Abstract
Interleukin (IL)-1 alpha treatment of a primate bone marrow stromal cell line, PU-34, transiently increased the steady state level of IL-11 mRNA. Nuclear run-on experiments showed that the transcription rate of the IL-11 gene was not affected appreciably by IL-1 alpha induction, but changes in the half-life of the IL-11 mRNA corresponded well with the changes in the steady state level of the IL-11 mRNA during the induction. Although transient transfection of PU-34 cells with IL-11 promoter constructs failed to respond to IL-1 alpha, a 10-base pair promoter region and JunD.AP-1 complex were found to be responsible for the basal level transcription of the IL-11 gene. The tyrosine kinase inhibitor genistein accelerated the degradation of the IL-11 mRNA without affecting the transcription rate of the IL-11 gene in IL-1 alpha stimulated cells. The insertion of DNA sequences corresponding to the 3'-untranslated region of the IL-11 gene into a rabbit beta-globin gene resulted in destabilization of the chimeric mRNA which failed to be induced by IL-1 alpha. Exogenous IL-11 expression generated from transient transfection with plasmid pCMV-IL-11, however, can be stabilized by IL-1 alpha. In contrast to the hypothesis that AUUUA motifs in the 3'-untranslated region are sufficient to regulate cytokine mRNA stability, our results suggest that IL-1 alpha induced stabilization of the IL-11 mRNA requires participation of RNA sequences from different regions of the IL-11 message.
Highlights
AULTUA motifs in the 3”untranslated region are suffi- cytokines such as GM-CSF, IL-3, and IL-6
IL-11 Gene Expression Induced by IL-la in PU-34 Cells Is Regulated at the Post-transcriptional Level-The kinetics of IL-11 gene expression following IL-la induction was determined by Northern analysis
Our data indicated that there contains both the 5'-promoter and the putativIeL-1 responsive was a basal level of IL-11 gene expression in PU-34 nuclei even element found in the 3'-flanking region of the IL-11 gene
Summary
Cell Culturesand Reagents-PU-34, a primate bone marrow derived [5]. The gene is 7 kb in length and consists of five exons and fibroblast cell line, was cultured as described previously [17]. Single-stranded DNA complementary to the I L l l mRNA was prepared by asymmetric polymerase chain reaction amplification according to standard procedures [21]. Single-stranded senseDNA, antisense DNA, and alkaline-denatured plasmid containing insert of human p-actin were slot-blotted onto nitrocellulose membrane for hybridization to the 32P-labeledrun-on products. Plasmid pNeoRBGATor pNeoRBGcc contains a 62-bp fragment (pNeoRBGATwith A'I"I"I'A repeats from the 3'-UTR of GM-CSF and pNeoRBGCCwith the corresponding AT to GC mutations) inserted into the noncoding region of rabbit p-globin gene. DNase I Footprinting Analysis-Nuclear extracts from PU-34 cells were prepared a s described previously [25]. The binding reaction was carried out with2.5 pg of the nuclear extract and 1 x 10' cpm of the probe a s described previously [27]. The cross-linked proteins were separated on a 10% SDS-polyacrylamide gelfor detection of the cross-linked bands
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