The STAT3 DNA-Binding Domain Mediates Interaction with NF-κB p65 and Inducible Nitric Oxide Synthase Transrepression in Mesangial Cells

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Abstract
Signal transducer and activator of transcription 3 (STAT3) and nuclear factor κB (NF-κB) are important transcription factors involved in glomerulonephritis and other inflammatory processes, including transcription of the inducible nitric oxide synthase (iNOS) gene. The ability of STAT3 to interact physically with NF-κB p65 in glomerular mesangial cells and thereby to inhibit NF-κB–mediated transactivation of the iNOS gene was demonstrated previously. STAT3 is a modular protein with several structurally and functionally defined domains. For defining STAT3 domains that interact with NF-κB p65, 35S-labeled proteins that corresponded to each STAT3α domain were synthesized, and their ability to bind specifically a GST-NF-κB p65 fusion protein in GST pulldown assays was tested. The coiled-coil and DNA-binding domains were specifically retained by GST-NF-κB p65, whereas the N-terminal, linker domain, Src homology 2 domain, and transcriptional activation domain failed to interact with NF-κB p65. Deletion of the region L358 through I369 of the STAT3 DNA-binding domain greatly reduced binding to GST-NF-κB p65. Alanine substitution mutations at four highly conserved residues—L358, N359, K363, and V366—in this region greatly abrogated the ability of STAT3 to bind NF-κB p65. Moreover, in contrast to the transrepression afforded by wild-type STAT3α, a STAT3α construct harboring these mutations, failed to suppress endogenous NO production and to transrepress iNOS promoter-reporter and κB element-reporter constructs in IL-β–stimulated mesangial cells. These data reveal a novel role for the DNA-binding domain in the physical and functional coupling of STAT3 to NF-κB p65 that is important for regulating the transcriptional activity of iNOS and likely other NF-κB p65 responsive genes that are important for mesangial cell responses.

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