ELSEVIER Biochimica et Biophysica Acta 1302 (1996) 110-116 BB Biochi ~mic~a et Biophysica A~ta Sterol carrier protein-2 expression in mouse L-cell fibroblasts alters cholesterol uptake D. Moncecchi a, E.J. Murphy b, D.R. Prows a, F. Schroeder b,* a Division of Pharmacology and Medicinal Chemisto', Unit,ersity of Cincinnati, College of Pharmacy, Cincinnati, OH 45267-0004, USA b Department of Physiology and Pharmacology, Texas A & M UniL'ersitv, TVMC College Station, TX 77843-4466, USA Received 5 October 1995; revised 16 January 1996; accepted 16 February 1996 Abstract Despite the progress made on the possible functions of sterol cartier protein (SCP-2) using assays in vitro, very little is known regarding the role of SCP-2 in intact cells. To further elucidate this role, mouse L-cell fibroblasts were transfected with the cDNA encoding for mouse 15 kDa or 13.2 kDa SCP-2. The data show for the first time, that SCP-2 expression increases cholesterol uptake into transfected L-cell fibroblasts. Untransfected L-cells expressed SCP-2 at levels near or below the lower limit of detectability. SCP-2 immunoreactive protein levels were 0.030 + 0.004% and 0.036 + 0.002% of total cytosolic proteins in the 15 and 13.2 kDa stable transfectants, respectively. Both the 15 and 13.2 kDa SCP-2 expressions products were found as 13.2 kDa proteins, consistent with rapid post-translational cleavage of the putative amino terminal mitochondrial targeting sequence from the 15 kDa SCP-2. The effect of expressing either form of SCP-2 on [3H]cholesterol uptake was determined. Expression of the 15 kDa form, but not the 13.2 kDa form of SCP-2, enhanced the rate and extent of [3H]cholesterol uptake compared to control or mock-transfected L-cells. The [3H]cholesterol uptake rate in 15 kDa SCP-2 expressing cells was increased 1.3-fold, while the extent of [3H]cholesterol uptake was increased 1.4-fold after 12 h of uptake compared to control L-cells. The differences in cholesterol uptake between the cells expressing the 13.2 versus the 15 kDa protein, suggest that the 15 kDa form of SCP-2 is functionally localized within the cell, while the 13.2 kDa product is not. Keywords: L-cell; Sterol carrier protein-2; Transfection; Cholesterol uptake 1. Introduction Although many studies in vitro suggests a role for sterol cartier protein-2 (SCP-2) in cholesterol transfer and metabolism, a similar effect has not been shown using a physiologically relevant system. SCP-2 has several differ- ent molecular weight forms, all originating from the same gene through differential expression at two initiation sites [1-3]. The 58 kDa form has sequence homology to thio- lase [1,3], contains a C-terminal peroxisomal targeting sequence [1,3] and appears to be located within the peroxi- somal matrix [4-8]. Levels of the 58 kDa form are induced by clofibrate, a peroxisomal proliferator [8], estradiol [9], and luteinizing hormone [10]. The 15 kDa SCP-2 contains a 20 amino acid sequence, homologous to a putative mitochondrial targeting sequence [ 11,12] that is post-trans- lationally cleaved to form the 13.2 kDa form. The 13.2 kDa form is found in the cytosol, mitochondria, and on the * Corresponding author. Fax: + 1 (409) 8624929. 0005-2760/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved. PII S0005-2760(96)00044-6 cytosolic face of the peroxisomal membrane [5,7,8]. The 13.2 kDa form is induced in peritoneal macrophages by exposure to 25-hydroxycholesterol or acetylated low den- sity lipoproteins [13]. Although the 15 and 13.2 kDa forms both transfer cholesterol similarly in vitro [14], very little is known regarding their physiological function in intact cells. Based on a number of different studies in vitro, SCP-2 is thought to function in cholesterol uptake and metabolism. SCP-2 binds cholesterol [15-18], cholesterol analogs [19,20], oxysterols [15], phospholipids [15,16], and free fatty acids [21]. Phosphatidylserine but not phosphatidic acid potentiates SCP-2 mediated sterol transfer [18,22,23], suggesting a charge interaction between the basic SCP-2, pl near 8.5, and the anionic phospholipid membrane. This effect is attenuated when SCP-2-facilitated sterol exchange is measured using natural membranes [17]. SCP-2 also mediates intramitochondrial cholesterol movement [24,25] and stimulates pregnenolone synthesis [26]. Collectively, these studies in vitro suggest that SCP-2 binds and facili-