Evidence for Coordinate Expression of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase and Low Density Lipoprotein Binding Activity*

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1 THE JOURNAL OF BIOLOGICAI. CHEMISTRY Vol. 256, No. 12, Isme of June 25. pp , 1981 Prrnled ~n I: S. A Evidence for Coordinate Expression of 3Hydroxy3methylglutaryl Coenzyme A Reductase and Low Density Lipoprotein Binding Activity* Jean Chin$ and TaYuan Changg (Received for publication, December 24, 1980, and in revised form, March 30, 1981) From the Department of Biochemistry, Dartmouth Medical School, Hanouer, New Hampshire A Chinese hamster ovary cell mutant that requires both cholesterol and unsaturated fatty acid for growth (Limanek, J. S., Chin, J., and Chang, T. Y. (1978) Proc. Natl. Acad Sci. U. S. A. 75, ) has been further characterized with respect to its dependence on cholesterol. Upon removal of serum lipids from the growth medium, the activity of the important cholesterogenic enzyme 3hydroxy3methylglutaryl coenzyme A (HMGCoA) reductase and the low density lipoprotein (LDL) binding activity both increase significantly in the normal cell. Both these increases were much less in the mutant cell. Studies in vitro with NaF indicate that the differences in reductase activities between normal and mutant cells are not due to differences in activation by a dephosphorylation mechanism. Heat inactivation profiles and K, for HMGCoA of both cell reductases were found to be identical, thus reducing the possibility that the mutant cell contains a mutation in the polypeptide chain of reductase. The fact that in lipiddeficient medium both reductase and LDL binding activities are low in the mutant strongly suggests that the expression of these activities is controlled in a coordinate manner. This conclusion is supported by parallel studies on a spontaneous rever tant of the mutant in which the expression of reductase and LDL binding activities have both reverted to normal. These results indicate that the phenotypic abnormalities seen in the mutant are probably caused by a single mutation. A common factor is postulated to me diate this coordinate expression, and the function of such a factor is altered in the mutant cell. Cell mutants are useful biological tools for unraveling important metabolic events. A recent example is the work by Goldstein and Brown (for reviews, see Refs. 1 and 2) using cultured human fibroblasts from normal subjects and patients with familial hypercholesterolemia to elucidate the low density lipoprotein receptor pathway. Normal cultured cells take up LDL from the serum in the medium and, thus, keep the * This research was supported by National Institutes of Health Grants HL and IP30 CA23108 to T.Y.C. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked adurrtisenenl in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. f Recipient of a predoctoral fellowship from the New Hampshire Heart Association. 3 To whom communications should be addressed. The abbreviations used are: LDL, low density lipoprotein; HMG CoA reductase, 3hydroxy3methylglutaryl coenzyme A reductase; CHO cells, Chinese hamster ovary cells; DeLM, 10% delipidated serum + F12 medium; FCSM, 10% fetal calf serum + F12 medium; 6304 activity of the important cholesterogenic enzyme 3hydroxy 3methylglutaryl coenzyme A reductase suppressed. In lipoproteindeficient serum or delipidated serum medium, there is a dramatic increase in HMGCoA reductase activity and in LDL receptor number (1,2). These events are coordinated in the cell in order to synthesize and maintain an adequate amount of cholesterol necessary for cell growth and division. The isolation and biochemical characterization of various sterol auxotrophs from Chinese hamster ovary cells (3, 4) should provide further insight into the intracellular biochemical events which control cholesterol synthesis. Previously, we described the isolation of a CHO cell mutant (designated mutant 1) which requires cholesterol and unsaturated fatty acid for growth (4). In fetal calf serumsupplemented medium the mutant grows nearly as well as the normal wild type cell. However, upon removal of serum lipids from the growth medium, the mutant cell, unlike the normal CHO cell, does not produce large increases in sterol and unsaturated fatty acid synthesis. As a consequence, the mutant stops dividing and dies in DeLM unless exogenous lipids are added. Furthermore, it was shown (4) that relatively low amounts of cholesterol (5 pg/ml) and oleic acid (5.7 pg/ml) supplemented in the DeLM could provide the sustained normal cell division of the mutant cell but did not correct the induction defects present in the mutant as shown by [ *C]acetate and [ CC] stearate pulse experiments; the control experiments showed that the same lipid supplements did not significantly suppress the induction of sterol and unsaturated fatty acid biosynthesis in the normal cell. These experiments ruled out the possibility that the observed abnormalities in the mutant cell might have been due to nonspecific consequence of arrest of cell growth in DeLM. In this paper, we focus on the regulation of cholesterol metabolism in CHO cells and its defect in the mutant. Specifically, we report large differences between the normal cell and the mutant cell in their abilities in DeLM to increase the activity of HMGCoA reductase and the activity of LDL binding. Parallel studies on a putative spontaneous revertant (4) of the mutant (designated revertant I) are also presented. EXPERIMENTAL PROCEDURES MaterialsBiochemicals and most lipids were from Sigma Chemical Co. 25Hydroxycholesterol was from Steraloids and judged to be >98% pure by gasliquid chromatography and by four thin layer Chromatography systems. 25OH was added to tissue culture medium from a stock 0.2% (w/v) solution in dimethylsulfoxide. Cholesterol (Sigma) was >99% pure by gasliquid and thin layer chromatography analyses and was added to medium from a stock 0.5% (w/v) solution in ethanol just prior to use. Control experiments have shown that UP to 1% ethanol or dimethylsulfoxide in growth medium for 24 h has no 25OH, 25hydroxycholestero1; zineethanesulfonic acid. Hepes, 4(2hydroxyethyl)lpipera

2 HMGCoA Reductase and LDL Binding Activities in CHO Cells 6305 effect on HMGCoA reductase activity in treated normal or mutant cells as previously reported (5). Oleic acidbovine serum albumin complex was prepared according to a published procedure (6). Human low density lipoprotein, d to g/ml, was prepared according to a published procedure (7). The composition of LDL and the relative distribution of its lipid components agreed with published data (8). The protein concentration of LDL was determined by the method of Lowry et al. (9) with bovine serum albumin as the standard. All concentrations of LDL reported in this paper are in terms of LDLprotein. ["H]Cholesterol (58 mci/mmol) was purchased from New England Nuclear. Sodium ["'I]iodide (17 mci/pg) was obtained from both New England Nuclear and Amersham/Searle. CellsAs previously described (4, 5, 10) CHO cell cultures were grown as monolayers in 25cm' or 75cm' Falcon tissue culture flasks in F12 medium (linoleic acid deleted) supplemented with either 10% fetal calf serum or 10% delipidated serum. Fetal calf serum was delipidated according to a published procedure (11). The protein concentration of the delipidated serum was adjusted to that of the fetal calf serum (40 mg/ml) after protein determination (9). The final concentration of cholesterol and fatty acid in the DeLM as determined by gasliquid chromatography was 0.3 pg/ml and 2.2 pg/ml, respectively. These values represent a 9870 extraction of cholesterol and a 92% extraction of fatty acids from the FCSM. Previously, the delipidated serum was prepared by extracting the serum lipids twice with organic solvents (4). Its purpose was to maximize the growth differences between the normal and mutant cell. The disadvantage was that the mutant was viable only for 24 h in DeLM. Later it was found that the once extracted delipidated serum was adequate in distinguishing the growth differences of the various cell types. All DeLM reported in this paper have employed delipidated serum extracted once with organic solvents. AssaysWhole cell homogenates prepared by modifications of a previously described method (4) were used to assay for HMGCoA reductase activity. The cells were broken by Dounce homogenization after hypotonic shock in 5 mm potassium phosphate, ph 7.4,0.5 mm sodium EDTA, and the homogenates (0.5 to 2.0 mg of protein/ml of hypotonic buffer containing 20 mm imidazole, ph 7.4, 5 mm dithio threitol) were preincubated at 37 "C for 20 min. The HMGCoA reductase activity of this cell homogenate was assayed for 60 min at 37 "C as previously described (4). A unit of activity is defined as 1 nmol of mevalonate formed per min per mgof cell homogenate protein. Protein for the HMGCoA reductase assay, LDL binding and uptake, and growth curve experiments was determined by a microbiuret method (12) using bovine serum albumin as the standard. For all assays described in this report, values shown in each figure or table represent average of results from duplicate flasks. Duplicate assays were performed using cell homogenate prepared from each flask. Variation of results between duplicate flasks was within 7% from the mean. Preparation of "'ZLDLOne to two mgof LDL was iodinated with 1 mci of sodium ["'Iliodide in 1 M glycinenaoh, ph 10, using the iodine monochloride method of McFarlane (13) as modified by Bilheimer et al. (14). After stopping the reaction with NaI and Na2S203 (15) and diluting to 1 ml with dialysis buffer, the labeled LDL was extensively dialyzed at 4 "C against Iliter volumes of20 mm TrisHCI, ph 7.4, 0.15 M NaCI, and 0.3 mm sodium EDTA. The buffer was changed every 30 min for 5 h and then every 60 min for 3 h. The labeled LDL was filtered through a 0.45pm Millipore filter and used in the assays without dilution by unlabeled LDL. Several lots of "'ILDL were prepared, with specific activities ranging from 3,ooO12,OoO cpm/pg of LDL protein, and each contained less than 1 atom of iodine per molecule of LDL. In these preparations 9294% of the radioactivity was precipitable by 16% trichloroacetic acid while of the counts was extractable with chloroformmethanol; these values agree with published data (16). Control experiments indicated that undiluted "'ILDL at various concentrations (5 to 20 pg/ml) was able to suppress HMGCoA reductase activity as effectively as unlabeled LDL. This suppression was prevented by heparin or chloroquine. Binding, Uptake, and Degradation of '?'ILDL by Cell Mono layersthese parameters were assayed following the incubation, washing, and harvesting procedures of Goldstein et al. (17) briefly described as follows. After 24 h in DeLM or FCSM with each medium supplemented with 2 mm CaCln and 10 mm Hepes, ph 7.5, the monolayers were preequilibrated with fresh medium for 30 min at 4 "C or 37 "C. Various amounts of "'ILDL were added, and the cells were further incubated for 1 h at 4 "C for binding or at 37 "C for uptake. Specific binding was determined after extensive washing and incubation for 1 h at 4 "C with 10 mg of heparin/ml of Hepes buffer by measuring the heparinreleasable "'ILDL. Cellular uptake was determined after extensive washing by measuring the '*'I in the NaOHdissolved cells. Degradation of "'1LDL was determined by measuring the trichloroacetic acidsoluble I2'I in the medium from cells that had been incubated with l2'1ldl for 5 h at 37 "C (16). The "'I was determined with a y counter at 80% efficiency. Preparation of [3H]Cholesteryl LinoleateLDL["HICholesteryl linoleate was prepared as described by Goodman (18) and purified according to Faust et al. (19). Following published procedures (19, 20), 1 mg of LDL protein was incubated with 40 pci of [3H]cholesteryl linoleate in dimethylsulfoxide for 2 h at 40 "C. After extensive dialysis for 48 h at 4 "C against 0.15 M NaCl, 0.3 mm EDTA, ph 7.0, the labeled LDL was filtered through a 0.45pm Millipore filter, stored at 4 "C, and used within a week of preparation. Several lots were prepared with specific activities of20,ooo30,ooo dpmlpg of LDL protein, and they were used without dilution by unlabeled LDL. Control experiments have indicated that the labeled LDL was as effective as unlabeled LDL in suppressing the activity of HMGCoA reductase in cells incubated in DeLM. RESULTS Growth Properties of the Mutant Cell in DeLMWe have optimized the procedure for the preparation of delipidated serum (described under "Experimental Procedures") so that the mutant cell can survive in DeLM without cholesterol and unsaturated fatty acid supplements for longer periods of time than was described previously (4). As shown in Fig. 1, the mutant cell grown in DeLM for 24 h was dividing nearly as rapidly as the mutant cell grown in FCSM; in fact, the mutant cell can be maintained viable and adherent to the growth surface in DeLM for 48 h. Addition of cholesterol and oleic acid to DeLM allowed the mutant to grow as well as the u O\ Time (days) FIG. 1. Growth curve of the mutant cell in FCSM, DeLM supplemented with lipids, and DeL". Mutant cells were seeded at 0.10 X IOfi cells/25cmy flasks in 3 ml of FCSM, while normal cells were seeded at 0.05 X lo6 cells/25cm2 flasks. After 2 days the monolayers were washed twice with 5 ml of phosphatebuffered saline, and then 6 ml of the indicated medium were added. Each medium was changed once a day up to 4 days. Cell growth was monitored at each time point by measuring cellular protein content as described previously (4) in duplicate flasks. In this experiment, the growth curve of normal cells in DeLM was found to be essentially identical with that of the mutant cell in FCSM; the normal cell growth curve was not presented. A, mutant cells in FCSM; A, mutant cells in DeLM + 10 pg of cholesterol/ml + 2 X M oleic acidbovine serum albumin complex; A, mutant cells in DeLM.

3 ~~ 6306 HMGCoA Reductase and LDL Binding Activities in CHO Cells Time (hours) FIG. 2. Time course of the change in normal, mutant, and revertant ceu HMGCoA reductase activity between cella incubated in FCSM and cells incubated in DeL". Normal and revertant cells were plated at 0.15 X IOF cells/25cm2 flask, and mutant cells were plated at 0.30 X 10' ceus/25cmz flask, all in 3 ml of FCS M. After 2 days of growth, the cells were washed twice with 5 ml of phosphatebuffered saline, and then 6 ml of either FCSM or DeLM were added. There were no further media changes. At time 0, 12, 24, 36, and 48 h, duplicate flasks were washed three times with phosphatebuffered saline, and whole cell homogenates were prepared to assay for HMGCoA reductase activity. 0 and 0, normal cell in FCSM and DeLM; W and 0, revertant cell in FCSM and DeLM; A and A, mutant cell in FCSM and DeLM, respectively. mutant cell in FCSM and as the normal cell in DeLM. All of the experiments reported in this paper involving cells grown in DeLM were completed within 48 h; most of them were completed shortly after 24 h in the lipiddeficient medium. Defect in the Increase in Activity of HMGCoA Reductase in the Mutant Cell Grown in DeL"As shown previously (4, 5, lo), removal of serum lipids from the medium caused a large and rapid increase in KMGCOG reductase activity in the normal CHO cell. Fig. 2 is a time course of this activity increase in the normal, revertant, and mutant cells. Both the normal and revertant cells showed a maximal increase of 1.35 units or a 1Cfold increase in enzyme activity in 24 h, whereas the mutant cell showed an increase of 0.20 units, or a 2fold increase in enzyme activity throughout the time course. The experiment indicates a defect in the expression of HMGCoA reductase in the mutant cell in DeLM.2z It is interesting to note that the reductase activity of the mutant cell grown in FCSM was 2 to 3fold higher than that. of the normal cell. This observation is explored later in Figs. 5 and 7. In addition, it should be pointed out that, as seen in Fig. 2, after 24 h in DeLM, there was a decline in HMGCoA reductase activity in all three cell types. This decrease was observed whenever there was no further medium change, as indicated in the legend or Fig. 2. We suspect that this could be due to the possibility that the concentration of cholesterol acceptors in the medium has become rate limiting, and that would prevent any further loss or exchange of cellular cholesterol into the In experiments not shown, cells grown in lipoproteindeficient serum medium gave the same results as those grown in DeLM. Experiments not presented here have shown that after removal of serum lipids from the medium for 24 h, the activity increase of NADHcytochrome bs reductase was found to be very similar between the normal and mutant celb. Also, using intact cells, control experiments have shown that, under the same growth condition, the glycolysis rate (as measured by the rate of lactate formation from glucose) as well as the respiration rate (as measured by the rate of "CC02 formation from [2'4C]pyruvate) were found to be very similar between the normal and mutant cells, indicating that the genera1 physiology of the mutant cell grown in DeLM for 24 h remained normal. medium. In numerous other experiments, we have found that. if the DeLM was changed every 12 h, the maximal levels 01 reductase activity in the normal and revertant cells were maintained. There was, however, no further or late increase in the mutant cell reductase activity. Properties of HMGCoA Reductase in Normal and Mutan CellsActivation of HMGCoA reductase by enzymatic de phosphorylation in vitro has been reported by a number o laboratories (2124). Although the physiological role of thi: phenomenon has yet to bestablished, it may be responsibb for the observed differences in reductase activity between thc normal and mutant cell as presented in Fig. 2. The work o Nordstrom et al. (21) has shown that there is an endogenou: activator (phosphatase) present in cell homogenates which ir inhibitable by NaF. Our results using whole cell homogenate! of CKO cells and presented in Table I confum these obser vations. Addition of NaF to the cell homogenate did decreast the reductase activity in both cell types incubated in eithe! FCSM or DeLM. However, NaF had essentially no effect or the increase in reductase activity between cells grown in FCS M and those grown in DeLM. The 7.8fold increase for thc normal cell and the 1.6fold increase for the mutant cell wen found with or without NaF treatment. In experiments no1 shown here, addition of excess exogenous alkaline phospha. tase to the cell homogenates of both cell types in the absencc of NaF resulted in no further increase of reductase activity indicating that the endogenous phosphatase activity was no1 rate limiting for the activation process. These data indicate that the low reductase activity of the mutant cell in DeLM is not due to a defect in the activation of HMGCoA reductase by a dephosphorylation mechanism. These data also indicatc that dephosphorylation does not play a major role in cont,rol. ling the fold increase in reductase activity in either cell type in vitro. Fig. 3 is a heat inactivation profile of HMGCoA reductase activity in normal and mutant cells grown in FCSM. The rate of loss in activity at 60 "C was essentially identical. Also, the K, for HMCCoA of reductases from the normal and mut,ant TABLE I Effect of NaF on HMGCoA reductase uctivity of normal and mutant cells in FCSM and DeLM Cells were grown as described in Fig. 2. After 24 h in either FCSM or DeLM, cell homogenates were prepared in the presence or absence of 50 mm NaF and used to assay for HMGCoA reductase activity with or without 50 mm NaF in the assay mixture. Samples were assayed in duplicate. Values shown represent average of results from duplicate flasks. Variation between duplicates was within 7% from the mean. A. Reductase activity in normal cells 50 mm NaF HMGCoA Reductase in Fold increase reductase activity Cell homo Assay FCSM DeLM from FCSM to eenate mixture DeLM nmol.rnin".rng" i i B. Reductase activity in mutant cells 50 RIM NaF HMGCoA Reductase in Fold increase in reductase activity Cell homo Assay from FCSM to FCSM DeLM genate mixture DeLM nmol.rnin".rng" t

4 HMGCoA Reductase and LDL Binding Activities in CHO Cells 6307 o L ~ 20 ' ' 40 ' ' 60 ' ' ' Time at 60 C (minl FIG. 3. Heat inactivation at 60 "C of HMGCoA reductase activity in normal and mutant cells grown in FCSM. Normal and mutant cells were plated at 0.3 X IO6 cells/75cm2 flask and at 0.4 X IO6 cells/75cm2 flask in 10 ml of FCSM, respectively. After 2 days of growth the cells were grown for 24 h in 10 ml of fresh FCSM and then harvested as described in Fig. 2. Cell homogenates were preincubated at 60 "C for the indicated amount of time and then assayed in duplicate for residual HMGCoA reductase activity. The control specific activity of reductase was 0.11 nmolmin". mg" in normal cells and 0.27 nmol.min". mg' in mutant cells. 0, normal cell in FCSM; A, mutant cell in FCSM. io 40 io io u O aj LDL (pglmll LDL (pgtml) (L C Normal + 25OH cells grown in FCSM had been compared and found to be identical (K, = 1.4 k 0.14 PM for the Disomer). Taken together, these results are evidence against the possibility that the normal and mutant cells possess structurally and catalytically different reductases. Suppression of HMGCoA Reductase Activity in Normal and Mutant Cells by LDL or by 25HydroxycholesterolAs shown in Fig. 2 the reductase activity of the mutant cell grown in FCSM is 2 to 3foJd elevated with respect to that of the normal cell. This observation suggested the possibility that the mutant enzyme activity may not be suppressible by sterols. To test this possibility, suppression of HMGCoA reductase with varying concentrations of LDL or 25hydroxycholesterol was done using cells incubated in DeLM, and the results are presented in Fig. 4. The normal cell reductase was suppressed by both LDL and 25OH, whereas the mutant cell reductase was suppressed by 25OH, but not by LDL. Since 25OH has been found to suppress reductase activity in a manner independent of the LDL receptor (25, 26), this result strongly suggests that there may be a defect in the LDL pathway in the mutant cell. To investigate this possibility further, an experiment was done to test theffect of chloroquine on the LDL suppression of HMGCoA reductase in both cell types. As shown by Goldstein et al. (27, 28) in human fibroblasts, chloroquine inhibits lysosomal function which is necessary for the hydrolysis of LDLcholesteryl ester. Inhibition of this process prevents the LDLmediated suppression of reductase. We have confiied these observations in CHO cells. In Fig. 5, LDL, in the presence of chloroquine, failed to suppress the normal cell reductase (Fig. 5A) while it had little effect on the mutant cell reductase in the presence or absence of chloroquine (Fig. 5C). Addition of chloroquine to FCSM caused a 3fold elevation in the normal cell reductase (Fig. 5B) but caused no change in the mutant cell reductase (Fig. 50) which as indicated earlier in Fig. 2 was already 2 to 3fold elevated. The O dl d.2 d.3 d4 d5 d l 012 d3 d4 oi 25OH (pg/mli 25OH (pq/mll FIG. 4. Suppression of HMGCoA reductase activity by addition of LDL or 25OH to DeL" in normal and mutant cells. Normal and mutant cells were plated at 0.10 X IO" cells/25cm2 flask and 0.15 X 10' cells/25cm2 flask in 3 ml of FCSM and grown as described in Fig. 2. After 24 h in DeLM, with media changes at 0 and 12 h, the indicated concentrations of LDL or 25OH were added, and the cells were further incubated at 37 "C for 6 and 12 h, respectively. (Incubation of the cells with LDL for 12 h instead of 6 h gave the same results.) Whole cell homogenates were prepared and used to assay for HMGCoA reductase activity. A, normal cell with LDL; B, mutant cell with LDL; C, normal cell w;$h %OH; D, mutant cell with 25OH. The control specific activitiep in.mol.min'. mg.' were: A, 1.47; B, 0.53; C, 1.06; D, LDL Binding and Uptake in Normal, Mutant, and Revertant C~~~S'~~ILDL was prepared according to published procedures (see under "Experimental Procedures") and used to measure LDL receptor binding and ~ptake.~ Fig. 6 indicates the amounts of Iz5ILDL bound by monolayers at 4 "C or the amounts taken up at 37 "C in 1 h by normal and mutant cells in either FCSM or DeLM. As can be seen, in normal cells there was a large increase (5.7fold) in LDL binding activity between cells incubated in FCSM and those incubated in DeLM (Fig. 6A). This increase in binding correlated well with the increase in uptake of LDL by the normal cell (Fig. 60. On the other hand, there was a much smaller increase (2fold) in LDL binding activity between mutant cells incu bated in FCSM and those incubated in DeLM (Fig. 6B). This small increase in binding again correlated well with the From experiments not presented, we have found that binding of '"ILDL by normal and mutant CHO cells was saturable, with halfmaximal saturation at approximately1012pg/ml. It was largely prevented by unlabeled LDL and inhibited by heparin and dextran sulfate. Degradation of '2sILDL and hydrolysis of [3H]cholesteryl linoleateldl were found to correlate well with the LDL binding and results presented in Figs. 4 and 5 indicate that a defect in LDL uptake data. These results, coupled with previously published findings (5) that suppression of HMGCoA reductase activity was specific for uptake may exist in the mutant cell. The next set of experi LDL and not for HDL, confirm the reports by Goldstein and Brown ments tested this possibility directly with radioactively labeled (29, 30) that the LDL receptor pathway is present and operational in LDL. CHO cells.

5 6308 HMGCoA Reductase and LDL Binding Activities in CHO Cells I A Norrnol m DeiM FIG. 5. Effect of chloroquine on the LDLmediated suppression of HMGCoA reductase activity in normal and mutant cells. Cells were prepared as in Fig. 4. After 24 h in FCSM or DeL M, 100 p~ chloroquine was added to certain flasks with or without LDL. Following a 6h incubation at 37 "C, whole cell homogenates were prepared and used to assay for HMGCoA reductase activity. A, normal cells in DeLM f LDL f chloroquine; B, normal cells in FCS M f chloroquine; C, mutant cells in DeLM f LDL f chloroquine; D, mutant cells in FCSM f chloroquine. The various media were: a, DeLM; b, DeLM + 42 pg of LDL/ml; c, DeLM + 84 pg of LDL/ml; d, DeLM + 42 pg of LDL/ml p~ chloroquine; e, DeLM + E3 pg of LDL/ml p~ chloroquine; f, FCSM; and g, FCSM p~ chloroquine. cedures"). The less dramatic difference in LDL uptake at 37 "C between normal and mutant cells probably reflects the fact that the total uptake of LDL is a combination of specific uptake via the LDL receptor and of nonspecific uptake independent of the LDL receptor (2). In human fibroblasts, the nonspecific uptake of LDL has been shown by Goldstein and Brown (16) to be due to cellular pinocytosis; its rate of uptake was linearly proportional to the LDL concentration in the medium and nonsaturable. The relative proportions of specific and nonspecific uptake of LDL by CHO cells have been determined by two different methods described by Goldstein and Brown (16), the "slope peeling" technique and the displacement of radioactive LDL by an excess of nonradioactive LDL. In the normal cell, the specific uptake at 25 pg of '251LDL/ml of DeLM accounted for about 70% of the total observed uptake at 37 "C, whereas the nonspecific uptake contributed about 30%. Comparison of the nonspecific uptake by normal and mutant cells indicated that it was similar for both cell types. These results indicate that the differences between normal and mutant cell uptake of LDL are due primarily to the differences in specific binding of LDL by the cells. It is interesting to note that, despite the substantial nonspecific uptake of LDL at high concentrations of LDL in DeLM, this LDL did not suppress the HMGCoA reductase activity in the mutant, as shown Fig. 4, confirming in the earlier work of Brown and Goldstein (2). Fig. 7 is the time course of the increase in LDL uptake in the normal, mutant, and revertant cells upon removal of serum lipids from the growth medium. As can be seen, in the normal and revertant cells, there was a 4 to 5fold increase in LDL uptake between cells grown in DeLM and those grown in FCSM; this increase peaks at 24 h. In contrast, such an increase was much less in the mutant cell. Since a nearly saturating concentration of '251LDL(25 pg/ml) for specific binding was used in this experiment, these data suggest that the differences in LDL uptake seen among the normal, mutant, and revertant cells probably reflect the differences in c 800 C Normal at 37 C \ m c 600 c 0) 500 FIG. 6. Surface binding and cellular content of TLDL in normal and mutant cells. Cells were plated and grown as described in Fig. 2. After 24 h in DeLM with media change at 0 and 12 h, cells were incubated for 1 h at 4 "C with 18 pg of "51LDL/ml (4196 cpm/ pg) and at 37 "C with 25 pg of "'ILDL/ml (2812 cpm/pg). The monolayers were then extensively washed. Cells incubated at 37 "C were harvested for cellular uptake of "'1LDL while those at 4 "C were further treated with 10 mg/ml of heparin for 1 h at 4 "C to determine surfacebound "'ILDL. smaller increase in uptake of LDL by the mutant cell (Fig. 6D). These results have been confiied by separate experiments using [3H]cholesteryl linoleateldl prepared according to published procedures (see under "Experimental Pro 0 d 400 J N 200 I 00L I C Time (hours) FIG. 7. Time course of '"ILDL uptake by normal, mutant, and revertant cells in FCSM and DeLM. Cells were plated and grown as described in Fig. 2. At time 0, 12, 24, 36, and 48 h, cells were incubated for 1 h at 37 "C with 25 pg of I2"ILDL/ml (2812 cpm/pg) and harvested for uptake of LDL as described in Fig and 0, normal cell in FCSM and DeLM; and 0, "revertant" cell in FCS M and DeLM; A and A, mutant cell in FCSM and DeLM, respectively.

6 HMGCoA Reductase and LDL Binding Activities in. CHO Cells 6309 number of LDL receptors present. Changes in the number of normal. These results indicate that the phenotypic abnormal LDL receptors on the cell surface upon addition or removal of LDL have been previously demonstrated in cultured human ities seen in mutation. the mutant are probably caused by a single fibroblasts (31). It is interesting to note that LDL uptake in FCSM by the mutant was lower than that by either the normal or revertant cell. This observation may explain why the HMGCoA reductase activity in the mutant cell grown in FCSM was 2 to 3 fold elevated with respect to that in the normal or revertant cell. The fact that the activity increases in LDL binding and in HMGCoA reductase were both shown to be defective in the mutant strongly suggest that these two activities are coordinately expressed. This conclusion is further substantiated by the fact thathese activities are both reverted to normal in a A common factor is postulated to mediate the coordinate expression of reductase and LDL binding activity; the function of this factor is altered in the mutant cell. It is unknown at present whether this factor is involved in the transmission of the signal of lipid depletion of the growth medium or in the responses of the cell to such a signal. This factor may be affecting the expression of other cholesterogenic enzymes. The existence of a common regulatory factor has been suggested by recent studies from this laboratory (5) on CHO cell mutants resistant to suppression by 25 hydroxycholesterol. Further investigation is needed to determine whether these factors have any functional or structural naturally occurring revertant of the mutant (Figs. 2 and 7). relationship to each other. This coordinate expression may be effected by a common factor, whose function is rendered abnormal by mutation in the mutant cell. Further biochemical characterization of the mutant presented in this paper should help to elucidate the mechanism for the regulation of cholesterol metabolism at the molecular level. It should also help to determine how this mutation is DISCUSSION The CHO cell mutant presented in this paper has been affecting the synthesis of unsaturated fatty acid in CHO cells. Current studies in this direction are in progress. characterized with respect to its requirement for cholesterol and compared with the normal cell. The data indicate that AcknowledgmentWe wish to acknowledge James S. Limanek of the activity of the putative ratelimiting cholesterogenic en this laboratory for determining the K, for HMGCoA. zyme HMGCoA reductase and the LDL binding activity both increased significantly in the normal cell upon removal of REFERENCES serum lipids from the growth medium. 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