In conf luent fibroblast monolayers, an increase in the selective uptake of free cholesterol (FC) from plasma low density lipoprotein (LDL) was accompanied by an increase in FC eff lux. The rate of FC eff lux was proportional to the FC content of the cell surface caveolae and to mRNA levels of caveolin, an FC-binding protein of caveolae. Inhibitors of LDL-FC internalization reduced the increase in caveolin mRNA levels and FC eff lux. Oxysterols reduced caveolin mRNA levels, as well as transport of FC to the cell surface and FC eff lux. DNA antisense to caveolin reduced caveolin mRNA and inhibited FC eff lux. These data suggest that regulation of FC eff lux can contribute to cellular FC homeostasis when LDL levels are modified over the physiological range, and they link this regulation to caveolin.

Caveolae form the terminus for a major pathway of intracellular free cholesterol (FC) transport. Caveolin mRNA levels in conf luent human skin fibroblasts were up-regulated following increased uptake of low density lipoprotein (LDL) FC. The increase induced by FC was not associated with detectable change in mRNA stability, indicating that caveolin mRNA levels were mediated at the level of gene transcription. A total of 924 bp of 5 f lanking region of the caveolin gene were cloned and sequenced. The promoter sequence included three G؉C-rich potential sterol regulatory elements (SREs), a CAAT sequence and a Sp1 consensus sequence. Deletional mutagenesis of individual SRE-like sequences indicated that of these two (at ؊646 and ؊395 bp) were essential for the increased transcription rates mediated by LDL-FC, whereas the third was inconsequential. Gel shift analysis of protein binding from nuclear extracts to these caveolin promoter DNA sequences, together with DNase I footprinting, confirmed nucleoprotein binding to the SRElike elements as part of the transcriptional response to LDL-FC. A supershift obtained with antibody to SRE-binding protein 1 (SPEBP-1) indicated that this protein binds at ؊395 bp. There was no reaction at ؊395 bp with anti-Sp1 antibody nor with either antibody at ؊646 bp. The cysteine protease inhibitor N-acetyl-leu-leu-norleucinal (ALLN), which inhibits SREBP catabolism, superinhibited caveolin mRNA levels regardless of LDL-FC. This finding suggests that SREBP inhibits caveolin gene transcription in contrast to its stimulating effect on other promoters. The findings of this study are consistent with the postulated role for caveolin as a regulator of cellular FC homeostasis in quiescent peripheral cells, and the coordinate regulation by SREBP of FC inf lux and eff lux.In most peripheral cells, the expression of low density lipoprotein (LDL) receptors is strongly down-regulated, even by the low levels of LDL present in extracellular fluid. As a result, these cells, which internalize free cholesterol (FC) selectively from LDL at a rate proportional to medium LDL concentration, respond to changes in FC content by modifying the rate of FC efflux (1). Plasma membrane caveolae represent a major site from which FC exits the cell for transfer to medium plasma lipoproteins, particularly high density lipoprotein (2, 3).Caveolae are invaginated cell-surface microdomains (60-80 nm diameter) expressed in many quiescent peripheral cells (4). LDL-FC, internalized through clathrin-coated pits, is transferred in endosomal vesicles to the region of the trans-Golgi network (3). This FC, together with newly synthesized sterol, is returned to the cell surface caveolae by a temperaturesensitive, nocodazole-dependent pathway, probably as part of the FC-glycolipid ''rafts'' also carrying GPI-anchored proteins (4).Caveolae contain FC-binding proteins (caveolins) that play a key role in the organization of these organelles at the cell surface. Transformed cells normally contain few if any caveolae, and caveolin i...

Transcription of the human caveolin gene, directed by a TATA-less promoter, is downregulated in actively dividing cells during S-phase, together with free cholesterol (FC) efflux. It is upregulated by medium low density lipoprotein FC levels in quiescent cells. In this study, a common mechanism has been identified to coordinate the growth- and FC-dependent expression of caveolin. In human skin fibroblasts, transcription factors E2F/DP-1 and Sp1 bound to adjacent consensus sites at -151 to -138 bp of the caveolin promoter DNA sequence in a complex stabilized by tumor suppressor protein p53. Wild-type p53 also bound directly to DNA to a caveolin promoter sequence containing two consensus half-sites (-292 to -283 bp and -273 to -264 bp) for this transcription factor. SREBP-1, previously identified as a transcriptional regulator of caveolin expression in response to FC, mediated its effect via the same E2F/Sp1 site. Overexpression of E2F or p53 increased E2F binding to the -148 to -141 bp site, increased FC efflux, and inhibited cell division. The mutant protein p53(143V-->A) was inactive. Okadaic acid, previously shown to inhibit growth, FC efflux, and caveolin expression, inhibited E2F/Sp1 binding, while higher concentrations of extracellular FC increased it. The present findings provide a molecular link between the cell cycle and FC homeostatic effects of caveolin. These results also describe a novel mechanism of action for p53 in a TATA-less gene promoter and provide further evidence for a significant regulatory role for FC in cell cycle progression.

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