Identification of an IL1A gene segment that determines aberrant constitutive expression of interleukin-1 alpha in...

To further explore the mechanisms of transcriptional regulation of the IL1A gene in fibroblasts from the affected skin of patients with systemic sclerosis (SSc).

The transcription activity of IL1A was estimated by nuclear run-on assay. A reporter construct (pLuc.-1437) was prepared that contained a 1.5-kb genomic fragment extending from -1437 to +39 relative to the transcription start site of the IL1A gene inserted upstream of the luciferase gene. The DNA protein binding activity for the cis-element of the IL1A gene was determined by electrophoretic mobility shift assay (EMSA).

Affected fibroblasts exhibited a strong nuclear run-on signal, indicative of constitutive transcription activity of the IL1A gene. Luciferase assay indicated high levels of transcription activity of pLuc.-1437 in transiently transfected fibroblasts from affected skin; this was absent in transiently transfected control fibroblasts. Analysis of constructs containing a series of 5' deletions of pLuc.-1437 revealed that the sequence between -96 and -82 (nuclear factor-interleukin-1 alpha [NF-IL-1 alpha]) was the most important for transcription of the IL1A gene in affected SSc fibroblasts. NF-IL-1 alpha contains a 5'-GGAA-3' sequence that is essential for specific binding of members of the Ets nuclear protein family. Using EMSA, a DNA protein complex was detected in samples containing nuclear extracts from affected fibroblasts and the NF-IL-1 alpha probe, whereas no specific band was detected in samples from normal fibroblasts. The DNA protein complex disappeared with the use of NF-IL-1 alpha probes that had mutations of the Ets binding sites.

These findings suggest that a DNA binding protein containing the Ets domain is constitutively expressed only in fibroblasts from the affected skin of SSc patients and regulates the transcription of the IL1A gene, which contributes to the fibrogenic phenotype of fibroblasts in SSc.