric Ig; SC, secretory component. Back

Received for publication June 29, 2001. Accepted for publication October 1, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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