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.
Highlights
A variety of stimuli have been demonstrated to regulate SR-BI expression
Cotransfection with either a constitutively active p21-activated protein kinase-1 (PAK1) construct (T423E) or a kinase-deficient p21-activated kinase-1 (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
It has been shown that LPS down-regulates SR-BI mRNA and protein levels in monocytes and macrophages [16]
Summary
A variety of stimuli have been demonstrated to regulate SR-BI expression. The hormones estrogen and adrenocorticotropic hormone have been observed to alter SR-BI expression [7, 13,14,15]. Pro-inflammatory mediators such as lipopolysaccharide (LPS) have been shown to down-regulate the mRNA and protein levels of SR-BI in the monocyte and macrophage [16]. It has been shown that the sterol response element-binding protein activates transcription of the rat SR-BI promoter in variety of cell lines [18] and that steroidogenic factor-1 binds to and activates the human SR-BI promoter in mouse adrenocortical cells [6]. LPS is known to signal through Toll-like receptor-4 [19] and to trigger the activation of nuclear factor-B (NF-B) plus several MAPKs, the ERK, p38, and JNK proteins (20 –24). I; LPS, lipopolysaccharide; NF-B, nuclear factor-B; MAPK, mitogenactivated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; PAK1, p21-activated kinase-1; DMEM, Dulbecco’s modified Eagle’s medium; NIK, NF-B-inducing kinase; RSV, Rous sarcoma virus; AP-1, activator protein-1; MZF-1, myeloid zing finger protein-1; HSF, heat shock factor. PAK1 Inhibits SR-BI Promoter Activity been shown to activate p21-activated kinase-1 (PAK1) in the macrophage, and PAK1 activation triggers nuclear accumulation of NF-B [25]
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