Transcriptional regulation of the rat sperm-associated antigen 11e ( Spag 11e ) gene during endotoxin challenge

The lipopolysaccharide (LPS) inducible expression of antimicrobial proteins of the Sperm-Associated Antigen 11 ( Spag11 ) family is dependent on nuclear factor-κB (NF-κB) activation and epigenetic factors. However, the regulatory mechanisms that govern their gene expression during endotoxin challenge are unknown. In this study, we demonstrate that the Spag11e gene upstream sequence contains binding sites for androgen receptor (AR), NF-κB, nuclear factor-1, E-twenty-six and activator protein 2. The role of these transcription factors in inducing Spag11e gene during LPS challenge was analysed by measuring luciferase activity in HEK cells transiently transfected with deletion constructs that lacked one or more of the binding sites. Deletion of AR-binding site resulted in loss of luciferase activity and no further decrease was observed when progressive deletions of the other transcription factor binding sites were made. Mutations in AR or NF-κB binding site resulted in loss of luciferase activity. Electrophoretic gel-mobility shift assays indicated that AR and NF-κB proteins bind to the synthesised radio-labelled oligomers used as probes and the mobility shifted when respective antibodies were added. Results of this study indicate the direct involvement of AR and NF-κB in LPS-induced Spag11e expression, thereby expanding our understanding of antimicrobial gene expression during endotoxin challenge.