Abstract
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-kappaB. 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.
Highlights
Interleukin-1 is a pro-inflammatory cytokine that plays multiple roles in the immune, nervous, and neuroendocrine systems [1]
The results of this study revealed three functionally relevant clusters of transcription start sites (TSS), each with a unique promoter, in the murine IL-1R1 gene
Previous studies have used large amounts of mRNA isolated from individual cell lines to determine the IL-1R1 TSS by primer extension assay [9]
Summary
Reagents and Cell Lines—Luciferase reporter vector pGL4.10 was obtained from Promega (Madison, WI). 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., 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. 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. 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 [14]. Variations in mRNA levels were evaluated by one-way analysis of variance followed by post-hoc analysis (Tukey test). p Ͻ 0.05 is considered statistically significant
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