Transcriptional regulation of Translocator protein (18 kDa) (TSPO) in microglia requires Pu.1, Ap1 and Sp factors

摘要

Mitochondrial Translocator protein (18 kDa) (TSPO) is strongly expressed in reactive microglia and serves as a therapeutic target for alleviation of neuronal degeneration. However, little is known about TSPO's transcriptional regulation in microglia. The aim of this study was to identify genetic elements and transcription factors required for basal and inducible TSPO expression in microglia. Murine Tspo promoter was cloned into the pGL4.10 luciferase vector and functionally characterized in BV-2 cells. Deletion mutagenesis indicated that 845 bases upstream were sufficient to reconstitute near maximal promoter activity in BV-2. Deletion of 593 to 520 sequences, which harbour an Apl, Ets.2 and Nkx3.1 site which also serves as a non-canonical binding site for Spl-family transcription factors, led to a dramatic decrease in both basal and LPS induced promoter activity. Further deletion of 168 to 39 sequences, which contains four GC boxes, also led to a significant decrease in promoter activity. Targeted mutations of Apl, Ets.2, Nkx3.1/Spl/3/4 and the GC boxes led to significant decreases in promoter activity. ChIP-qPCR revealed that Pu.1, Apl, Stat3, Spl, Sp3 and Sp4 bind to the endogenous Tspo promoter. Notably, binding of these factors, with the exception of Stat3, was significantly enhanced upon LPS treatment. RNAi silencing of Pu.1, cJun, cFos, Spl, Sp3, Sp4 and Stat3 strongly lowered Tspo promoter activity while Apl silencing inhibited LPS induced increase in Tspo protein levels. These findings demonstrate that consensus binding sequences for Apl, Ets.2, distal as well as proximal Sp1/3/4 sites regulate basal and LPS induced Tspo promoter activity in microglia.