Three Promoters Regulate Tissue- and Cell Type-specific Expression of Murine Interleukin-1 Receptor Type I
2009; Elsevier BV; Volume: 284; Issue: 13 Linguagem: Inglês
10.1074/jbc.m808261200
ISSN1083-351X
AutoresQun Chen, Hao Zhang, Qiming Li, Ying An, Miles Herkenham, Wenmin Lai, Phillip G. Popovich, Sudha Agarwal, Ning Quan,
Tópico(s)NF-κB Signaling Pathways
ResumoThe type 1 interleukin-1 receptor (IL-1R1) mediates diverse functions of interleukin-1 (IL-1) in the nervous, immune, and neuroendocrine systems. It has been suggested previously that the versatile functions of IL-1 may in part be conferred by the multiple promoters of IL-1R1 that have been identified for the human IL-1R1 gene. Promoters for murine IL-1R1 (mIL-1R1) gene have not been studied in detail. We performed 5′-rapid amplification of cDNA ends to determine the transcription start sites (TSS) in mIL-1R1, using mRNAs derived from 24 different tissues. The results revealed three putative TSSs of mIL-1R1. Three full-length cDNAs containing these distinct TSSs were recovered in screens of cloned cDNA libraries. Translation of these cDNAs produced IL-1R1 proteins that were verified by Western blot analysis. IL-1 stimulation of the individual IL-1R1 proteins resulted in the activation of NF-κB. Promoter-reporter assay for genomic DNA sequences immediately upstream of the three TSSs validated that the sequences possess promoter activity in a cell type-specific manner. These promoters are termed P1, P2, and P3 of the mIL-1R1, in 5′ to 3′ order. Quantitative PCR analysis of P1-, P2-, and P3-specific mIL-1R1 mRNAs showed that there is tissue-specific distribution of these mRNAs in vivo, and there are distinct patterns of P1, P2, and P3 mRNA expression in different cell lines. In the brain, P3 mRNA is expressed preferentially in the dentate gyrus. Further, glucocorticoids differentially regulate these promoters in a cell type-specific manner. Together, these results suggest that the different IL-1R1 promoters contribute to the discrete and diverse actions of IL-1. The type 1 interleukin-1 receptor (IL-1R1) mediates diverse functions of interleukin-1 (IL-1) in the nervous, immune, and neuroendocrine systems. It has been suggested previously that the versatile functions of IL-1 may in part be conferred by the multiple promoters of IL-1R1 that have been identified for the human IL-1R1 gene. Promoters for murine IL-1R1 (mIL-1R1) gene have not been studied in detail. We performed 5′-rapid amplification of cDNA ends to determine the transcription start sites (TSS) in mIL-1R1, using mRNAs derived from 24 different tissues. The results revealed three putative TSSs of mIL-1R1. Three full-length cDNAs containing these distinct TSSs were recovered in screens of cloned cDNA libraries. Translation of these cDNAs produced IL-1R1 proteins that were verified by Western blot analysis. IL-1 stimulation of the individual IL-1R1 proteins resulted in the activation of NF-κB. Promoter-reporter assay for genomic DNA sequences immediately upstream of the three TSSs validated that the sequences possess promoter activity in a cell type-specific manner. These promoters are termed P1, P2, and P3 of the mIL-1R1, in 5′ to 3′ order. Quantitative PCR analysis of P1-, P2-, and P3-specific mIL-1R1 mRNAs showed that there is tissue-specific distribution of these mRNAs in vivo, and there are distinct patterns of P1, P2, and P3 mRNA expression in different cell lines. In the brain, P3 mRNA is expressed preferentially in the dentate gyrus. Further, glucocorticoids differentially regulate these promoters in a cell type-specific manner. Together, these results suggest that the different IL-1R1 promoters contribute to the discrete and diverse actions of IL-1. Interleukin-1 is a pro-inflammatory cytokine that plays multiple roles in the immune, nervous, and neuroendocrine systems (1Quan N. Herkenham M. Histol. Histopathol. 2002; 17: 273-288PubMed Google Scholar). The biological activity of IL-1 2The abbreviations used are: IL, interleukin; IL-1R1, IL-1 receptor type I; IL-1R2, IL-1 receptor type II; TSS, transcription start site(s); RACE, rapid amplification of cDNA ends; GSP, gene-specific primer(s); Dex, dexamethasone; UTR, untranslated region; SVE, SV40 enhancer; CREB, cAMP response element binding; m, murine; RT, reverse transcription; COX, cyclooxygenase; NFR, NF-κB reporter; contig, group of overlapping clones. is mediated by distinct IL-1 receptors. A receptor for both IL-1α and IL-1β was first cloned from mouse T cells (2Sims J.E. March C.J. Cosman D. Widmer M.B. MacDonald H.R. McMahan C.J. Grubin C.E. Wignall J.M. Jackson J.L. Call S.M. Friend D. Alpert A.R. Gillis S. Urhal D.L. Dower S.K. Science. 1988; 241: 585-589Crossref PubMed Scopus (710) Google Scholar). This receptor is the type I IL-1 receptor (IL-1R1). In B cells (3Bomsztyk K. Sims J.E. Stanton T.H. Slack J. McMahan C.J. Valentine M.A. Dower S.K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8034-8038Crossref PubMed Scopus (150) Google Scholar) and monocytes (4Spriggs M.K. Lioubin P.J. Slack J. Dower S.K. Jonas U. Cosman D. Sims J.E. Bauer J. J. Biol. Chem. 1990; 265: 22499-22505Abstract Full Text PDF PubMed Google Scholar), a second type of IL-1 receptor was discovered. This receptor was later cloned and named the type II IL-1 receptor (IL-1R2) (5McMahan C.J. Slack J.L. Mosley B. Cosman D. Lupton S.D. Brunton L.L. Grubin C.E. Wignall J.M. Jenkins N.A. Brannan C.I. Copelad N.G. Huebner K. Croce C.M. Cannizzarro L.A. Benjamin D. Dower S.K. Spriggs M.K. Sims J.E. EMBO J. 1991; 10: 2821-2832Crossref PubMed Scopus (619) Google Scholar). IL-1R2 was later shown to serve as a decoy receptor for IL-1, negatively regulating the activity for IL-1 (6Sims J.E. Dower S.K. Eur. Cytokine Netw. 1994; 5: 539-546PubMed Google Scholar). In some cells, IL-1R1 is exquisitely sensitive to IL-1 stimulation, requiring only 10 molecules/cell to mediate potent stimulatory effects (6Sims J.E. Dower S.K. Eur. Cytokine Netw. 1994; 5: 539-546PubMed Google Scholar). In a B-cell cell line 70Z/3, IL-1R2 but not IL-1R1 could be detected, yet IL-1 signaling in these cells is mediated exclusively by IL-1R1, suggesting that trace amounts of IL-1R1 mediate the effect of IL-1 (7Sims J.E. Gayle M.A. Slack J.L. Alderson M.R. Bird T.A. Giri J.G. Colotta F. Re F. Mantovani A. Shanebeck K. Grabstein K.H. Dower S.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6155-6159Crossref PubMed Scopus (546) Google Scholar) despite the presence of large quantities of IL-1R2. In general, it is widely accepted, with rare exceptions (8Chou H.H. Takashiba S. Maeda H. Naruishi K. Nishimura F. Arai H. Lu H. Murayama Y. J. Dent. Res. 2000; 79: 1683-1688Crossref PubMed Google Scholar), that IL-1R1 is the receptor that mediates the biological effects of IL-1. The promoters for the IL-1R1 gene have not been studied in depth. Ye et al. (9Ye K. Dinarello C.A. Clark B.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2295-2299Crossref PubMed Scopus (68) Google Scholar) were the first to identify multiple transcription start sites (TSS) in the human IL-1R1 gene by primer extension analysis. They later provided evidence that human IL-1R1 gene expression may be controlled by three distinct promoters (10Ye K. Vannier E. Clark B.D. Sims J.E. Dinarello C.A. Cytokine. 1996; 8: 421-429Crossref PubMed Scopus (21) Google Scholar). Clinically, a PstI polymorphism near the human IL-1R1 promoter 2 (P2) was found to have significant association with insulin-dependent diabetes mellitus (11Bergholdt R. Karlsen A.E. Johannesen J. Hansen P.M. Dinarello C.A. Nerup J. Pociot F. Cytokine. 1995; 7: 727-733Crossref PubMed Scopus (51) Google Scholar). A polymorphism within Exon 1B has also been associated with protective effects against endometriosis development (12D'Amora P. Sato H. Girao M.J. Silva I.D. Schor E. Am. J. Reprod. Immunol. 2006; 56: 178-184Crossref PubMed Scopus (16) Google Scholar). Exon 1B expression is driven specifically by the P2. Therefore, the heretofore under-characterized IL-1R1 promoters may play critical roles in IL-1 biology. We chose to characterize the murine IL-1R1 (mIL-1R1) promoters to permit in-depth analysis of their structure and function in vivo and in vitro. Thus far, only two putative TSSs for mIL-1R1 mRNA can be found in the GenBank™, but no promoter analysis has been reported. In this study, we identified and characterized the mIL-1R1 promoters and determined that they contribute to the differential expression and regulation of mIL-1R1 mRNA. Reagents and Cell Lines-Luciferase reporter vector pGL4.10 was obtained from Promega (Madison, WI). Lipofectamine LTX with Plus reagents and Steadylite HTS for luciferase assay were purchased from PerkinElmer Life Sciences. Rabbit polyclonal antibody against mIL-1R1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Power SYBR Green PCR Master Mix was purchased from Applied Biosystems (Foster City, CA). RAW 264.7 (macrophage), D10.G4.1 (Th2 cell), Neuro-2a (neuroblast cell), SVEC4-10 (peripheral endothelial cell), C8-DIA (type 1 astrocyte), C8-D30 (type 3 astrocyte), C8-S (type 2 astrocyte), bEnd.3 (brain endothelial cell), and LADMAC (macrophage progenitor) were purchased from ATCC (Manassas, VA). These cell lines were maintained according to the instructions of the ATCC protocols. 5′-Rapid Amplification of cDNA Ends-The Mouse Sure-RACE kit (OriGene, Rockville, MD) containing PCR-ready mouse cDNAs (generated from 24 different tissues) was used. The 5′ end of mRNA in this kit has been modified to contain 5′ adaptor sequences (ADP) to facilitate PCR amplification of the 5′ ends of mRNAs. Two gene-specific primers (GSP) for mIL-1R1 were designed from the published mIL-1R1 mRNA sequence in GenBank™ (accession number M20658). The sequence for GSP1 is 5′-TACGTCAATCTCCAGCGACAGCAGAG, and the sequence for GSP2 is 5′-AGCAGAGGCACCATGAGACAAATGAG. These primers are located in Exon 2 of the published mouse IL-1R1 mRNA sequence, after the putative start codon. Nested PCR was performed with a pair of outer anchor primers, ADP1 and GSP1, followed by a pair of inner anchor primers, ADP2 and GSP2. A graphic depiction of the RACE design is shown in Fig. 1A. The PCR products were separated by electrophoresis using a Bioanalyzer (Agilent, Santa Clara, CA). For the RACE-PCR amplicons with a single major band, the products were directly cloned into the PCR 2.1-TOPO vector by TOPO TA cloning (Invitrogen). For the RACE-PCR amplicons containing multiple bands, all of the visible bands were resolved by 1.0% agarose gel electrophoresis and purified. The isolated bands were subsequently cloned into the PCR 2.1-TOPO vector. The cloned cDNAs were sequenced by an automatic sequencer (Plant-Microbe Genomics Facility at Ohio State University). The sequence data were aligned to the mouse genomic data base of the National Center for Biotechnology Information. mRNA Library Screening-Sequence data from the RACE assay revealed three different alternative first exons of mIL-1R1. We annotated these findings in the context of known genomic structure of mIL-1R1 in Fig. 1C and designated these alternative first exons as Exon1A, Exon1B, and Exon1C. We then designed PCR primers to target these exons. The primer pairs for Exon1A, Exon1B, and Exon1C are 5′-GATGTCATCAGAGTTCCCAGTG/5′-CATTCTGCTGATGAATCCTG, 5′-CTGCTGGATTGTTGAACATCG/5′-CATTCTGCTGATGAATCCTG, and 5′-AGGACACTGAGACATTCGCTGG/5′-CATTCTGCTGATGAATCCTG, respectively. These primers were sent to OriGene for PCR-screening using the OriGene Rapid-Screen Master plates containing full-length mRNA libraries generated from adult brain (MAB-1001), embryonic day 19 (MEA-1001), embryonic day 12.5 (MEB-1001), adult liver (MLI-1001), thymus (MTM-1001), and adult testis tissues (MTS-1001). Screening from a total number of three million clones yielded three full-length cDNAs containing Exon1A, Exon1B, or Exon1C. These cDNAs were cloned into the expression vector pCMV6-XL4. These clones were sequenced to verify that the Exon1A, Exon1B, and Exon1C indeed exist in different full-length IL-1R1 mRNA species. Because the three exon 1 alternatives suggest that there are three different promoters for the mIL-1R1, we designated these clones as P1-, P2-, and P3-cDNA clones. Western Blot-To study protein products of these three different mIL-1R1 cDNA clones, the cDNAs were transfected into Neuro-2a cells. Transfection was performed with Lipofectamine LTX and Plus reagents (Invitrogen) according to the Invitrogen protocol. Twenty-four h after the transfection, the cells were harvested and washed twice with cold phosphate-buffered saline, and 2 × 106 cells were resuspended in 250 μl of phosphate-buffered saline. Then 50 μl of 6× loading buffer (0.35 m Tris-HCl, pH 6.8, 10.28% SDS, 36% glycerol, 0.6 m dithiothreitol, and 0.01% phenol red) was added. The samples were briefly sonicated and heated to 95 °C for 5 min in preparation for Western blotting. Twenty-microliter solution from each sample was resolved by SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride membranes. The blot was then blocked with 10% milk in TBS and probed with a rabbit polyclonal antibody against mouse IL-1R1 (sc-689;Santa Cruz Biotechnology) in 1% milk TBS-T followed by the IRDye 680 Goat anti-rabbit antibody (LI-COR Inc. Lincoln, NE). The signal was detected using the LI-COR infrared imaging system. NF-κB Reporter Assay-To study the function of proteins translated from the P1-, P2-, and P3-initiated mRNAs of mIL-1R1, we tested the ability of these proteins to mediate IL-1-induced NF-κB activation, a well known IL-1 activity. The MHC-3XκB-Luc luciferase reporter, an NF-κB reporter construct, was obtained from Dr. Denis Guttridge (Ohio State University, Columbus, Ohio). In a 24-well culture plate, P1-, P2-, or P3-cDNA (0.1 μg) was co-transfected with MHC-3XκB-Luc (0.4 μg) in a molar ratio of 1:5 into the Neuro-2a cells. Twenty-four h after the transfection, IL-1α was added to each well (the resulting concentration of IL-1α was 100 pg/ml), and the cells were incubated for additional 6 h. Luciferase activity was measured by the SteadyLite HTS assay system and a VICTOR3 Multi-Label Reader (both from PerkinElmer Life Sciences). Promoter-Reporter Constructs-After aligning the sequences of P1-, P2-, and P3-mRNA to the genomic mIL-1R1 sequence, three putative transcription start sites (TSS) were identified. To determine promoter activities upstream of these TSSs, ∼500-800-bp (core promoter region, CP) and 2-kb (long promoter region, LP) upstream genomic sequences were PCR-amplified. The following primer pairs were used: 5′-TGTGACAGCCATTCTGAGTTG/5′-TCAAGTCAGCCCAGGACAG (CP1); 5′-AATTCCATCCCGAAGATAACC/5′-TGGGAACTCTGATGACATCCAG (LP1); 5′-TGTCCTCCTTGCACCCTGTC/5′-ACCTTCTGAGCAGCACGCTG (CP2); 5′-CACCAAGTGACAGTGTCAAG/5′-AGTGGCGATGTTCAACAATCC (LP2); 5′-CAGCCACAAGAACTGACTG/5′-CTCTGGACCTCCCTAGCAAG (CP3); and 5′-TCAGCTCCATGTGCCACAAG/5′-CTCTGGACCTCCCTAGCAAG (LP3). Each resulting amplicon (CP1, LP1, CP2, LP2, CP3, and LP3) was sequenced and cloned separately into the pGL4.10 vector between the restriction sites KpnI/XhoI, immediately before the luciferase reporter sequence. To study the influence of potential enhancers on the activities of these putative promoters, an SV40 enhancer was added to each promoter-reporter construct downstream of the luciferase reporter sequence to generate the promoter-reporter-enhancer constructs (a graphic depiction of the promoter-reporter constructs is shown in Fig. 3, A and B). Promoter-Reporter Assay-Promoter-reporter constructs were transfected into the Neuro-2a (neuron), RAW 264.7 (macrophage), and SVEC4-10 (endothelial cell) cells using the Lipofectamine LTX and Plus reagents as described above. These cells were always grown to be 50-80% confluent at the time of transfection. Transfected cells were incubated at 37 °C in a CO2 incubator for 24 h before the analysis of luciferase activity using the same method described above. Analysis of P1-, P2-, and P3-mRNA Expression Patterns in Various Cell Lines and in Different Tissues-Total RNA was isolated from nine different cultured cells (RAW 264.7, D10.G4.1, Neuro-2a, SVEC4-10, C8-DIA, C8-D30, C8-S, bEnd.3, and LADMAC) and nine major tissues (heart, spleen, lung, pituitary, liver, kidney, adrenal gland, thymus, and testis) followed by the generation of cDNAs by reverse transcription. The mRNA levels were analyzed by SYBR Green-based quantitative PCR. The following primer sets for quantitative PCR analysis of P1-, P2-, and P3-mRNA transcripts were used. The forward primers for P1-, P2-, and P3-mRNAs were 5′-GATGTCATCAGAGTTCCCAGTG, 5′-CTGCTGGATTGTTGAACATCG, and 5′-AGGACACTGAGACATTCGCTGG, respectively. These different primers reside in Exon1A, Exon1B, and Exon1C, respectively. A common reverse primer 5′-TGGAGTAAGAGGACACTTGC was used. This sequence is complementary to a sequence in the Exon 3 of the mIL-1R1 mRNA. This common reverse primer was used because P1-, P2-, and P3-mRNAs share this common downstream sequence, and Exon 3 is separated from all the alternative exons 1 by Intron 2. This design minimizes the possibility that genomic DNA of mIL-1R1 will be amplified in the PCR. These primers were validated by a test in which cloned P1-, P2-, or P3-cDNAs were used as templates. The mRNA levels of the housekeeping gene G3PDH were measured to provide an internal control. The procedure for mouse tissue sample collection in this experiment was conducted in accordance with the National Institutes of Health guide on the care and use of animals for research and an in-house protocol approved by the Ohio State University Animal Care and Use Committee. Laser Capture Microdissection-Laser capture microdissection was used to capture specific brain structures. Brains from 8-10-week-old FVB mice were rapidly frozen in supercooled 2-methylbutane. Tissue was sectioned at 10 μm on a Microm cryostat (HM 505 E) and mounted on RNase-free P.A.L.M. Membrane Slides (Zeiss, Thornwood, NY). The slides were stained with Cresyl Violet and Acridine Orange following Ambion’s laser capture microdissection staining kit protocols. Stained slides were stored in a closed box with desiccant until microdissection. At the time of microdissection, the sections were rapidly thawed and the desired brain structures: dentate gyrus, choroid plexus and striatum, were circumscribed using the P.A.L.M. Microbeam laser capture system fit with a 5× cutting objective. After microdissection, RNA from different brain structures was isolated using the RNAqueous micro kit (Ambion, Inc., Austin, TX). DNase treatment was performed on the purified RNA using 2 units of DNase I for 20 min. The DNase I was subsequently removed using the DNase Inactivation Reagent from Ambion’s DNA-free system. Quantitative RT-PCR was performed to analyze P1-, P2-, and P3-mRNA levels as described above. In Situ Hybridization Histochemistry-The novel mRNA sequence for mIL-1R1 discovered in this study is the P3-mRNA. The P3-mRNA contained a 1.7-kb 5′ mRNA sequence that is different from the published IL-1R1 mRNA sequence. To study the expression patterns of P3-mRNA in the brain, in situ hybridization histochemistry was performed. First, a 400-bp fragment of Exon 1C was PCR-amplified (the PCR primers were 5′-TATAGACTGTTAACCTCCTGAAAACCATG and 5′-ATTCTCCTGGGGAAACAGAAGAAG) and cloned into the TOPO 2.1 vector. The resulting plasmids were examined for the orientation of the insert. One plasmid containing the Exon1C sequence with desired insert orientation was selected and linearized by SacI digestion. The antisense riboprobe against Exon1C was transcribed using T7 RNA polymerase. The same plasmid was linearized with Kpn1 and transcribed with SP6 RNA polymerase to generate a sense control probe. The ribonucleotide probes were labeled with the isotope S-35, and in situ hybridization histochemistry was performed as described previously (13Quan N. Whiteside M. Herkenham M. Neuroscience. 1998; 83: 281-293Crossref PubMed Scopus (275) Google Scholar). The results were examined by autoradiography. The presence of P3-mRNA was determined if the intensity of the labeling in a brain region by the antisense probe was at least 2-fold higher than the labeling intensity by the sense probe. Dexamethasone Treatment-To study differential regulation of P1, P2, and P3 activity by glucocorticoids, Neuro-2a, SVEC4-10, and RAW246.7 cells were incubated with varying concentrations of dexamethasone (Dex) or vehicle overnight. Changes in P1-, P2-, and P3-IL-1R1 mRNA levels were analyzed by quantitative RT-PCR as described above. To study the effect of glucocorticoids in vivo, the animals were injected intraperitoneally with Dex (5 mg/kg). Tissue RNA was extracted 2 h after the injection, and levels of promoter-specific IL-1R1 mRNA were analyzed by quantitative RT-PCR. The responsiveness of SVEC4-10 and RAW246.7 cells to IL-1 stimulation after these cells were treated with Dex or vehicle was compared. Briefly, the cells were incubated with 10-6 m of Dex or vehicle overnight and then washed with culture medium three times before IL-1α (final concentration of IL-1α in the cultures was 1 ng/ml) was added to the cultures. RNA was extracted from these cultured cells 2 h later and analyzed for the expression of cyclooxygenase 2 (COX-2) mRNA levels using quantitative RT-PCR as we described previously (14Zhang H. Ching S. Chen Q. Li Q. An Y. Quan N. Neuroscience. 2008; 157: 895-907Crossref PubMed Scopus (24) Google Scholar). Statistical Analysis-The data are presented as the means ± S.E. Variations in mRNA levels were evaluated by one-way analysis of variance followed by post-hoc analysis (Tukey test). p < 0.05 is considered statistically significant. Fig. 1B shows results of electrophoresis of 5′-RACE PCR products from various mouse tissues listed in the Fig. 1 caption. Multiple band patterns were obvious when PCR products from different tissues were compared. In some tissues, e.g. brain, stomach, and skin, multiple bands (indicated by asterisks) were generated by the RACE-PCR. These bands were isolated, cloned into the TOPO 2.1 vector, and sequenced. In other tissue, e.g. thymus and muscle, only one major band was generated. The PCR products from these tissues were directly cloned into TOPO vector and sequenced. The sequences generated from the RACE clones were aligned to the genomic DNA sequence of mIL-1R1 by two-sequence BLAST. The results are summarized in Table 1. Only the unique sequences are listed. Sequences found in the existing data base with GenBank™ accession numbers as references were also included. Many TSS were identified in the different tissues. Three major groups of TSS in IL-1R1 were found. The first group of TSSs (the numbers for the start sites are in regular font in Table 1) spans 271 bp, aligning to nucleotide positions 17808499-17808670 of the reference contig sequence NT_039170.7. This group of TSSs is close to two published mRNA start sites (accession numbers NM_008362 and BC109135), although some of the TSSs discovered in the present studies are further upstream, whereas others are downstream to the published TSSs. The second group of TSSs (the numbers for the start sites are in bold type in Table 1) was found clustered close to the position of 17850082 of the NT_039170.7 sequence. These TSSs are ∼41 kb downstream of the first group of TSS sites. These TSSs are slightly downstream of the published TSSs at this locus (accession numbers AK054357 and AK048550). The third type of TSS was cloned from the involuting breast tissue (sample 24 in the RACE kit). This rare TSS (italic in Table 1) is located immediately in front of the second exon of the IL-1R1 gene. In fact, the mRNA sequence of this subspecies of IL-1R1 mRNA lacks the entire first intron defined in the GenBank™. No reported TSS matched this TSS.TABLE 1Sequence analysis of murine IL-1R1 TSSExon IExon II start siteTranscripts in different tissuesStart siteEnd1780849917808655178657296, 2417808523178088031786572911780852317808655178657296, 51780855817808655178657291, 4, 11, 23178085761780880317865729NM_0083621780862717808803178657294, 20178086701780880317865729BC109135178500821785037817865729AK054137178501641785037817865729AK0485501785030917850378178657291, 11, 6, 231786355217865792No intron gap24 Open table in a new tab To verify that these three groups of TSSs, resulting in three different Exons 1 (designated as Exon 1A, Exon 1B, and Exon 1C), can be found in full-length mIL-1R1 mRNAs, PCR primers were designed to screen the full-length mRNA libraries from OriGene (three million randomly cloned full-length cDNA from six different tissues). We obtained three clones after the screening that confirmed the existence of three alternative Exons 1 in mIL-1R1 mRNA. Because these three alternative Exons 1 suggest three different promoters, we designated these putative promoters as the P1, P2, and P3 of the mIL-1R1, in the 5′ to 3′ order. The mRNAs driven by these promoters are denoted as the P1-, P2-, and P3-mRNA. The positions of the IL-1R1 promoters are annotated in Fig. 1C in the context of known genomic structure of the mIL-1R1 gene. The complete P1-, P2-, and P3-mRNA sequences are presented in the supplemental Fig. S1. Simplified exon structure diagrams for P1-, P2-, and P3-mRNAs are shown in Fig. 2A. The only difference found among these three mRNAs is at the 5′ end. Exon 1A, Exon 1B, and Exon 1C are distinct. Analysis of open reading frames showed that whereas the predicted start codon for P2-mRNA resides in exon 2, the predicted start codon for P1-mRNA resides in Exon 1A, resulting in an addition of two more amino acids at the N terminus. The Exon 1C is the longest exon 1, shifting the predicted open reading frame significantly toward the 5′ end and resulting in the addition of 43 additional amino acids at the N terminus. P1-, P2-, and P3-cDNA were transfected into the Neuro-2a cells. Expression of these cDNAs was examined by Western blotting using an antibody targeted at the C terminus of the mIL-1R1. Fig. 2B shows that IL-1R1 protein was not detected in Neuro-2a cells transfected with an empty vector. IL-1R1 protein was found in Neuro-2a cells transfected with either P1-, P2-, or P3-cDNA. No difference in molecular weight was found among the detected IL-1R1 proteins extracted from P1-, P2-, and P3-cDNA transfected cells. Two bands were noted in the Western blot. This band pattern is consistent with that published by the manufacturer of the antibody used in this study; this antibody probably detects two IL-1R1 isoforms resulting from different glycosylation of the IL-1R1 protein. Fig. 2C shows results from the NF-κB reporter assay. Neuro-2a cells were co-transfected with P1-, P2-, or P3-cDNA together with the NF-κB reporter (NFR). The cells were then stimulated with 100 pg/ml of IL-1α. In a control experiment, the cells were transfected with NFR together with the empty vector for the IL-1 cDNAs (V). IL-1 stimulation did not induce NF-κB activation in cells transfected with the empty vector. In co-transfected cells that contained both IL-1 cDNA and the NFR, IL-1 stimulation resulted in significant NF-κB activation as indicated by the increased luciferase activity. No difference was detected among P1-, P2-, and P3-cDNA transfected cells. Fig. 3 shows promoter activity detected by the promoter-reporter assay. 2-kb DNA sequences immediately upstream of the TSSs found in the P1-, P2-, and P3-mRNAs were studied. These sequences were designated as the long promoters. Hence they are denoted as LP1, LP2, and LP3. The 3′ end of the long promoters also included 100-150 bp of the 5′-untranslated region. We also investigated the promoter activity in shorter DNA sequences (∼500 bp) near the TSSs. These sequences are called core promoters and are hence designated as CP1, CP2, and CP3. The positional relationship between LP1 and CP1, LP2 and CP2, and LP3 and CP3 is shown in Fig. 3A. These promoters were studied in the peripheral endothelial (SVEC4-10), macrophage (RAW264.7), and neuronal (Neuro-2a) cell lines. Fig. 3B shows the summary of promoter activity determined in this study. LP2 exhibits the strongest activity in all three cell lines, whereas LP1 and LP3 showed detectable but small promoter activities. The activity of the LPs was also tested when a SVE sequence was added. LP1 activity was increased by the addition of SVE in all three cell lines (LP1 versus LP1+SVE, p < 0.05). SVE increased LP2 and LP3 activity in both the endothelial and macrophage cell lines but did not increase LP2 activity in the neuronal cell line. CP1 showed stronger promoter activity than LP1 in both the endothelial cells and macrophage cells, but CP1 was not active in the neuronal cell line. CP2 showed stronger promoter activity than LP2 in all three tested cell lines. CP3 activity was not greater than LP3 activity in any of the cell lines tested (CP3 versus LP3, p > 0.05). Because the promoter activity of CP3 was weak (the activity was barely stronger than the promoter-less construct), we tested the promoter activity of CP3+SVE. Fig. 3D shows that SVE increased CP3 activity in both endothelial cells and macrophage cells but not in the neuronal cells. Fig. 4A shows patterns of P1- and P2-mRNA expression in various cell lines. P2 mRNA was the dominantly expressed IL-1R1 in D10.G4.1, LADMAC, and SVEC4.10 cells. P1-mRNA was the dominantly expressed IL-1R1 in b.End3 cells. Very little IL-1R1 mRNA was detected in C8-DIA, C8-S, C8-D30, RAW264.7, and Neuro-2a cells. P3-mRNA was expressed at very low levels in all of the cells tested (data not shown). Fig. 4B shows the tissue distribution of P1-, P2-, and P3-IL-1R1. In the liver, P1 is the dominantly expressed IL-1R1. In the lung, spleen, heart, thymus, testis, adrenal gland, and kidney, P2 is the dominantly expressed IL-1R1 mRNA. P3-mRNA was clearly detected in many tissues. In the liver, P3-mRNA was expressed at a higher level than P2-mRNA. Among the three tissues tested in the brain, the highest P1-mRNA expression was found in the striatum, whereas the highest P2-mRNA expression was found in the dentate gyrus. P3-mRNA was preferentially expressed in the dentate gyrus (Fig. 4C). Because the P3-mRNA sequence is significantly different from the P1- and P2-mRNA sequ
Referência(s)