Contribution of single nucleotide polymorphisms of the IL1A gene to the cleavage of precursor IL-1α and its transcription activity

We previously demonstrated the association of IL1A gene single nucleotide polymorphisms (SNPs) with susceptibility to systemic sclerosis (SSc) patients. In this study, we explored the effects of SNP on the transcriptional activity and processing of the precursor IL-1 alpha (pre-IL-1 alpha) in skin fibroblasts. Two kinds of promoter regions of the IL1A gene were prepared including C or T at -889, referred to C/IL1A and T/IL1A, and inserted into a luciferase reporter vector (pGL3). Skin fibroblasts were explanted from two SSc patients whose genomic DNA contained GG and TT genotypes at +4845 of the IL1A gene, respectively. Cell lysates were collected and reacted with various concentrations of calpain, and then the processing of pre-IL-1 alpha was analyzed by Western blotting using monoclonal anti-IL-1 alpha antibody. A SNP was determined by the allelic discrimination method using fluorescence-labeled Taq-Man probes. Significant differences in the luciferase activities were not detected between pGL3 (C/IL1A) and pGL3 (T/IL1A) in SSc fibroblasts. Calpain required a 100-fold higher concentration to process the pre-IL-1 alpha containing Ala at the 114th amino acid than that to do containing Ser. The frequency of the GG genotype was significantly higher in SSc patients than that in healthy donors, whereas the frequency of TT genotype was significantly higher in RA patients than that in healthy donors. Our observation showed that the SNP at +4845 affected the enzymatic efficiency of the protease in cleaving pre-IL-1 alpha. Pre-IL-1 alpha with Ala, which was high in frequency in SSc patients, was more resistant to be cleaved by proteases in human sera than pro-IL-1 alpha with Ser.