L‐Arginine Inhibits a Critical Structural Contact Required for Transcription of the IL1B Gene Coding for Human Interleukin 1β

Abstract

Interleukin 1β (IL‐1β) is a proinflammatory cytokine that is normally expressed and released by monocytes in response to infection and injury. Chronic expression of IL‐1β has been associated with numerous inflammatory diseases. We previously reported chromatin looping for the IL1B gene that is mediated by a mutual DNA‐binding domain (DBD) interaction between two transcription factors, Spi‐1/PU.1 (Spi‐1) at the promoter and C/EBPβ at a distal enhancer. The nature of this critical interaction between the Spi‐1 wHTH domain and the C/EBPβ leucine zipper was investigated by using the existing X‐ray structures of these two proteins bound to DNA as a guide in designing mutants for in vitro protein‐protein interaction studies. This generated surface interaction maps for the two proteins that in concert with computational techniques supported an association mode similar to, but distinct from, the C/EBPβ·c‐Myb X‐ray structure reported by others. The distinction was that, in addition to the C/EBPβ leucine zipper interaction with the c‐Myb wHTH domain, Spi‐1 utilizes an additional pseudo‐symmetrical bidentate interaction between the carboxyl‐tail extensions of each of the C/EBPβ leucine zipper monomers with the two ends of the Spi‐1 DNA recognition helix. This is consistent with our previous report that the carboxyl‐tail of C/EBPβ is critical for Spi‐1 association, suggesting that pocket‐like surfaces at the ends of the Spi‐1 DNA recognition helix might be targets for inhibition. Candidate small molecules were generated using a virtual screening approach. L‐arginine, a known anti‐inflammatory molecule, was identified from this approach as having potential to bind to each end of the Spi‐1 recognition helix, possibly inhibiting IL1B gene expression. Studies in 2.5 h LPS‐treated human THP‐1 macrophages in the presence of L‐arginine revealed a concentration‐dependent blunting of transcription in parallel with a decrease in C/EBPβ association with Spi‐1 on the IL1B gene promoter, while Spi‐1 binding to DNA was unaffected. In the same experiment, there was no significant change in direct binding of C/EBPβ to DNA at a well‐characterized site in the IL6 gene. In conclusion, these studies are consistent with an L‐arginine‐dependent decrease in IL1B gene expression that is due to its ability to inhibit the protein‐protein interaction between C/EBPβ and Spi‐1 in the absence of any effect on direct DNA binding for either factor. This is consistent with the predicted structure of the C/EBPβ·Spi‐1 interaction.