p21-activated Kinase-1 (PAK1) Inhibition of the Human Scavenger Receptor Class B, Type I Promoter in Macrophages Is Independent of PAK1 Kinase Activity, but Requires the GTPase-binding Domain

Abstract

Scavenger receptor class B, type I (SR-BI), is a high density lipoprotein receptor that mediates the flux of cholesterol between high density lipoprotein and cells. Recent evidence suggests that SR-BI plays a role in atherosclerosis and that inflammatory mediators down-regulate SR-BI in the macrophage. The purpose of this study was to evaluate the ability of lipopolysaccharide (LPS) to down-regulate the activity of the human SR-BI promoter in the macrophage and to delineate the mechanisms involved. Experiments with cultured cells and in vivo derived macrophages showed that LPS has a powerful suppressive effect on SR-BI expression both in vitro and in vivo. Transient transfection studies demonstrated that LPS represses SR-BI promoter activity in the macrophage cell line RAW 264.7. Cotransfection with either a constitutively active p21-activated protein kinase-1 (PAK1) construct (T423E) or a kinase-deficient PAK1 construct (K299R) resulted in repression of the SR-BI promoter, similar to LPS. These results demonstrate that PAK1-mediated down-regulation of the SR-BI promoter is independent of PAK1 kinase activity and suggest that PAK1 mediates the LPS-induced decrease in promoter activity. Cotransfection with constitutively active Cdc42 or Rac expression constructs also resulted in down-regulation of the promoter; whereas the dominant-negative Cdc42 and Rac constructs elevated basal promoter activity and blunted the LPS response. Cotransfection of PAK1 constructs containing mutations in both the kinase domain and the Cdc42/Rac-binding domain attenuated the PAK1-mediated down-regulation of the promoter, suggesting that Rac and Cdc42 are required for PAK1-mediated decreases in SR-BI promoter activity. 5′-Deletion analysis and gel shift data suggest that LPS inhibits binding of a novel transcription factor to a myeloid zing finger protein-1 like element (-476 to -456) in the human SR-BI promoter. These results demonstrate that the PAK1 pathway down-regulates the SR-BI promoter and suggest that activation of this pathway may play an important role in cholesterol trafficking in the vessel wall.