University Children's Hospital, University of Freiburg, Mathildenstrasse 1, 79106 Freiburg, Germany
Correspondence to: K. A. Deichmann
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Abstract |
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Keywords: genomic structure, IL-4 receptor, soluble, splicing
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Introduction |
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With respect to the numerous functions in IgE regulation, as well as B and T cell differentiation, the IL-4R gene represents a candidate gene for atopy. Human IL-4R
exists in at least two forms: the membrane-bound form (huIL-4R
) and a soluble one (shuIL-4R
). In mice two distinct pathways lead to the soluble forms. These involve alternative mRNA-splicing and limited proteolysis (shedding) of IL-4R
(7). So far, only the genomic structure of the mouse IL-4R
gene has been described (8,9). The mRNA coding for the smaller soluble muIL-4R
contains a 114 bp insert that terminates the open reading frame upstream of the predicted transmembrane region. Both mRNAs of the muIL-4R
originate from the same gene and the 114 bp insert corresponds to an alternatively spliced exon (exon 8) (9). The production of the soluble receptor by proteolysis seems to be regulated in a different way from the mRNA form in mouse and can be stimulated by, for example, TCR (7). A recombinant soluble human IL-4R
receptor form was found to either enhance or inhibit IL-4 signaling in a dose-dependent manner in supernatants of activated T cells (10), similar to previous findings in mouse (11). In humans, it was postulated that the soluble receptor is only produced by proteolytic shedding (4). This is supposed to be accomplished under the control of metalloproteinases (12).
Our interest was to deduce the complete genomic structure of the huIL-4R, including the promotor region. We investigated the existence of an additional form of soluble receptor in man, produced by mRNA splicing (shuIL-4R
/splice).
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Methods |
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Preparation of cDNA
mRNA was isolated from human whole blood by using the RNeasy blood mini-kit (Qiagen, Hilden, Germany). The cDNA was synthesized by the First-strand cDNA synthesis kit (Amersham/Pharmacia, Freiburg, Germany).
Exon 8
Exon 8 was detected by performing a nested PCR technique. First PCR: 5'-TCTGCTGCTGACCTGGAGCA-3' and oligo(dT) primer (resulting in a DNA fragment of 454 bp in length). Second PCR: 5'-GCACCCTGAAGTCTGGGATT-3' and oligo (dT) primer (resulting in a DNA fragment of 302 bp in length). PCR was carried out in a volume of 10 µl containing 30 ng cDNA, 5 pmol of each primer, 0.06 U Taq polymerase (Pharmacia) and 2 mmol dNTP mix with the buffer recommended by the supplier. Annealing temperatures were 35°C. The detection of exon 8 was confirmed by sequencing and comparison with the genomic sequence of huIL-4R. PCR was performed with cDNA templates derived from whole blood of probands using the primers: exon 7: 5'-GCACCCTGAAGTCTGGGATT-3' and exon 8: 5'-TCTCCCTCCAGAATGTCAGC-3'.
Northern blot
Total RNA was isolated from human whole blood by using the RNeasy blood mini-kit (Qiagen, Hilden, Germany). Then 15 µg RNA was separated on a 1% agarose gel containing 2.2 M formaldehyde and afterwards transferred onto a Hybond N membrane (Amersham, Braunschweig, Germany). The blot was hybridized with a 32P-labeled 330 bp PCR fragment coding for an extracellular part of the IL-4 receptor. Primers: 5'-CTTGCGAGTGGAAGATGAAT-3' and 5'-GTAATTGTCAGGGGGATACG-3'.
Cell culture and IL-4 stimulation for characterization of a STAT6 binding site
Characterization of the STAT6 binding site followed the technique as described by Matsuno et al. (13). Peripheral blood mononuclear cells were derived from whole blood and were grown for 4872 h at 37°C and 5% CO2 in the presence of 3 µg/ml phytohemagglutinin (Gibco/BRL, Eggenstein, Germany) in RPMI 1640 containing 2 mM L-glutamine, 20 mM HEPES, 100 U/ml penicillin, 50 mg/ml streptomycin and 10% FCS (PAN Systems, Aidenbach, Germany).
Cells were made quiescent for 516 h in RPMI 1640/glutamine/HEPES/penicillin/ streptomycin and 1% FCS. T cells (2x107) were stimulated with 100 nM human IL-4 (PAN Systems) for 515 min at 37°C. Cell pellets were then stored at 80°C.
After thawing, cells were suspended in lysis buffer (10 mM Tris, pH 7.8, 5 mM EDTA, 50 mM NaCl, 30 mM pyrophosphate, 50 mM sodium fluoride, 20 µM sodium orthovanadate, 1% Triton X-100, 1 mM PMSF, 5 µg/ml aprotinin, 1 µg/ml pepstatin A and 10 µg/ml leupeptin; 108 cells/ml buffer) and incubated for 60 min at 4°C. Insoluble material was removed by centrifugation. Part of the cell extract was separated on 12% native PAGE in several lanes and transferred onto PVDF filters (Millipore, Bedford, UK) (cell extract-blot). The residual binding sites on the filters were blocked overnight with TPBS (150 mM sodium chloride, 3 mM potassium chloride, 1 mM potassium dihydrogen phosphate, 7 mM disodium hydrogen phosphate and 0.05 % Tween 20) and 5% non-fat dried milk. One part of the filter was incubated with monoclonal anti-STAT6 (Transduction, Lexington, KY), the appropriate horseradish peroxidase-coupled secondary antibodies (Dako, Hamburg, Germany) and developed with ECL (Amersham). The other part of the filter was incubated with the rest of the cell extract and a 32P-radiolabeled PCR fragment bearing the potential STAT6 binding site (position intron 2: 4959; primers: 5'-CTGGCCCTTGGTGTACATTT-3' and 5'-GACACCACCTTCACCAAGTG-3'). As a negative control, the same experiment was done using a 32P-radiolabeled PCR fragment bearing the potential STAT but not STAT6 binding site from the promotor region of the gene (position 2328 of the promotor region, see Fig. 2, primers: 5'-GCCTGGGATGAAGCATCAAC-3' and 5'-GGCACTGACAAGTGAGCAGA-3').
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Results |
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Discussion |
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Due to the increasing importance of huIL-4R in the context of IgE regulation as well as its role in the Th1/Th2 shift and in order to allow further studies on variants in regulatory elements of the gene, the whole genomic structure has been characterized in this work.
Genomic structure
In general, the genomic structure of the human IL-4R gene resembles that of the mouse gene (9). It consists of 12 exons being interrupted by 11 introns. As the size of the mouse and human proteins slightly differ, the number of amino acids coded by the individual exons is slightly different. For example, we detected a signal peptide of 25 amino acids in human, in contrast to the mouse showing a signal peptide of 23 amino acids. Furthermore, the intracellular part of the human protein is bigger in size (exon 12).
Introns
Regarding the intron sequences, they are on the whole larger than the mouse introns, so that the complete huIL-4R gene spans ~51 kb on chromosome 16p11.212.1 (19), which is double in size compared to 26 kb on mouse chromosome 7.
Promotor analysis
Analyzing the promotor region, we could detect binding sites for those transcription factors having an influence on genes of the inflammatory processes (Fig. 2). These include NF
B, AP-1, NF-AT, CREB and STAT (20). It may well be that huIL-4R
is differentially controlled by these factors, which still has to be investigated.
Strikingly, we could detect several potential TATA boxes in the promotor region and intron 1. There is no TATA box directly preceding the proposed cap site (mRNA start) (4) in human, whereas there is one at position 29 in mouse (9). We therefore postulate multiple transcription initiation sites in human, which have already been postulated for the muIL-4R gene (9).
As the mouse IL-4R chain is known to be up-regulated by IL-4 through STAT6 binding (21), we were interested in a potentially similar function of STAT6 in humans. By using a combined Western/Southern blot technique we could detect a typical STAT6 binding site at position 4959 of intron 2. It is situated about 5 kb 5' of the start codon (ATG) of the huIL-4R
gene. The sequence TTCTCAGGAA and the position is completely different from the mouse IL-4R
gene, where the STAT6 binding site has been located to position 402 in the promotor and the sequence was shown to be TTCATCTGAA (21). STAT6 is an important factor for the activation of genes belonging to the IL-4 pathway (22). There is strong evidence for STAT6 not only being a direct effector substrate of the IL-4R
protein itself (23), but also influencing its transcription.
From our findings we conclude that due to strong variations in structure from human IL-4R to that of the mouse, transcription is probably organized in a different way. Further studies are planned to verify this hypothesis.
Exon 8; shuIL-4R produced by alternative splicing
Here we present for the first time evidence for the existence of an additional exon (exon 8) being located in intron 7/8 of huIL-4R. Due to the observations in the mouse system (9), exon 8 is very likely to code for a soluble form of huIL-4R
. A corresponding mRNA coding for the soluble receptor form could be shown in Northern blotting (see Fig. 5
).
Likewise in mice we found a stop codon in the sequence after additional amino acids. This soluble form is lacking transmembrane and intracellular domains. Therefore the shorter receptor form is likely to be excreted into the cytoplasm. Whereas in mice six amino acids are added to the cytoplasmic domain of IL-4R (4), in human we find three (Asn, Ile and Cys).
As proposed in mice (9), the shorter form of IL-4R mRNA is produced by alternative splicing mechanisms. We could detect shuIL-4R
/splice mRNA in several samples derived from human whole blood. As the samples were collected randomly (not only from atopics), we suspect that shuIL-4R
/splice is expressed in relatively equal amounts in mononuclear cells, regardless of the stimulation status. Our hypothesis is supported by similar findings in mice (24); however, it has to be further investigated.
shu IL-4R produced by limited proteolysis (shedding)
A soluble receptor form produced by the process of proteolytic shedding has already been described (12). It remains to be shown if both forms of shuIL-4R exhibit similar functions and if they are regulated by distinct processes.
It has been found previously that in atopics and asthmatics, as well as other inflammatory states of the respiratory epithelium, the concentration of shuIL-4R is much lower compared to healthy subjects (25). If we assume that the mRNA concentration is the same in all cases, it may well be that either altered post-translational regulations of the mRNA spliced form occur or that the shedding is somehow reduced, probably due to different surface conditions of huIL-4R
, due to influences on the activity of metalloproteinases itself or due to increased degradation of shuIL-4R
forms. In order to deduce the exact regulatory mechanisms it will be also necessary to identify the specific metalloproteinase responsible for the huIL-4R
shedding.
The exact function of shuIL-4R forms still has to be elucidated. Inhibitory effects and co-stimulatory effects on IL-4 signaling as well as depot effects for IL-4 have been discussed (10).
We suggest careful discrimination between the different shuIL-4R forms regarding their two distinct origins. Furthermore, the meaning of the polymorphism Ile50Val (17), which appears in all three forms, has to be further evaluated.
Our findings indicate complex regulatory mechanisms for the expression of membrane bound as well as soluble IL-4R. This opens a wide spectrum of further studies investigating the regulation either on the genomic, mRNA or protein level.
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Acknowledgments |
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Abbreviations |
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IL-4R![]() ![]() |
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Notes |
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Received 18 May 1999, accepted 24 August 1999.
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References |
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