REGULATION OF HEPATIC LIPASE EXPRESSION BY AN INTERMEDIATE OF THE CELLULAR CHOLESTEROL BIOSYNTHETIC PATHVAY.

REGULATION OF HEPATIC LIPASE EXPRESSION BY AN INTERMEDIATE OF THE CELLULAR CHOLESTEROL BIOSYNTHETIC PATHVAY

INTRODUCTION

Two lipolytic enzymes, hepatic triglyceride lipase (H-TGL) and lipoprotein lipase (LpL), are responsible for the catabolism of lipoproteins in the circulation (1). H-TGL is bound to endothelial cells lining the sinusoidal cavities of the liver and has been identified on the adrenals and ovaries as well. These steroidogenic tissues utilize cholesterol derived from receptor-mediated and receptor-independent pathways (2-7). Recent studies suggest that only the hepatocyte is capable of synthesizing H-TGL, and that extrahepatic H-TGL is derived directly from hepatic secretion (8). The level of hepatic secretion of H-TGL has been shown to be partially responsive to specific hormonal concentrations in the plasma and several factors in situ. However, no evidence has yet linked H-TGL expression to regulation of the cholesterol biosynthetic pathway. In t is study we demonstrate that the expression of H-TGL in the transformed hepatic cell-line, HepG2, is induced under conditions in which the cholesterol biosynthetic pathway is inhibited. Ve demonstrate that by depriving the cell of a biosynthetic intermediate prior to cholesterol, H-TGL expression is substantially induced.

MATERIALS AND METHODS

Materials. [2-14C]Acetate, sodium salt (53 mCi/mmol), tri[1- 14C]oleoylglycerol (54.3 mCi/mmol), [a-32P]dATP (400 Ci/mmol), and [a- 32P]UTP (400 Ci/mmol) were purchased from Amersham Corp. (Arlington Heights, IL). Mevinolin was obtained from Merck Sharpe and Dohme Research Laboratories (Rahway, NJ). LDL and HDL were isolated from normal human plasma by ultracentrifugation in KBr between densities 1.019-1.063 and 1.063-1.210 g/ml, respectively.

Hepatoma cells. HepG2 cells were obtained from American Type Culture Collection (Rockville, MD). They were maintained in Eagle's MEM supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine (maintenance medium) at 37°C in a humidified air atmosphere of 5% C02 • Lipoprotein-deficient serum (LPDS) was prepared from FBS by ultracentri­ fugation in KBr at density 1.21 g/ml. After centrifugation for 48 h at 214,000 x g the lipoprotein layer was removed by aspiration. The bottom fraction was dialyzed against phosphate buffered saline (PBS, 10 mM potassium phosphate, pH 7.4, containing 120 mM NaCl and 2.7 mM KCl) and sterile filtered through a Corning 0.22 micron cellulose acetate membrane (Corning Glass Yorks, Corning, NY) prior to use.

Measurement of cellular cholesterol biosynthesis. Cholesterol biosynthesis was determined in cells by measuring the incorporation of [14C]acetate into total cholesterol. Confluent monolayers of HepG2 cells were established in 36 mm well plates (Falcon) and were fed with maintenance media. Prior to each experiment cells were incubated with 10% LPDS-MEM media for 4 h. Then mevinolin was added to the cell cultures and incubated for 2 h. Cells were pulsed with 1 Ci of [14C]acetate per well for 4 h. The medium was removed, and cell mono­ layers were washed 2x with PBS. Lipids were extracted by adding 2 ml of hexane:isopropanol (3:2, v/v) to each well and allowing the extraction to proceed for 2 h at room temperature, taking care to avoid evaporation of the solvent. After removing the solvent, each well as washed 2x with 1 ml of the same solvent. The combined lipid extracts were dried under a stream of nitrogen, and each pellet was then resolubilized in 500 l of the same solvent. Ten 1, containing 10 g of unlabeled cholesterol and 5 g of unlabeled cholesterol oleate added as carrier, were spotted on silica gel TLC strips and developed in heptane:diethyl ether:acetic acid (85:14:1, v/v/v). TLC strips were air dried and stained with iodine vapor to locate cholesterol and cholesterol oleate; spots corresponding to these standards were cut out and radioactivity determined. Following lipid extraction, cell protein was solubilized in 1 ml of 1 N NaOH and quantitated by the method of Lowry et al. (9)using bovine serum albumin (BSA) as a standard.

Assay media was substrate described for H-TGL activity. H-TGL quantitated by measuring of tri[l-14C]oleoylglycerol previously (10, 11).

activity in the HepG2 cell culture the lipolytic activity towards a emulsified with Triton NlOl as Cell total cholesterol determination. HepG2 cells (approximately 2 x 10°) were trypsinized and transferred to conical 50 ml centrifuge tubes. Cells were washed with two sequential washes of 15 ml Hank's Buffer and were extracted with 5 ml of hexane:isopropanol (3:2, v/v) overnight at 4°C. After centrifugation, the cells were re-extracted with 2 ml of solvent for 30 min at room temperature. The two extractions were combined, and total cholesterol was determined by gas chromatography after saponification and extraction with hexane; free cholesterol was determined by omitting the saponification step.

Measurement of H-TGL mRNA transcript levels. Clone pST668, used for solution hybridization to quantitate H-TGL mRNA transcripts, is a 668 bp Sstl-Sstl insert (from nucleotides 115 to 783) of H-TGL pHL220 (10), ligated into the polylinker Sstl site of the vector pSP6/T7-19 (BRL, Bethesda, MD). Antisense mRNA (cRNA) probes were synthesized on a linear Pvull-EcoRl isolated fragment from the pST668 plasmid, containing the entire 668 bp internal H-TGL eDNA insert and the T7 polymerase binding site. These labeled transcripts were synthesized by the manufacturers recommendation. Tracer excess solution hybridization was carried out essentially as described by Lee et al. (12). Total cellular RNA was isolated from HepG2 cells by the guanidinium isothiocyanate procedure followed by cesium chloride centrifugation (13). The total cellular RNA pellet was resolubilized in water and passed through a RNase free G-50 Sephadex column prior to determining the nucleic acid concentration by UV absorbance at 260 nm. H-TGL transcripts were calculated per HepG2 cell based on the mass of total RNA per cell of 6.9 pg (14).

Measurement of H-TGL and HMG-CoA reductase mRNA by Northern-blot analysis. Northern-blot analysis for H-TGL was performed as described previously (10) using hybridization conditions of Busch et al. (11). For the Northern-blot analysis for HMG-eoA reductase a 2.5 kb Bgl 11 fragment containing the majority of the human HMG-eoA reductase eDNA (15) was cut from pHRED-102 (ATeei 57042) and isolated by gel electrophoresis. This

fragment was labeled with [«-32P)ATP and [«-32P]eTP to a specific

activity of 1-2 x 109 cpm/ g. Prehybridization and hybridization were performed at 42°e in 0.2% BSA, 0.2% polyvinylchloride, 0.2% ficoll, 50 mM Tris-Hel, pH 7.5, 0.1% sodium pyrophosphate, 1.0% SDS, and 50% formamide; prehybridization was performed for 4 h and hybridization for 48-72 h.

The final wash of the blots was at 65°e for 45 min in 0.1 X SSe (1 X SSe = 0.15 M Nael, 0.015 M sodium citrate, pH 7.0), 1% SDS.

RESULTS

The human hepatoma cell line, HepG2, when treated with lipoprotein deficient serum, was inducible for cholesterol biosynthesis. Incorporation of [14e]acetate into cholesterol increased approximately 2- fold from 16,654 to 35,553 cpm/mg of cell protein. This treatment did not result in altered levels of H-TGL secretion. Simultaneous addition of a cholesterol biosynthetic inhibitor, mevinolin, blocked 85% of the cholesterol biosynthesis and induced the level of secreted H-TGL activity by approximately 5-fold. H-TGL-specific mRNA levels were increased 2- fold by mevinolin treatment, whereas HMG-eoA reductase mRNA levels were induced 14-fold. The addition of LDL or HDL, in the absence of mevinolin treatment, had no significant effect on H-TGL expression. In contrast, simultaneous addition of LDL and mevinolin induced H-TGL expression 2- fold over mevinolin alone. This also resulted in a strong decrease in HMG-eoA reductase expression. These results demonstrate that a decrease in the flow of intermediates into the cholesterol biosynthetic pathway resulted in an increase in both H-TGL and HMG-eoA reductase whereas LDL feeding strongly suppressed HMG-eoA reductase. In contrast, this treatment did not suppress but instead further enhanced H-TGL expression induced by mevinolin.

Accumulation of cellular cholesterol was stimulated by mevalonic acid feeding of cells. In Table 1, the addition of mevalonic acid to cells alone caused an elevation in cellular cholesterol relative to cell protein and a decrease in the basal levels of H-TGL secretions. hen mevalonic acid was added with mevinolin, cholesterol levels were also elevated and H-TGL secretion again decreased to below controls. These results show a differential response of H-TGL regulation to mevinolin + LDL compared to mevinolin + mevalonic acid and suggest that an intermediate in the cholesterol biosynthetic pathway other than cholesterol regulates H-TGL expression.

The results in which LDL feeding in the presence of mevinolin resulted in HMG-eoA reductase repression suggested that oxidized cholesterol was produced during the metabolism of LDL and that it .caused this repression. In contrast, H-TGL expression was enhanced by this treatment. A similar effect was observed in cells treated simultaneously with mevinolin and 25-hydroxycholesterol. HMG-eoA reductase mRNA levels were reduced to below detectable levels as a result of treatment with 25- hydroxycholesterol while H-TGL mRNA levels were increased 4-fold with a concomitant increase in H-TGL secretion. e suggest that H-TGL expression is regulated by an intermediate of the cholesterol biosynthetic pathway and that maximum induction due to mevinolin and 25-hydroxycholesterol treatment results from complete blockage of this cholesterol biosynthetic pathway.

HepG2 cell cultures were fed LPDS-MEM and subsequently refed with the indicated additions at the following concentrations; mevalonic acid, 1 mM; mevinolin, 37 M. Medium was replaced every 24 h and the second change was recovered and quantitated for H-TGL activity as described in Materials and Methods. Cell protein and cholesterol were also quantitated for each culture plate as described. H-TGL activity is expressed as nmol oleic acid released/h/15 ml culture medium± SD (n=3).

DISCUSSION

In this study the hepatic mesenchymal cell-line, HepG2, was utilized to demonstrate that H-TGL expression can be induced by reducing cellular concentrations of metabolites in the cholesterol biosynthetic pathway. Addition of mevinolin to HepG2 cells clearly reduced cellular cholesterol content, induced H-TGL mRNA levels and induced the level of H-TGL secreted activity. This suggests that H-TGL expression may be regulated by the level of cellular cholesterol or intermediates in the cholesterol biosynthetic pathway. Since mevalonic acid treatment reduced H-TGL expression, we suggest that H-TGL expression is regulated by a product at or beyond the level of mevalonic acid. Both LDL-cholesterol feeding and 25-hydroxycholesterol feeding in the presence of mevinolin enhanced the mevinolin-induction of both H-TGL mRNA and secreted lipolytic activity and simultaneously reduced HMG-CoA reductase expression. This suggests a differential regulatory point between HMG-CoA reductase and H-TGL expression. The mechanism of H-TGL regulation is not at present clear. Regulation is apparent at both the level of H-TGL-specific mRNA and H-TGL protein. Post-transcriptional regulation may also be suggested by the relatively modest increases in H-TGL secretion accompanying the 4-fold increase in H-TGL-specific mRNA levels due to mevinolin and 25-hydroxy­ cholesterol treatment. Perhaps synthesis of an effector which influences either H-TGL mRNA turnover or transcription is blocked by lack of mevalonic acid. Transcriptional induction of H-TGL might occur as a consequence of decreased maturation of a repressor protein. In this regard, Beck et al. (1988) have described the requirement of mevalonic acid as a precursor for the formation of isoprenylated nuclear proteins. This isoprenyl-protein form is itself an obligate intermediate necessary for proper maturation of the nuclear laminins. Mevalonic acid depletion due to strong inhibition of HMG-CoA reductase by a combined treatment of mevinolin + 25-hydroxycholesterol would decrease the availability of mature repressor protein. Mevalonic acid feeding would therefore prevent induction by mevinolin (Table 1) and even lower basal expression when added in excess of normal cellular levels. Identification of the pathway intermediate(s) which is responsible for H-TGL repression will help to clarify the mechanism involved in H-TGL regulation.

ACKNOWLEDGEMENTS

The authors would like to thank Ms. Anjalee Jaganathen and Ms. Mary Lynn Points for help in preparation of this manuscript.

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