Sterol Regulation of Fatty Acid Synthase Promoter

Abstract

The gene encoding fatty acid synthase, the essential multi-functional enzyme of fatty acid biosynthesis, is shown to be regulated by cellular sterol levels similar to genes that encode important proteins of cholesterol metabolism. We show that expression of the endogenous FAS gene is repressed when regulatory sterols are included in the culture medium of HepG2 cells and that the FAS promoter is subject to similar regulation when fused to the luciferase reporter gene. Mutational studies demonstrate that sterol regulation is mediated by binding sites for the sterol regulatory element-binding protein (SREBP) and transcription factor Sp1, making it mechanistically similar to sterol regulation of the low density lipoprotein receptor gene. It is also demonstrated that SREBP and Sp1 synergistically activate the FAS promoter in Drosophila tissue culture cells, which lack endogenous Sp1. These experiments provide key molecular evidence that directly links the metabolism of fatty acids and cholesterol together. Coordinate regulation of fatty acid and cholesterol accumulation is essential for balanced membrane biosynthesis and turnover to accommodate metabolic fluctuations that occur during normal cellular growth. Also, these two important lipids are simultaneously required in the liver for regular-ordered assembly of very low density lipoprotein particles, which deliver their lipid load of cholesterol and fatty acids from the liver to other sites in the body to maintain lipid homeostasis (1, 2). Co-regulation of genes that encode important enzymes of both fatty acid and cholesterol metabolism would be an ideal way to coordinate lipid regulation, and recent work has identified a family of transcriptional regulatory proteins that could link the two pathways together (3-5). Sterol regulatory element binding proteins 1 and 2 (SREBP 1 and 2) 1 are highly related proteins that bind to the same cis-acting elements in the LDL receptor and HMG-CoA synthase promoters and activate expression only when cellular sterol stores are depleted (3, 5, 6). The cDNAs for both were cloned based on amino acid sequence information obtained from the purified proteins (3, 5). Independently, the rat equivalent of SREBP-1 was cloned from an adipocyte cDNA expression library (4). The rat mRNA was expressed at very high levels in brown fat and was also abundant in white fat and liver. The rat SREBP-1 mRNA was also induced during adipocyte differentiation in cell culture, so it was named the adipocyte determination-and differentiation-dependent factor 1 (ADD1). These observations suggested that SREBP-1/ADD1 is a regulator of genes that are important for lipid accumulation in the adipocyte. The above studies indicate that the activity of SREBP-1/ ADD1 may provide a direct link between the regulation of cholesterol and fatty acid metabolism. In the present paper we demonstrate that expression of the mRNA for fatty acid syn-thase (FAS), an essential enzyme of fatty acid biosynthesis, is regulated by sterols in a manner similar to genes that encode important proteins of cholesterol metabolism. Moreover, in the region of the FAS promoter that is required for sterol regulation there are two binding sites for SREBP-1. One of them is located close to a binding site for the generic factor Sp1, and we show this site is crucial for sterol regulation. The involvement of SREBP-1 and Sp1 in activation of the FAS promoter is reminiscent of the LDL receptor promoter where both proteins are essential for sterol regulation as well. These experiments provide strong evidence for a direct molecular connection between the regulation of two different classes of cellular lipids that are both required for cellular growth and normal lipopro-tein metabolism. EXPERIMENTAL PROCEDURES Cells and Media-CV-1 cells were obtained from Dr. K. Cho (Uni-versity of California, Irvine). HepG2 cells were obtained from the ATCC. All cell culture materials were obtained from Life Technologies Inc. Lipoprotein-deficient serum was prepared by ultracentrifugation as described (7). Cholesterol and 25-OH cholesterol were purchased from Steraloids Inc., and stock solutions were dissolved in absolute ethanol. Cell Culture-HepG2 cells were plated at 225,000 cells/100-mm dish on day 0. Two days later the dishes were washed three times with phosphate-buffered saline and split into two groups. One set of dishes were refed induced medium (DMEM containing 10% lipoprotein-depleted serum) and one set was fed suppressed medium (same but containing 10 g/ml cholesterol and 1 g/ml 25-OH cholesterol) media. Following a 24-h incubation the cells were harvested for RNA by the guanidine lysis procedure, followed by cesium chloride gradient sedimentation (8). PCR Cloning of FAS cDNA-cDNA was prepared from HepG2 RNA with the aid of a kit from Invitrogen. The cDNA was used as a template with PCR primers designed to two highly conserved regions of the fatty acid synthase mRNA that were chosen by aligning the rat and chicken FAS cDNA sequences. The 5 primer (5-GACACAGCCTGCTCCTC(C/ T)AG-3) was designed to hybridized to a 20-base pair region beginning at nucleotide number 249 of the rat cDNA (where the A of the ATG start