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The Journal of Immunology, 2001, 166: 6149-6155.
Copyright © 2001 by The American Association of Immunologists

Transcriptional Regulation of Intracellular IL-1 Receptor Antagonist Gene by IL-1{alpha} in Primary Mouse Keratinocytes

Eunhye La and Susan M. Fischer1

Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX 78957


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The inflammatory cytokine IL-1{alpha} mediates inflammatory reactions in skin and up-regulates the expression of other proinflammatory genes. We previously found that IL-1{alpha} also increases steady state mRNA levels for intracellular IL-1 receptor antagonist (icIL-1Ra) in primary mouse keratinocytes; however, the mechanism for this was unknown. Here we show that increased expression in primary keratinocytes is due to increased rates of transcription. To study the transcriptional regulation of icIL-1Ra expression induced by IL-1{alpha}, we functionally characterized 4.5 kb of the 5'-flanking region of the human icIL-1Ra gene. Deletion analysis showed that regulatory elements were contained in the -598- and -288-bp region upstream of the transcription start site. Then we investigated cis- and trans-acting factors required for icIL-1Ra expression and found that a NF-IL-6 site and a NF-{kappa}B site in the icIL-1Ra promoter were responsible for IL-1{alpha}-induced icIL-1Ra expression. Moreover, gel shift assays and cotransfection experiments showed that CCAAT/enhancer-binding proteins {alpha}, {beta}, and p65 bind to the NF-IL-6 site and NF-{kappa}B site, respectively, and functionally trans-activate the icIL-1Ra promoter. Finally, mutational analysis confirmed that these elements were both essential for maximal transcription induced by IL-1{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
When skin comes in contact with irritants, epidermal keratinocytes can act as initiators of subsequent inflammation and hyperplasia. IL-1{alpha} is a cytokine that mediates much of the inflammatory reaction in skin. The activity of IL-1{alpha}, however, is dependent on the other IL family members, including IL-1R types I and II (IL-1R1 and IL-1R2), IL-1R antagonist (IL-1Ra),2 and an accessory protein (AcP) (1). Whereas human keratinocytes express both isoforms of IL-1, IL-1{alpha} and IL-1{beta}, murine keratinocytes express only IL-1{alpha} (2). Of the two receptors, only IL-1R1 is thought to be capable of transducing a signal (3). An important component of the IL-1 system is the endogenous IL-1Ra, which competes with IL-1 for binding to its receptors, and abrogates its signaling. We have previously demonstrated IL-1 itself up-regulates IL-1Ra, suggesting the existence of a negative feedback mechanism (4). The question addressed in this study is how IL-1{alpha} alters the steady state levels of IL-1Ra mRNA.

We previously showed that IL-1{alpha} induced the intracellular form of the receptor antagonist (icIL-1Ra) mRNA in a time- and dose-dependent manner in keratinocytes (4), in agreement with the work on the secretory form (sIL-1Ra) in other cell types, including monocytes (5), hepatocytes (6), and fibroblasts (7). The two isoforms of IL-1Ra are produced by alternative splicing of two distinctive first exons. The first exon for icIL-1Ra is located 9.6 kb upstream of the first exon for sIL-1Ra (8) and is spliced at an internal site with the sequences encoding the sIL-1Ra signal peptide. Thus, unlike sIL-1Ra, icIL-1Ra is controlled by the activity of its own distinct promoter region, which imparts both stimulus and cell type-restricted expression (9). The sIL-1Ra protein is produced by monocytes, macrophages, neutrophils, hepatocytes, and fibroblasts, whereas icIL-1Ra is produced primarily by keratinocytes and other epithelial cells (10).

Studies on the transcriptional regulation of icIL-1Ra have been limited but have shown marked cell type specificity (9). Several cis-acting DNA-regulatory elements and corresponding nuclear proteins have been identified. These include an upstream induction sequence, which binds the transcription factor NF-IL-6, cAMP response element-binding protein, NF-1, NF-{kappa}B, and AP-1 (9). Little is known, however, about how this gene is controlled in keratinocytes, particularly in response to IL-1{alpha}.

Several transcription factors have been shown to be IL-1 inducible, including NF-{kappa}B, NF-IL-6, AP-1 Egr-1, NAK-1, and Myc (11). IL-1 has been shown to activate NF-{kappa}B and CCAAT/enhancer-binding proteins (C/EBPs) in numerous cell types (12, 13). NF-{kappa}B is an inducible enhancer of many inflammatory genes, and its DNA binding activity consists of homo- and heterodimers of Rel proteins, such as RelA (p65), RelB, cRel, NF-{kappa}B1 (p105/p50), and NF-{kappa}B2 (p100/p52) (14). C/EBP transcription factors comprise a family of related basic region leucine zipper DNA-binding proteins that regulate transcription, including C/EBP{alpha}, C/EBP{beta}, C/EBP{delta}, C/EBP{gamma}, GADD153 (C/EBP-homologue protein-10 (CHOP-10)), and liver-enriched transcriptional activating protein (LAP). C/EBP factors have been shown to differentially modulate transcription and differentiation in several cell types including adipocytes and myelomonocytic cells (15, 16, 17).

In this report, we show that IL-1{alpha} regulates its receptor antagonist primarily at the level of increased transcription by enhanced binding of C/EBP and NF-{kappa}B proteins to their consensus sequences in the 5'-flanking region.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture of primary keratinocytes

Mouse epidermal keratinocytes were isolated from newborn mice (1–2 days old) by trypsinization overnight at 4°C, using a modification of a described method (18). Cells were plated in enriched Waymouth’s medium containing 10% FCS and allowed to attach to the plate for 2 h. The plating medium was changed to enriched MCDB 151 containing 0.1% BSA but no serum and is referred to as SPRD-111 (0.04 mM Ca2+) (19). After plating, the cultures were incubated at 37°C in 5% CO2 for 2 days.

Northern analysis

Primary keratinocytes were cultured for 2 days and treated with actinomycin D (5 µg/ml) and IL-1{alpha} (300 U/ml) for various time periods. Total RNA was isolated using TriReagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s protocol. Twenty micrograms of RNA were separated on a formaldehyde-containing 1% agarose gel, transferred onto nylon membranes (Micron Separations, Westboro, MA), and UV cross-linked to the membrane using a Stratalinker (Stratagene, La Jolla, CA). cDNA for the C/EBPs and GAPDH were radiolabeled with [{alpha}-32P]dCTP using the Decaprime II random prime kit (Ambion, Austin, TX). The blot was prehybridized (5x SSC phosphate/EDTA (SSPE), 5x Denhardt’s solution, 150 µg/ml denatured salmon sperm DNA, 0.5% SDS, and 10% dextran sulfate) for 3 h and hybridized for 20 h at 68°C. The blot was washed in 2x SSPE, 0.1% SDS for 15 min, twice at 42°C, then twice in 0.1x SSPE, 0.1% SDS for 30 min at 68°C. Specific bands were detected by autoradiography. The autoradiographs were scanned by densitometry, and the results were integrated and normalized to GAPDH cDNA.

Plasmids

The luciferase reporter vector (pIC4555.Luc) containing the promoter region of the human icIL-1Ra gene (-4555/+38 from the transcription initiation site) was kindly provided by Cem Gabay (University of Colorado Health Science Center, Denver, CO) and was used as a template plasmid. By using the unique restriction sites within the promoter, a series of 5'-deletion and internal deletion constructs were made (9). The icIL-1Ra promoter insert was cut out of the template plasmid with HindIII digestion. The promoter was gel purified using Qiagen Kit II (Qiagen, Chatsworth, CA) and then cut at -1423, -909, -598, 288, and -156 bp by NcoI, SmaI, StuI, NdeI, and NheI, respectively. The -49 to +38 construct was made by PCR using a 3'-primer corresponding to bases +19 to +38 containing a HindIII site distal to +38; the 5'-primer corresponded to bases -49 to -31 containing a HindIII site at the 5'-end (9). The digested inserts were ligated back into the reporter vector. CHOP-10 expression vector was gift from David Ron (New York University, New York, NY) and plasmids expressing C/EBP{alpha}, -{beta}, and -{delta} were provided by Steven McKnight (Tularik, San Francisco, CA). The I{kappa}B mutant expression vector was a gift from G. Tim Bowden (University of Arizona, Tucson, AZ). Plasmids expressing p65 and p50 were provided by Irina Budunova (University of Texas M. D. Anderson Cancer Center, Smithville, TX).

Transient transfection

Cells were plated in 35-mm dishes 40 h before transfection. Luciferase reporter vector (8 µg) and pCMV-{beta}-gal vector (0.5 µg) as an internal control per dish were transfected into cells when they were ~80% confluent. The luciferase vector was complexed with 10 µl lipofectin (Life Technologies, Gaithersburg, MD) and transfected into the cells following the manufacturer’s protocol. The amount of DNA per dish was made constant in cotransfection experiments by adding pA3.Luc, the promoterless luciferase vector. After 4 h transfection, cells were washed with PBS twice and incubated in EMEM for 40 h. Cells were then stimulated with 300 U/ml IL-1{alpha} for an additional 18 h. Proteins were extracted according to the manufacturer’s protocol (Tropix, Bedford, MA). Luciferase and {beta}-galactosidase activities were measured with a luminometer from Tropix. Promoter activity was normalized by {beta}-galactosidase activity.

Preparation of nuclear extracts

Nuclear extracts were prepared as described previously (20) with the following modification. Briefly, cells were incubated in serum-free medium for 24 h and treated with 300 U/ml IL-1{alpha} for an additional 3 h. Washed cells were scraped and pelleted with microcentrifugation and incubated in 2 packed cell volumes of buffer A (10 mM HEPES (pH 8.0), 0.5% Nonidet P-40, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 200 mM sucrose) for 5 min at 4°C. The crude nuclei were collected by microcentrifugation, rinsed, resuspended in 1 packed cell volume of buffer B (20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, and 1.0 mM DTT) and incubated on a rocking platform for 30 min at 4°C. Nuclei were clarified by microcentrifugation for 5 min, and the supernatants were diluted 1/1 with buffer C (20 mM HEPES (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT). Protease inhibitors (1 mM PMSF, 50 µg of both aprotinin and leupeptin/ml) and phosphatase inhibitors (10 mM NaF, 10 mM {beta}-glycerophosphate, 0.1 mM sodium orthovanadate, and 1 mM EGTA/ml) were added to each type of buffer. Nuclear extracts were kept at -80°C until used.

EMSA

Synthetic oligonucleotides (IDT, Santa Clara, CA) or restriction fragments containing the appropriate promoter region of the icIL-1Ra gene were end-labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase (Promega, Madison, WI). Assays were performed by incubating 2 µg nuclear extracts in the binding buffer (4 mM Tris-HCl, 12 mM HEPES-KOH (pH 7.9), 150 mM KCl, 12% glycerol, 0.5 mM EDTA, and 1 µM DTT) containing 1 µg poly(dI-dC) and 20,000 cpm labeled probe for 25 min at room temperature. To assure the specific binding of transcription factors to the probe, the probe was chased by a 50-fold molar excess of cold wild-type or mutant oligonucleotide. For the supershift experiments, Abs (Santa Cruz Biotechnology, Santa Cruz, CA) were incubated with nuclear extracts on ice for 30 min before addition to the binding reaction. Samples were then electrophoresed on 5% nondenaturing polyacrylamide gels (0.5 x TBE as running buffer), and the gels were dried and subjected to autoradiography.

Site-directed mutagenesis

The NF-IL-6 and NF-{kappa}B sites were mutated on the pIC4555.Luc by PCR-based site-directed mutagenesis (21). Complementary overlapping oligonucleotides containing specific mutations were generated as follows. The NF-{kappa}B mutation site was incorporated into primers spanning -442 to -422; the NF-IL-6 mutation site was incorporated into a primer spanning -430 to -408. The NF-IL-6 mutant was made by a TTGCGCAA to GACTAGTC mutation, and NF-{kappa}B mutant was made by a G to C point mutation. For each construct, two separate PCR were conducted with either the -713 to -697 upstream primer or the -157 to -140 upstream primer. The products were separated on 0.5% agarose gel and purified with a Qiagen kit. The products were mixed, denatured, and allowed to reanneal. Amplification of the heteroduplex with overlapping 3'-ends was conducted by 3'-extension in the absence of specific primers followed by amplification using outside primers (-713 to -697 primer and -157 to -140 primer) in a second round of PCR. Final PCR products were digested with NheI and HindIII, gel purified, and ligated into the StuI and NheI site of pIC4525.Luc. Oligonucleotides used for mutagenesis are shown in Fig. 1Go. Site-specific mutations were confirmed by DNA sequencing.



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  		FIGURE 1. Sequences of the oligonucleotides used in EMSA and site-directed mutagenesis. Sequences of NF-IL-6, NF-{kappa}B, and NF-1 of the human icIL-1Ra promoter are shown as bold letters, and mutated sequences are shown as lower case letters.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-1Ra expression is transcriptionally regulated by IL-1{alpha} in mouse primary keratinocytes

IL-1{alpha} regulates many genes both transcriptionally and posttranscriptionally. The activity of the icIL-1Ra promoter was assessed by transfecting primary keratinocytes with a luciferase-reporter vector. Stimulation of cultures with IL-1{alpha} treatment increased luciferase activity 2.5-fold (Fig. 2Go), indicating that icIL-1Ra expression is transcriptionally regulated by IL-1{alpha} in primary keratinocytes. To assess the possible involvement of changes in mRNA stability to the increased steady-state icIL-1Ra mRNA after IL-1{alpha} treatment, we measured the rate of decay of these transcripts following IL-1{alpha} treatment after inhibiting new transcription with actinomycin D treatment. The level of IL-1Ra mRNA was decreased to basal levels by 6 h (data are not shown).



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  		FIGURE 2. Analysis of icIL-1Ra promoter deletion constructs. Mouse newborn keratinocytes were transfected with various luciferase reporter vectors (10 µg/35-mm dish) containing different regions of the icIL-1Ra promoter along with the {beta}-galactosidase expression plasmid, pCMV-{beta}-gal (0.5 µg/dish), as an internal control, using lipofectin. Numbers indicate distance in base pairs from the transcription start site. The transfected cells were incubated with ({square}) or without ({blacksquare}) 300 U/ml IL-1{alpha} for 18 h. The luciferase activity was standardized to the {beta}-galactosidase activity and expressed as a percentage of the -4555-bp construct, which was defined as 100. Data are the average of three independent experiments.

 
Identification of the cis-acting regions of the icIL-1Ra promoter required for IL-1{alpha} induced icIL-1Ra in keratinocytes

We next attempted to identify the cis-acting elements responsible for the expression of the icIL-1Ra gene in keratinocytes. Transient transfection experiments with a series of 5'-flanking region deletion constructs showed positive regulatory sequences for the expression of icIL-1Ra (Fig. 2Go). Deletion of the region spanning from -598 to -288 showed significant reduction in promoter activity (<10% of pIC4555.Luc). The deletion disrupts the consensus sequences for NF-{kappa}B, NF-IL-6, and NF-1. Further deletion (-288 to -49 bp) of the icIL-1Ra promoter led to nearly the complete loss of promoter activity.

To begin to determine whether the -598 to -288 region binds NF-{kappa}B, NF-IL-6 and/or NF-1, nuclear extracts were prepared from unstimulated (control) or IL-1{alpha}-treated (300 U/ml for 3 h) primary keratinocytes. When nuclear extracts were incubated with the radiolabeled -598/-288 oligonucleotides, a gel shift was observed such that two bands were observed. When unlabeled consensus oligonucleotides for NF-{kappa}B, NF-IL-6, and NF-1 (shown in Fig. 1Go) were included in the binding assay, the upper band denoted by an arrow was lost when using oligonucleotides for NF-{kappa}B and NF-IL-6, but not when using oligonucleotides for NF-1 (Fig. 3Go, lanes 3–5). The lower band appears to represent nonspecific binding.



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  		FIGURE 3. EMSA to determine the protein-binding sites on the icIL-1Ra promoter. To delineate the subregion responsible for protein-DNA interaction on the icIL-1Ra promoter, EMSA was performed with a 32P-labeled -598/-288 DNA fragment, and nuclear extract (2 µg) was isolated from IL-1{alpha}-treated primary keratinocytes. For the competition experiment, labeled probes were chased with 50-fold molar excess of the cold oligonucleotides shown in Fig. 1Go. The binding reactions were resolved by 5% nondenaturing polyacrylamide electrophoresis.

 
NF-{kappa}B and NF-IL-6 can mediate the response to IL-1{alpha} in keratinocytes

To show a role for NF-{kappa}B and NF-IL-6 in the expression of icIL-1Ra induced by IL-1{alpha}, DNA-protein complexes were subjected to competition and supershift experiments with consensus and mutated oligonucleotides and specific Abs. Because C/EBP, a basic leucine-zipper DNA-binding protein, has been shown to bind to the consensus NF-IL-6 site, C/EBP consensus oligonucleotide and each C/EBP isoform-specific Ab were used for EMSA. As shown in Fig. 4GoA, the complexes were competed out by a C/EBP self-oligonucleotide, representing the promoter sequences (lane 4), and consensus oligonucleotides (lane 6), whereas a mutated oligonucleotide had no effect (lane 5). Abs against C/EBP{alpha} cleared the DNA-protein binding significantly (Fig. 4GoB, lane 4), whereas Abs against C/EBP{beta} clear the binding partially (lane 5). Ab against C/EBP{delta} did not changed the binding (lane 6).



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  		FIGURE 4. C/EBP transcription factors bind to the NF-IL-6 site of the icIL-1Ra promoter. An oligonucleotide containing a NF-IL-6 site was end-labeled with 32P and incubated with nuclear extract (2 µg) from primary keratinocytes treated with IL-1{alpha} (300 U/ml for 3 h) (lanes 3–6) or without treatment (lane 2). Top, For competition assays, molar excess (50-fold) of self (lane 4), mutant (lane 5), and consensus (lane 6) oligonucleotides were added to the binding reaction. Bottom, Clearing of the complexes was achieved by adding Abs for C/EBP{alpha} and -{beta} (lane 4–6) to the binding reaction. Lanes are numbered from left to right.

 
When nuclear extracts of keratinocytes treated with IL-1{alpha} were incubated with the NF-{kappa}B oligonucleotide probe, competition for binding was observed when unlabeled wild-type and consensus oligonucleotides were included (Fig. 5GoA, lanes 4 and 6, respectively). Supershift experiments using Abs against p50 and p65 were performed because those are the major NF-{kappa}B-binding proteins in keratinocytes. When extracts were preincubated with p65 Ab, the formation of two complexes was markedly decreased, whereas the p50 Ab only partially cleared the complex (Fig. 5GoB, lanes 4 and 5). Because NF-{kappa}B binding proteins bind to a target sequence as either a homo- or heterodimer, the complexes appear to be composed of p65 homodimer.



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  		FIGURE 5. EMSA targeting the NF-{kappa}B binding element. 32P-labeled NF-{kappa}B oligonucleotide was incubated with nuclear extract (2 µg) from primary keratinocytes treated with IL-1{alpha} (300 U/ml for 3 h) or without treatment. Top, Competition assays were performed by preincubating with a 50-fold molar excess of the unlabeled NF-{kappa}B probe (lane 4), mutant (lane 5), or consensus oligonucleotides (lane 6). Bottom, Supershift assays were performed by preincubating with Abs for p50 and p65 (lane 4 and 5). Lanes are numbered from left to right.

 
C/EBPs trans-activate the icIL-1Ra promoter activity in an isoform-specific manner

To further explore the role of C/EBPs in the expression of icIL-1Ra, C/EBP{alpha}, -{beta}, and -{delta} expression vectors were transfected into keratinocytes along with the -598 icIL-1Ra reporter vector. Promoter activity was increased by all three isoforms in a dose-dependent manner (Fig. 6GoA). Among the isoforms, C/EBP{alpha} and -{beta} showed a slightly stronger trans activation effect on icIL-1Ra than did C/EBP{delta}. To obtain further evidence for a role for C/EBPs in IL-1{alpha}-dependent icIL-1Ra promoter activation, keratinocytes were treated with IL-1{alpha} in the presence of CHOP-10, a C/EBP family member that lacks the trans activation and DNA-binding domains while possessing intact dimerization domains (22). As shown in Fig. 6GoB, CHOP-10 produces a concentration-dependent inhibition of icIL-1Ra promoter activity.



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  		FIGURE 6. Top, C/EBPs regulate icIL-1Ra promoter activity in an isoform-specific manner. Various amounts of the C/EBP expression plasmids MSV-C/EBP{alpha}, MSV-C/EBP{beta}, and MSV-C/EBP{delta} were cotransfected with the -598 reporter construct into primary keratinocytes. The total amount of DNA was made constant by using empty pMSV vector. Data are expressed as fold induction by C/EBPs relative to the -598 construct; values are the means of triplicate. Bottom, CHOP-10, a dominant negative trans activator of C/EBPs, inhibits icIL-1Ra promoter activity. Increasing amounts of CHOP-10 expression plasmid were cotransfected with the -598 reporter construct into primary keratinocytes. The total amount of plasmid DNA was kept constant by supplementing with empty pcDNA3luc vector. Data are expressed as a percentage of the -598 reporter construct luciferase activity which was defined as 100.

 
Differential expression of C/EBP isoforms induced by IL-1{alpha} was confirmed by Northern analysis (Fig. 7Go). There was a significant induction of C/EBP{alpha} mRNA 6 h after IL-1{alpha} treatment. The level of C/EBP{beta} also peaked at 6 h, but the level remained elevated up to 12 h after IL-1{alpha} treatment. The basal level of C/EBP{delta} was much higher than that of both {alpha} and {beta}; however, it was also induced by IL-1{alpha} and peaked at 6 h.



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  		FIGURE 7. Regulation of the expression of C/EBP isoforms by IL-1{alpha} in primary keratinocytes. Newborn primary keratinocytes were treated with IL-1{alpha} for the indicated times. Total RNA (20 µg) was isolated, separated by electrophoresis, and transferred to a nylon membrane. The blot was probed with 32P-labeled C/EBP cDNAs, stripped, and reprobed with 32P-labeled GAPDH cDNA as a control for loading.

 
p65 involvement in IL-1a-induced icIL-1Ra promoter activity

To assess which NF-{kappa}B-binding proteins are capable of regulating the transcription of the icIL-1Ra, p65, and p50 expression vectors were transfected into primary keratinocytes along with the -598 icIL-1Ra reporter vector. Promoter activity was increased significantly by overexpressed p65 (3.8-fold), whereas p50 showed only a slight trans activation effect (Fig. 8GoA). To further investigate whether transcription of icIL-1Ra is mediated by IL-1{alpha}-induced NF-{kappa}B, the I{kappa}BM expression vector, a dominant negative NF-{kappa}B, was transfected into IL-1{alpha}-treated keratinocytes along with the reporter vector. icIL-1Ra promoter activity decreased significantly (27% of control) with 1 µg I{kappa}BM expression vector, confirming the important role of NF-{kappa}B in IL-1{alpha}-induced icIL-1Ra transcription (Fig. 8GoB).



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  		FIGURE 8. Top, trans activation activity of the NF-{kappa}B subunits p65 and p50 on icIL-1Ra promoter activity. Increasing amounts of p65 and p50 expression plasmids were cotransfected with the -598 IL-1Ra promoter reporter construct into primary keratinocytes. The total amount of DNA per dish was held constant by adding empty pCMV vector. Data are expressed as fold induction by p65 and p50 relative to the luciferase activity of the -598 reporter construct. Bottom, I{kappa}BM, a dominant negative trans activator of NF-{kappa}B, inhibits icIL-1Ra promoter activity. Various amounts of I{kappa}BM expression plasmid were cotransfected with the -598 reporter construct into primary keratinocytes. Transfected cells were incubated with (columns 2–5) or without (column 1) IL-1{alpha} (300 U/ml) for 18 h. Columns are numbered from left to right. The total amount of plasmid DNA was kept constant by addition of the empty pCMV vector. Data are expressed as a percentage of the -598 reporter construct luciferase activity which was defined as 100.

 
Mutational analysis of the icIL-1Ra promoter

To further understand the role of the NF-{kappa}B and NF-IL-6 sites on the expression of icIL-1Ra induced by IL-1{alpha} in the context of the intact icIL-1Ra promoter, site-directed mutations were made at either the NF-{kappa}B or the NF-IL-6 sites. As shown in Fig. 9Go, the activity of the wild-type icIL-1Ra promoter was increased by 2.5-fold on stimulation with IL-1{alpha}. A 5-bp mutation introduced into the NF-IL-6 site reduced the activity of the icIL-1Ra promoter in response to IL-1{alpha}. This mutation did not reduced the basal icIL-1Ra promoter activity. A point mutation introduced into the NF-{kappa}B element of the icIL-1Ra also had little effect on basal activity, but abolish the IL-1{alpha} inducible activity completely.



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  		FIGURE 9. Effects of mutations in the NF-{kappa}B and NF-IL-6 sites in icIL-1Ra promoter activity. The -4525 reporter construct was used as templates for site-directed mutagenesis. Reporter vectors harboring mutations at the NF-{kappa}B or NF-IL-6 site were transfected into primary keratinocytes. The transfected cells were incubated with ({blacksquare}) or without ({square}) IL-1{alpha} (300 U/ml) for 18 h. The luciferase activity was expressed as a percentage of the -598 reporter construct defined as 100.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously demonstrated that IL-1{alpha} increases IL-1Ra mRNA in primary cultures of keratinocytes; however, the mechanism by which this occurs had not been elucidated (4). Here we show that the steady state level of icIL-1Ra mRNA observed after IL-1{alpha} treatment was due to increased transcription rather than changes in message stability. We also identified the cis- and trans-acting factors involved in IL-1{alpha}-induced transcription of IL-1Ra and showed that the NF-{kappa}B and NF-IL-6 response elements are the two major regulatory sequences. By supershift assay and cotransfection experiments with C/EBP expression vectors, we found that C/EBP{alpha} and -{beta} are the predominant binding proteins for the NF-IL6 site in the -598 to -288 region of the promoter.

The function of the different C/EBP isoforms appears to be quite different given that their expression is cell specific and differentiation stage specific. C/EBP{alpha} is a transcriptional activator involved in late differentiation in adipocytes while C/EBP{beta} and -{delta} are involved in early adipocyte differentiation (23). C/EBP{alpha} can induce growth arrest in various cell types including liver (24) and fibroblasts (25). C/EBP{beta} is also involved in the expression of several cytokines (26, 27).

The expression of C/EBPs in keratinocytes has been previously reported. C/EBP{alpha} and {beta} are expressed cultured human foreskin keratinocytes (28). Suprabasal cells of human epidermis also expressed C/EBP{alpha} (29). C/EBP{alpha} and -{beta} are highly expressed in mouse epidermis particularly in the suprabasal keratinocytes (20, 30). Recent reports showed that C/EBP{alpha} and -{beta} were differentially expressed during keratinocyte differentiation (30, 31) such that C/EBP{alpha} mRNA and especially protein levels increase with differentiation. In our study, C/EBP{alpha} and -{beta} mRNA levels were significantly induced by IL-1{alpha} by 6 h. In accord with these results, icIL-1Ra promoter activity was increased when C/EBP{alpha} and C/EBP{beta} expression vectors were transfected. In the supershift EMSA experiments, we did not expect to see clearing or shifting with the Ab against C/EBP{delta}. It has been previously reported that whereas C/EBP{delta} is expressed at the mRNA level, no protein expression can be detected in murine keratinocytes (30). We have also failed to detect C/EBP{delta} protein expression in normal skin, where as C/EBP{delta} and C/EBP{beta} are abundantly expressed (20).

It appears that icIL-1Ra induction by IL-1{alpha} via NF-{kappa}B binding is also an important regulatory mechanism. We identified p65 as the transcription factor binding to the NK-{kappa}B site on the icIL-1Ra promoter in keratinocytes treated with IL-1{alpha}. Smith et al. (32) demonstrated that the NF-{kappa}B site is the regulatory sequence responsible for the induction of sIL-1Ra by LPS. Supershift experiments showed that in keratinocytes the binding complex contains the p65/p65 homodimer. In unstimulated cells, NF-{kappa}B resides in the cytoplasm in an inactive state as a dimer, complexed with an inhibitory protein, inhibitory protein I{kappa}B (I{kappa}B).

We were unable to directly demonstrate whether or how the two different classes of transcription factors binding to the NF-{kappa}B and NF-IL-6 site interact with each other. Because mutation of either site alone completely abolished the IL-1{alpha}-induced promoter activity, the icIL-1Ra promoter appears to require both sites for promoter activity. Several reports have suggested that physical interaction between C/EBP and NF-{kappa}B leads to trans activation of several genes, including IL-6 (33) and IL-8 (34). The basic region leucine zipper domain of C/EBP is able to interact directly with the Rel homology domain of NK-{kappa}B proteins, which may have some bearing on the expression of genes containing adjacent C/EBP and NF-{kappa}B binding sites. In addition, p65 enhances the binding of C/EBP proteins to its response element (35).

It has been shown that a potent trans activation domain located in the C-terminal portion of p65 is necessary for the trans activation of NF-{kappa}B and that IL-1 up-regulates the p65 subunit (33). Reddy et al. (36) previously reported that phosphatidylinositol 3-kinase (PI3K) plays a role in transducing the IL-1 signal to NF-{kappa}B such that IL-1 stimulates interaction of IL-1R accessory protein (IL-1RAcP) with the p85 regulatory subunit of PI3K, leading to the activation of the p110 catalytic subunit. Thus, IL-1 simulates the PI3K-dependent phosphorylation and trans activation of NF-{kappa}B (37). IL-1 receptor associated kinase also has been shown to mediate the activation of NF-{kappa}B by IL-1. On binding of IL-1 to the type I receptor, IL-1RAcP docks to the receptor/ligand complex, which causes recruitment of adaptor proteins leading to activation of NF-{kappa}B-inducing kinase and NF-{kappa}B activation (38, 39). IL-1 also activates the mitogen-activated protein kinase cascade which both phosphorylates I{kappa}B followed by the translocation of NF-{kappa}B complexes into the nucleus (40) and phosphorylates and trans activates C/EBP (41).

The ability of IL-1 to induce its own antagonist is of interest in understanding the regulation and dysregulation of IL-1 signaling in normal and diseased tissue. Up-regulation of the receptor antagonist represents a mechanism for turning off IL-1 signaling and creating a temporary state of refractoriness. The basis for the elevated expression of icIL-1Ra that we observed in murine skin tumors, in which IL-1 was not appreciably up-regulated, suggests that in tumors the IL-1-signaling system may be dysregulated. Whether the altered profile of C/EBP isoforms in murine skin tumors is responsible for up-regulated IL-1Ra and how this affects IL-1 signaling in these tumors remains to be determined (20).

In summary, our results demonstrate that IL-1{alpha} regulates icIL-1Ra at the level of transcription. We identified the NF-IL-6 and NF-{kappa}B sites on the icIL-1Ra promoter as the major positive regulatory sequences. Furthermore, C/EBP{alpha}, C/EBP{beta}, and p65 were found to be the transcription factors binding to those sites. Finally, we demonstrated that increased levels of icIL-1Ra induced by IL-1{alpha} correlate with an increase in the expression of C/EBP{alpha} and C/EBP{beta} in normal murine keratinocytes.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Susan M. Fischer, Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX 78957. Back

2 Abbreviations used in this paper: IL-1Ra, IL-1R antagonist; C/EBP, CCAAT/enhancer-binding protein; CHOP-10, C/EBP homologous protein-10; icIL-1Ra, intracellular IL-1R antagonist; I{kappa}B, inhibitory {kappa}B; AcP, accessory protein; PI3K, phosphatidylinositol 3-kinase; sIL-1Ra, secretory IL-1R antagonist; SSPE, SSC phosphate/EDTA; LAP, liver-enriched transcriptional activating protein. Back

Received for publication July 24, 2000. Accepted for publication March 7, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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