2 STEROL REGULATORY ELEMENT-LIKE SEQUENCES MEDIATE UP-REGULATION OF CAVEOLIN GENE-TRANSCRIPTION IN RESPONSE TO LOW-DENSITY-LIPOPROTEIN FREE-CHOLESTEROL

Caveolae form the terminus for a major pathway of intracellular free c holesterol (FC) transport, Caveolin mRNA levels in confluent human ski n fibroblasts were up-regulated following increased uptake of low dens ity lipoprotein (LDL) FC, The increase induced by FC was not associate d with detectable change in mRNA stability, indicating that caveolin m RNA levels were mediated at the level of gene transcription, A total o f 924 bp of 5' flanking region of the caveolin gene were cloned and se quenced. The promoter sequence included three G+C-rich potential stero l regulatory elements (SREs), a CAAT sequence and a Sp1 consensus sequ ence. Deletional mutagenesis of individual SRE-like sequences indicate d that of these two (at -646 and -395 bp) were essential for the incre ased transcription rates mediated by LDL-FC, whereas the third was inc onsequential. Gel shift analysis of protein binding from nuclear extra cts to these caveolin promoter DNA sequences, together with DNase I fo otprinting, confirmed nucleoprotein binding to the SRE-like elements a s part of the transcriptional response to LDL-FC, A supershift obtaine d 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- Spl antibody nor with either antibody at -646 bp. The cysteine proteas e 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 influx and efflux.