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.
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.
Hullinger
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Department of Cardiovascular Pharmacology, Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, MI 48105, USA. Thomas.Hullinger@Pfizer.com
Panek
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Xu
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Karathanasis
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D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D018955:CD36 Antigens / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D015536:Down-Regulation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005799:Genes, Dominant; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011971:Receptors, Immunologic; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; D051122:Scavenger Receptors, Class B; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000235:genetics; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins / Q000235:genetics
D018955:CD36 Antigens; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D012333:RNA, Messenger; D011971:Receptors, Immunologic; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; C495124:SCARB1 protein, human; C454739:Scarb1 protein, mouse; D051122:Scavenger Receptors, Class B; D012313:RNA; D002784:Cholesterol; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D020558:GTP Phosphohydrolases; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins