Loss of transcriptional activation of three sterol-regulated genes in mutant hamster cells.

104

The synthesis and uptake of cholesterol requires transcription factors designated sterol regulatory element-binding proteins (SREBPs). SREBPs are bound to membranes in a hairpin orientation with their transcriptionally active NH 2 -terminal segments facing the cytosol. The NH 2 -terminal segments are released from membranes by two-step proteolysis initiated by site 1 protease (S1P), which cleaves in the luminal loop between two membrane-spanning segments. Next, site 2 protease (S2P) releases the NH 2 -terminal fragment of SREBP. The S2P gene was recently isolated by complementation cloning using Chinese hamster ovary cells that require cholesterol for growth, due to a mutation in the S2P gene. A similar approach cannot be used for S1P because all previous cholesterol auxotrophs manifest defects in S2P, which is encoded by a single copy gene. To circumvent this problem, in the current studies we transfected Chinese hamster ovary cells with the S2P cDNA, assuring multiple copies. We mutagenized the cells, selected for cholesterol auxotrophy, and identified two mutant cell lines (SRD-12A and -12B) that fail to cleave SREBPs at site 1. Complementation analysis demonstrated that the defects in both cell lines are recessive and noncomplementing, indicating a mutation in the same gene. These cells should now be useful for expression cloning of the sterol-regulated S1P gene.Studies in tissue culture cells have defined a feedback regulatory system that controls the synthesis and uptake of cholesterol and fatty acids. The central agents are membrane-bound transcription factors designated as sterol regulatory elementbinding proteins (SREBPs) 1 (reviewed in Ref. 1). These proteins, which average 1,150 amino acids in length, consist of three segments. The NH 2 -terminal segment of ϳ480 amino acids is a transcription factor of the basic-helix-loop-helixleucine zipper family. This is followed by a membrane attachment segment consisting of two membrane-spanning sequences separated by a 31-amino acid hydrophilic loop that projects into the lumen of the endoplasmic reticulum and nuclear envelope. The COOH-terminal segment of ϳ580 amino acids performs a regulatory role. The SREBPs are inserted into the membranes of the endoplasmic reticulum and nuclear envelope in a hairpin fashion with the NH 2 -terminal and COOH-terminal segments projecting into the cytosol (2).In sterol-depleted cells, a two-step proteolytic process releases the NH 2 -terminal segments (designated nSREBPs) from the membranes, allowing them to enter the nucleus, where they stimulate transcription of multiple genes encoding enzymes of cholesterol synthesis, including HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate synthase, squalene synthase, and others (1, 3-6). The nSREBPs also enhance transcription of genes encoding enzymes of fatty acid and triglyceride biosynthesis (acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase-1 and -2, glycerol-3-phosphate acyltransferase) (5-10) and for the low density lipoprotein (LDL) receptor (1, 6). ...

Section: Discussionmentioning

confidence: 99%

Isolation of Cholesterol-requiring Mutant Chinese Hamster Ovary Cells with Defects in Cleavage of Sterol Regulatory Element-binding Proteins at Site 1

Rawson

Cheng

Brown

et al. 1998

104

“…Steroid hormones were purchased from Steraloids Inc. RU486 was the kind gift of Dr. Raymond Daynes (University of Utah). [9, (20,21,23,24).…”

Section: Methodsmentioning

confidence: 99%

Role of Multidrug Resistance P-glycoproteins in Cholesterol Esterification

Debry

Nash²,

Neklason³

et al. 1997

132

Cholesterol esterification, catalyzed by acyl-CoA:cholesterol acyltransferase (ACAT), plays a central role in cellular cholesterol homeostasis and in physiologic processes that lead to coronary heart disease. Although ACAT resides in the endoplasmic reticulum (ER), the cholesterol substrate for esterification originates in the plasma membrane and must be transported to the ER for esterification. Progesterone inhibits esterification, possibly by blocking the transport of cholesterol to the ER. Recent studies suggest that progesterone acts by inhibiting the activity of one or more of the multidrugresistant (MDR) P-glycoproteins. In the current manuscript, we demonstrate that progesterone's ability to inhibit esterification is not mediated through the progesterone receptor. We evaluate a series of steroid hormones and find a strong correlation between a steroid hormone's hydrophobicity and its ability to inhibit both cholesterol esterification and MDR-catalyzed drug efflux. We also find that cholesterol esterification is inhibited by nonsteroidal MDR inhibitors, and that this inhibition specifically affects the esterification of cholesterol derived from the plasma membrane. MDR inhibitors also inhibit cholesterol esterification in a wide range of cultured human cell lines. These observations suggest that MDR activity normally functions in a general process of intracellular cholesterol transport. Cells maintain exquisite control over the level of free cholesterol through a complex set of homeostatic mechanisms that control the rates of cholesterol biosynthesis, cholesterol esterification, and receptor mediated endocytosis of cholesterol-rich low density lipoproteins (LDL 1 ; reviewed by Goldstein and Brown (1)). Cholesterol esterification is catalyzed by acyl-CoA: cholesterol acyltransferase (ACAT) in the endoplasmic reticulum (ER) and involves the covalent attachment of fatty acids to the 3-position hydroxyl group of cholesterol to form cholesteryl esters. In a homeostatic process, excess free cholesterol stimulates esterification resulting in the storage of excess cholesterol in the form of cytosolic lipid droplets. While free cholesterol can be toxic to cells, cholesteryl esters can accumulate to relatively high levels. Under most physiologic conditions, the rate of cholesterol esterification is not limited by ACAT levels but rather by the availability of cholesterol substrate in the ER (2, 3). Very little cholesterol is found in the ER; 90% of all cholesterol resides in the plasma membrane (4), and most of the remainder is found in endocytic vesicles derived from the plasma membrane (5). Under conditions of excess cholesterol, cholesterol is transported from the plasma membrane to the ER where it is esterified by ACAT. The mechanism by which this transport occurs is poorly understood.The delivery of plasma membrane cholesterol to the ER is also required for a number of other processes involved in cholesterol homeostasis. HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis, resides in the ER e...

“…[2][3][4][5][6][7][8][9][10][11][12][13][14] C]Acetic acid (52-55 mCi/mmol) and [1␣,2␣-3 H]cholesterol (47 Ci/mmol) were purchased from Amersham. Other materials were obtained from previously reported sources (16,19,20).…”

Section: Methodsmentioning

confidence: 99%

Progesterone Inhibits Cholesterol Biosynthesis in Cultured Cells

Metherall

Waugh

1996

Cells acquire cholesterol through endogenous synthesis and through receptor-mediated uptake of cholesterol-rich low density lipoprotein (LDL). Esterification of LDL-derived cholesterol is catalyzed by acyl-CoA:cholesterol acyltransferase (ACAT) in the endoplasmic reticulum (ER). Progesterone inhibits esterification, and, although the mechanism of inhibition is not completely understood, this inhibition results from progesterone's ability to inhibit the activity of multiple drug resistance (MDR) P-glycoproteins (P. DeBry and J. E. Metherall, submitted for publication). In the current manuscript, we demonstrate that progesterone inhibits cholesterol biosynthesis resulting in the accumulation of a number of sterol precursors. In Chinese hamster ovary (CHO) cells, high concentrations (100 M) of progesterone completely blocked cholesterol production, resulting in the accumulation of lanosterol and a lanosterol precursor. Lower concentrations (40 M) of progesterone cause plasma membrane accumulation of several sterol products. The majority of these sterols are precursors of cholesterol since they were efficiently converted to cholesterol upon removal of progesterone from the culture medium. Although very high concentrations (>200 M) of progesterone killed CHO cells, their growth was restored by the addition of cholesterol to the growth medium, indicating that progesterone toxicity resulted from cholesterol auxotrophy. The effect of progesterone was not unique to CHO cells; progesterone also inhibited cholesterol biosynthesis in all human cell lines tested. These observations suggest that a common progesterone-sensitive pathway is involved in both cholesterol biosynthesis and the processing of LDL-derived cholesterol.