361 Biochimica et Biophysics Acta, 574 (1979) o Elsevier/North-Holland Biomedical Press
361-365
BBA Report BBA 51256
REGULATION OF STEROL SYNTHESIS IN HUMAN LYMPHOCYTES: EVIDENCE FOR POST-TRANSCRIPTIONAL CONTROL BY LOW DENSITY LIPOPROTEIN
WILHELM
KRONE*,
D. JOHN BETTERIDGE
Diabetes and Lipid Research Laboratory, (U.K.) (Received
and DAVID J. GALTON
St. Bartholomew’s
Hospital, London EClA
7BE
June lst, 1979)
Keywords: Hydroxymethylglutaryl-CoA control; LDL; (Human lymphocyte)
reductase; mRNA synthesis; Post-transcriptional
Summary The effect of cordycepin (3’-deoxyadenosine), an inhibitor of messenger RNA synthesis, on the induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase mediated by lipid-depleted serum was studied in isolated human lymphocytes. 50 pg/ml cordycepin, although inhibiting messenger RNA synthesis by more than 50%, had no inhibitory effect on the two and four-fold induction of hydroxymethylglutaryl-CoA reductase when cells were incubated in a medium containing lipid-depleted serum for 8 and 16 h, respectively. This result suggests that newly synthesises messenger RNA is not required for the effect of lipid-depleted serum on the induction of hydroxymethylglutaryl-CoA reductase in human lymphocytes.
The activity of the microsomal enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase determines the rate of cholesterol synthesis in a wide variety of mammalian cells [l]. The enzyme activity is regulated primarily by cholesterol which enters the cell bound to low density lipoprotein (LDL). Thus when isolated human leucocytes and cultured human fibroblasts are incubated in the presence of LDL the activity of hydroxymethylglutaryl-CoA reductase is suppressed. However, incubation of cells for several hours in a medium containing lipid-depleted serum leads to a substantial increase in the activity of the enzyme. Subsequent exposure to cholesterol either as LDL or in an ethanolic *Present address: Klinisches Institut fiir Herzinfarktforschung an der Medizinischen Universit;itskllnik Heidelberg. Bergheimerstrasse 58. D-6900 Heidelberg, F.R.G.
362
solution is followed by a suppression of the enzyme activity [2-41. Since under these conditions sterol synthesis is strictly proportional to the activity of hydroxymethylglutaryl-CoA reductase, the incorporation of [14C]acetate into sterols can be taken as a measurement of enzyme activity [5, 61. Four lines of evidence indicate that the rise in hydroxymethylglutarylCoA reductase activity in the presence of lipid-depleted serum is due to an increased de novo synthesis of the enzyme and not due to activation of preformed enzyme molecules or decreased degradation of the enzyme: (i) incubation of cells in a medium containing lipid-depleted serum, after a lag period of l-2 h, results in a linear rise in enzyme activity for at least 20 h; (ii) neither whole serum, LDL or cholesterol added to cell free extracts of fibroblasts produce any inhibitory effect on hydroxymethylglutaryl-CoA reductase activity; (iii) when extracts with low enzyme activity obtained from cells grown in whole serum are mixed with extracts from cells with high enzyme levels having been grown in lipoprotein-deficient serum the total combined activity of hydroxymethylglutaryl-CoA reductase is equal to the sum of the individual activities of the extracts when measured separately and (iv) when leucocytes or fibroblasts are incubated in lipid-depleted serum, the subsequent stimulation of the conversion of [14C]acetate into sterols [7] or the rise in hydroxymethylglutaryl-CoA reductase activity [8], respectively, is prevented by cycloheximide. Furthermore, in the presence of cycloheximide, the activity of the enzyme declines with first order kinetics and a half-life of about 3 h, the half-time of decay being similar in cells with depressed or elevated enzyme levels. An attractive model for the regulation of hydroxymethylglutaryl-CoA reductase activity by LDL-cholesterol has been proposed by Goldstein and Brown (for review, see Ref. 9). According to this plasma-LDL binds with high affinity to specific cell surface receptors. The membrane-bound LDL is then internalised by endocytosis and delivered to lysosomes, where the lipoprotein is degraded. Free cholesterol released from the lipoprotein reduces endogenous cholesterol synthesis by suppression of the activity of HMG-CoA reductase. However, the mechanism by which LDL-cholesterol acts on the enzyme is not known. We therefore studied the effect of cordycepin (3’-deoxyadenosine), an inhibitor of RNA synthesis, on the induction of sterol synthesis by lipiddepleted serum in isolated human lymphocytes and wish to report that newly synthesised mRNA is not required for the effect of lipid-depleted serum on the induction of sterol synthesis suggesting that hydroxymethylglutaryl-CoA reductase is regulated by LDL-cholesterol at a post-transcriptional or translational level. Isolation and incubation of lymphocytes. Peripheral human blood lymphocytes were isolated by the method of Boyiim [lo]. Cells were incubated at 37°C in 2 ml Krebs-Ringer phosphate buffer (pH 7.4), supplemented with aminoacids, vitamins, 15 mM glucose, 0.5 mM sodium acetate, 100 units penicillin/ml, 100 ,ug streptomycin/ml and 40% serum (which was depleted of lipid by the method of McFarlane [ll]). Incorporation of [jH]adenosine into cy toplasmic poly(A)-containing RNA. 2 X lo6 lymphocytes were labelled with AOOpCi [3H]adenosine (23 Ci/mmol)
for 40 min. rRNA synthesis and mtRNA synthesis were first suppressed with 0.4 pg/ml actinomycin D [12] and 1 pg/ml ethidium bromide [13], respectively.
363
Actinomycin D, ethidium bromide and cordycepin were added 30 min prior to labelling. Cytoplasmic extracts were prepared by harvesting, washing and resuspending cells in 2 ml of hypotonic buffer (10 mM NaCl, 1.5 mM MgCl?, 10 mM Tris-HCI, pH 7.4), adding the detergent Nonidet (NP40) to a final concentration of 0.5% and agitating with a vortex mixer for 30 s; nuclei were removed by centrifugation at 800 X g for 2 min [14]. The cytoplasmic RNA was extracted by a conventional phenol/CHC& procedure [15]. Poly(A)-containing RNA was isolated by binding to Oligo(dT)-cellulose. The bound RNA (poly(A)’ RNA), eluted with a NaCl-free buffer, and the unbound RNA (poly(A)- RNA) .were precipitated with 2 volumes of ethanol at -20°C for 18 h. The precipitates were dissolved in water and radioactivity in both types of RNA was measured by liquid scintillation spectrometry using a toluene-based scintilla&. The recovery of 32P-labelled ribosomal RNA was used to correct for procedural losses of [3H]RNA. Incorporation of [14C]acetate into sterols. 5 X lo6 cells were incubated at 37°C for the appropriate time when 1OpCi [2-14C]acetate (55 Ci/mol) was added and the incubation continued for 2 h. Incubations were terminated by the addition of CHC13/CH30H (1:2) and [1,2-3H]cholesterol was added as an internal standard. Lipids were extracted by the method of Bligh and Dyer [16] and were saponified by methanolic 2M KOH for 6 h at 70°C. The nonsaponifiable fraction was extracted with hexane and radioactivity was measured by liquid scintillation spectrometry using a toluene based scintillant. The recovery of [3H]cholesterol was used to correct for procedural losses of 14Clabelled nonsaponifiable lipids. Since changes in sterol synthesis are strictly proportional to changes in activities of hydroxymethylglutaryl-CoA reductase, the incorporation of [14C]acetate into sterols can be taken as a measure for the enzyme activity in lymphocytes [5, 61. Actinomycin D has previously been reported to prevent the increase in hydroxymethylglutaryl-CoA reductase after removal of LDL from the incubation medium in cultured human fibroblasts [8]. Similarly, we found that this antibiotic (0.4 E.cg/ml) blocked totally the subsequent rise in sterol synthesis from [14C]acetate in freshly isolated human lymphocytes following incubation in lipid-depleted serum. It should however be noted that actinomycin D, in addition to blocking transcription, can produce non-specific effects such as inhibition of the initiation of protein synthesis [17]. Although the mechanism by which cordycepin acts at the molecular level is still debated [18, 191, the effect of the drug in HeLa cells [14], mouse sarcoma 180 cells [20] and in cultured human fibroblasts [21] is to inhibit mRNA synthesis. The results shown in Fig. 1 demonstrate that addition of cordycepin to isolated lymphocytes 30 min prior to the addition of [3H]adenosine inhibits the appearance of the isotope in the cytoplasmic poly(A)containing RNA while it has no effect on the labelling of cytoplasmic RNA containing no poly(A)-sequences. As poly(A)-containing RNA represents most of the mammalian mRNA [22], our data indicates that cordycepin can substantially inhibit mRNA synthesis in lymphocytes. Although 50pg/ml cordycepin inhibited poly(A)-containing RNA synthesis by more than 50% (Fig. 1) the drug had no inhibitory effect on the two- and four-fold increase in sterol synthesis from [14C]acetate in cells incu-
364
. ” 0 c
x
E
B
0
d
0
25 CORDYCEPIN
50 (fig/ml)
1
1
I
0
a
16
TIME (HOURS)
Fig. 1. Effect of varying concentrations of cordycepin on the incorporation of r3Hladenosine into cytoplasmic poly(A>containing RNA (o---O) and RNA without poly(A)_sequences (o-o) in isolated human lymphocytes. Values are given as means f S.E. for 3-4 experiments. Fig. 2. Effect of varying concentrations of cordycepin on the induction of sterol synthesis from [“Cl acetate by lipid-depleted serum in isolated human lymphocytes. Controls without cordycepin (o--O), cordycepin 12.5 pglml (e-e), 25 pg/ml (A-a) and 50 pg/ml (m-m). Values are means + S.E. for 3-4 experiments.
bated in a medium containing lipid-depleted serum for 8 and 16 h, respectively (Fig. 2). Assuming that cordycepin inhibits synthesis of mRNA for the enzyme to a similar extent as total mRNA synthesis, our results suggest that newly synthesised mRNA is not required for the effect of lipid-depleted serum on the induction of hydroxymethylglutaryl-CoA reductase in lymphocytes. From the time course of the induction of hydroxymethylglutaryl-CoA reductase (Fig. 2) it appears that the half-life of the mRNA for the enzyme is relatively long. This is supported by the following experiment : lymphocytes were preincubated with full serum for up to 40 h in the presence of 50pg/ml cordycepin. This should significantly reduce the level of mRNA with a short half-life. Transfer of the cells from full to lipid-depleted serum and further incubation for 10 h again in the presence of 50 fig/ml cordycepin, resulted in a three-fold increase in sterol synthesis from [14C]acetate and this increase was the same in cells preincubated in the absence of cordycepin. These results suggest that in human lymphocytes neither the induction of hydroxymethylglutaryl-CoA reductase by lipid-depleted serum nor the subsequent repression of the enzyme by LDL-cholesterol, which occurs with a half-time of approx. 3 h [6], can be accounted for by a corresponding increase or decrease in the level of mRNA coding for the enzyme. LDL-cholesterol may
365
therefore regulate hydroxymethylglutaryl-CoA scriptional or translational level.
reductase
at a post-tran-
We gratefully acknowledge advice and assistance from Drs. P.S. Rudland and P. Piper (Imperial Cancer Research Fund). W. Krone is in receipt of a grant from the Deutsche Forschungsgemeinschaft. D.J. Betteridge is a R.D. Lawrence Research Fellow of the British Diabetic Association.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
RodweII, V.W.. Nordstrom, J.L. and Mitsehelen. J.J. (1976) Adv. Lipid Res. 14. l-74. Brown, M.S., Dana, S.E. and Goldstein, J.L. (1974) J. Biol. Chem. 249. 789-796. Higgins, M.J.P.. Lecamwasam. D.S. and G&on. D.J. (1975) Lancet ii, 737-739. Ho, Y.K., Faust, J.R.. Bilheimer. D.W., Brown, M.S. and Goldstein, J.L. (1977) J. Exp. Med. 145, 153 l-l 549. Fogelman. A.M., Edmond. J.. Seager. J. and Popjak. G. (1975) J. Biol. Chem. 250, 2045-2055. Higgins, M.J.P. and Galton, D.J. (1977) Eur. J. CIin. Invest. 7, 301-305. Fogehnan, A.M., Edmond. J.. Polito. A. and Popjak. G. (1973) J. Biol. Chem. 248. 6928-6929. Brown. M.S.. Dana. S.E. and Goldstein. J.L. (1973) J. Biol. Chem. 70. 2162-2166. Golds&, J.‘L. and’Brown, M.S. (1976) Cur: Top..CeIL Regal. 11,147-181. Boy;m, A. (1968) Stand. J. Clin. Lab. Invest. 97. Suppl. 21. 77-89. McFarlane, A.S. (1942) Nature 149, 439. Perrv. R.P. (1963) Exu. Cell Res. 29.400-406. Zylbei, E.. cesco; C. and Penman, S.. (1969) J. MoL Biol. 44. 195-204. Penman, S.. Rosbach. M. and Penman, M. (1970) Proc. Natl. Acad. Sci U.S.A. 67, 1878-1885. RudIand. P.S., We& S. and Hunter, A.R. (1975) J. Mol. Biol. 96. 745-766. Bligh. E.G. and Dyer. W.J. (1959) Can. J. Biochem. Phvsiol. 37. 911-917. Singer, R.H. and tie&an, Sl (19i2) Nature 240. IOO-i02. Maale, G.. Stein, G. and Mans, R. (1975) Nature 255, 80-82. Horowitz, B., Goldfinger. B.A. and Marmur, J. (1976) Arch. Biochem. Biophys 172. 143-148. Mendecki, J., Lee, S.Y. and Brawerman, G. (1972) Biochemistry 11, 792-798. Cholou. J.J. and Stud&&i, G.P. (1974) Science 184, 160-161. Lewin. B. (1975) Cell 4. 11-20.
361-365
BBA Report BBA 51256
REGULATION OF STEROL SYNTHESIS IN HUMAN LYMPHOCYTES: EVIDENCE FOR POST-TRANSCRIPTIONAL CONTROL BY LOW DENSITY LIPOPROTEIN
WILHELM
KRONE*,
D. JOHN BETTERIDGE
Diabetes and Lipid Research Laboratory, (U.K.) (Received
and DAVID J. GALTON
St. Bartholomew’s
Hospital, London EClA
7BE
June lst, 1979)
Keywords: Hydroxymethylglutaryl-CoA control; LDL; (Human lymphocyte)
reductase; mRNA synthesis; Post-transcriptional
Summary The effect of cordycepin (3’-deoxyadenosine), an inhibitor of messenger RNA synthesis, on the induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase mediated by lipid-depleted serum was studied in isolated human lymphocytes. 50 pg/ml cordycepin, although inhibiting messenger RNA synthesis by more than 50%, had no inhibitory effect on the two and four-fold induction of hydroxymethylglutaryl-CoA reductase when cells were incubated in a medium containing lipid-depleted serum for 8 and 16 h, respectively. This result suggests that newly synthesises messenger RNA is not required for the effect of lipid-depleted serum on the induction of hydroxymethylglutaryl-CoA reductase in human lymphocytes.
The activity of the microsomal enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase determines the rate of cholesterol synthesis in a wide variety of mammalian cells [l]. The enzyme activity is regulated primarily by cholesterol which enters the cell bound to low density lipoprotein (LDL). Thus when isolated human leucocytes and cultured human fibroblasts are incubated in the presence of LDL the activity of hydroxymethylglutaryl-CoA reductase is suppressed. However, incubation of cells for several hours in a medium containing lipid-depleted serum leads to a substantial increase in the activity of the enzyme. Subsequent exposure to cholesterol either as LDL or in an ethanolic *Present address: Klinisches Institut fiir Herzinfarktforschung an der Medizinischen Universit;itskllnik Heidelberg. Bergheimerstrasse 58. D-6900 Heidelberg, F.R.G.
362
solution is followed by a suppression of the enzyme activity [2-41. Since under these conditions sterol synthesis is strictly proportional to the activity of hydroxymethylglutaryl-CoA reductase, the incorporation of [14C]acetate into sterols can be taken as a measurement of enzyme activity [5, 61. Four lines of evidence indicate that the rise in hydroxymethylglutarylCoA reductase activity in the presence of lipid-depleted serum is due to an increased de novo synthesis of the enzyme and not due to activation of preformed enzyme molecules or decreased degradation of the enzyme: (i) incubation of cells in a medium containing lipid-depleted serum, after a lag period of l-2 h, results in a linear rise in enzyme activity for at least 20 h; (ii) neither whole serum, LDL or cholesterol added to cell free extracts of fibroblasts produce any inhibitory effect on hydroxymethylglutaryl-CoA reductase activity; (iii) when extracts with low enzyme activity obtained from cells grown in whole serum are mixed with extracts from cells with high enzyme levels having been grown in lipoprotein-deficient serum the total combined activity of hydroxymethylglutaryl-CoA reductase is equal to the sum of the individual activities of the extracts when measured separately and (iv) when leucocytes or fibroblasts are incubated in lipid-depleted serum, the subsequent stimulation of the conversion of [14C]acetate into sterols [7] or the rise in hydroxymethylglutaryl-CoA reductase activity [8], respectively, is prevented by cycloheximide. Furthermore, in the presence of cycloheximide, the activity of the enzyme declines with first order kinetics and a half-life of about 3 h, the half-time of decay being similar in cells with depressed or elevated enzyme levels. An attractive model for the regulation of hydroxymethylglutaryl-CoA reductase activity by LDL-cholesterol has been proposed by Goldstein and Brown (for review, see Ref. 9). According to this plasma-LDL binds with high affinity to specific cell surface receptors. The membrane-bound LDL is then internalised by endocytosis and delivered to lysosomes, where the lipoprotein is degraded. Free cholesterol released from the lipoprotein reduces endogenous cholesterol synthesis by suppression of the activity of HMG-CoA reductase. However, the mechanism by which LDL-cholesterol acts on the enzyme is not known. We therefore studied the effect of cordycepin (3’-deoxyadenosine), an inhibitor of RNA synthesis, on the induction of sterol synthesis by lipiddepleted serum in isolated human lymphocytes and wish to report that newly synthesised mRNA is not required for the effect of lipid-depleted serum on the induction of sterol synthesis suggesting that hydroxymethylglutaryl-CoA reductase is regulated by LDL-cholesterol at a post-transcriptional or translational level. Isolation and incubation of lymphocytes. Peripheral human blood lymphocytes were isolated by the method of Boyiim [lo]. Cells were incubated at 37°C in 2 ml Krebs-Ringer phosphate buffer (pH 7.4), supplemented with aminoacids, vitamins, 15 mM glucose, 0.5 mM sodium acetate, 100 units penicillin/ml, 100 ,ug streptomycin/ml and 40% serum (which was depleted of lipid by the method of McFarlane [ll]). Incorporation of [jH]adenosine into cy toplasmic poly(A)-containing RNA. 2 X lo6 lymphocytes were labelled with AOOpCi [3H]adenosine (23 Ci/mmol)
for 40 min. rRNA synthesis and mtRNA synthesis were first suppressed with 0.4 pg/ml actinomycin D [12] and 1 pg/ml ethidium bromide [13], respectively.
363
Actinomycin D, ethidium bromide and cordycepin were added 30 min prior to labelling. Cytoplasmic extracts were prepared by harvesting, washing and resuspending cells in 2 ml of hypotonic buffer (10 mM NaCl, 1.5 mM MgCl?, 10 mM Tris-HCI, pH 7.4), adding the detergent Nonidet (NP40) to a final concentration of 0.5% and agitating with a vortex mixer for 30 s; nuclei were removed by centrifugation at 800 X g for 2 min [14]. The cytoplasmic RNA was extracted by a conventional phenol/CHC& procedure [15]. Poly(A)-containing RNA was isolated by binding to Oligo(dT)-cellulose. The bound RNA (poly(A)’ RNA), eluted with a NaCl-free buffer, and the unbound RNA (poly(A)- RNA) .were precipitated with 2 volumes of ethanol at -20°C for 18 h. The precipitates were dissolved in water and radioactivity in both types of RNA was measured by liquid scintillation spectrometry using a toluene-based scintilla&. The recovery of 32P-labelled ribosomal RNA was used to correct for procedural losses of [3H]RNA. Incorporation of [14C]acetate into sterols. 5 X lo6 cells were incubated at 37°C for the appropriate time when 1OpCi [2-14C]acetate (55 Ci/mol) was added and the incubation continued for 2 h. Incubations were terminated by the addition of CHC13/CH30H (1:2) and [1,2-3H]cholesterol was added as an internal standard. Lipids were extracted by the method of Bligh and Dyer [16] and were saponified by methanolic 2M KOH for 6 h at 70°C. The nonsaponifiable fraction was extracted with hexane and radioactivity was measured by liquid scintillation spectrometry using a toluene based scintillant. The recovery of [3H]cholesterol was used to correct for procedural losses of 14Clabelled nonsaponifiable lipids. Since changes in sterol synthesis are strictly proportional to changes in activities of hydroxymethylglutaryl-CoA reductase, the incorporation of [14C]acetate into sterols can be taken as a measure for the enzyme activity in lymphocytes [5, 61. Actinomycin D has previously been reported to prevent the increase in hydroxymethylglutaryl-CoA reductase after removal of LDL from the incubation medium in cultured human fibroblasts [8]. Similarly, we found that this antibiotic (0.4 E.cg/ml) blocked totally the subsequent rise in sterol synthesis from [14C]acetate in freshly isolated human lymphocytes following incubation in lipid-depleted serum. It should however be noted that actinomycin D, in addition to blocking transcription, can produce non-specific effects such as inhibition of the initiation of protein synthesis [17]. Although the mechanism by which cordycepin acts at the molecular level is still debated [18, 191, the effect of the drug in HeLa cells [14], mouse sarcoma 180 cells [20] and in cultured human fibroblasts [21] is to inhibit mRNA synthesis. The results shown in Fig. 1 demonstrate that addition of cordycepin to isolated lymphocytes 30 min prior to the addition of [3H]adenosine inhibits the appearance of the isotope in the cytoplasmic poly(A)containing RNA while it has no effect on the labelling of cytoplasmic RNA containing no poly(A)-sequences. As poly(A)-containing RNA represents most of the mammalian mRNA [22], our data indicates that cordycepin can substantially inhibit mRNA synthesis in lymphocytes. Although 50pg/ml cordycepin inhibited poly(A)-containing RNA synthesis by more than 50% (Fig. 1) the drug had no inhibitory effect on the two- and four-fold increase in sterol synthesis from [14C]acetate in cells incu-
364
. ” 0 c
x
E
B
0
d
0
25 CORDYCEPIN
50 (fig/ml)
1
1
I
0
a
16
TIME (HOURS)
Fig. 1. Effect of varying concentrations of cordycepin on the incorporation of r3Hladenosine into cytoplasmic poly(A>containing RNA (o---O) and RNA without poly(A)_sequences (o-o) in isolated human lymphocytes. Values are given as means f S.E. for 3-4 experiments. Fig. 2. Effect of varying concentrations of cordycepin on the induction of sterol synthesis from [“Cl acetate by lipid-depleted serum in isolated human lymphocytes. Controls without cordycepin (o--O), cordycepin 12.5 pglml (e-e), 25 pg/ml (A-a) and 50 pg/ml (m-m). Values are means + S.E. for 3-4 experiments.
bated in a medium containing lipid-depleted serum for 8 and 16 h, respectively (Fig. 2). Assuming that cordycepin inhibits synthesis of mRNA for the enzyme to a similar extent as total mRNA synthesis, our results suggest that newly synthesised mRNA is not required for the effect of lipid-depleted serum on the induction of hydroxymethylglutaryl-CoA reductase in lymphocytes. From the time course of the induction of hydroxymethylglutaryl-CoA reductase (Fig. 2) it appears that the half-life of the mRNA for the enzyme is relatively long. This is supported by the following experiment : lymphocytes were preincubated with full serum for up to 40 h in the presence of 50pg/ml cordycepin. This should significantly reduce the level of mRNA with a short half-life. Transfer of the cells from full to lipid-depleted serum and further incubation for 10 h again in the presence of 50 fig/ml cordycepin, resulted in a three-fold increase in sterol synthesis from [14C]acetate and this increase was the same in cells preincubated in the absence of cordycepin. These results suggest that in human lymphocytes neither the induction of hydroxymethylglutaryl-CoA reductase by lipid-depleted serum nor the subsequent repression of the enzyme by LDL-cholesterol, which occurs with a half-time of approx. 3 h [6], can be accounted for by a corresponding increase or decrease in the level of mRNA coding for the enzyme. LDL-cholesterol may
365
therefore regulate hydroxymethylglutaryl-CoA scriptional or translational level.
reductase
at a post-tran-
We gratefully acknowledge advice and assistance from Drs. P.S. Rudland and P. Piper (Imperial Cancer Research Fund). W. Krone is in receipt of a grant from the Deutsche Forschungsgemeinschaft. D.J. Betteridge is a R.D. Lawrence Research Fellow of the British Diabetic Association.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
RodweII, V.W.. Nordstrom, J.L. and Mitsehelen. J.J. (1976) Adv. Lipid Res. 14. l-74. Brown, M.S., Dana, S.E. and Goldstein, J.L. (1974) J. Biol. Chem. 249. 789-796. Higgins, M.J.P.. Lecamwasam. D.S. and G&on. D.J. (1975) Lancet ii, 737-739. Ho, Y.K., Faust, J.R.. Bilheimer. D.W., Brown, M.S. and Goldstein, J.L. (1977) J. Exp. Med. 145, 153 l-l 549. Fogelman. A.M., Edmond. J.. Seager. J. and Popjak. G. (1975) J. Biol. Chem. 250, 2045-2055. Higgins, M.J.P. and Galton, D.J. (1977) Eur. J. CIin. Invest. 7, 301-305. Fogehnan, A.M., Edmond. J.. Polito. A. and Popjak. G. (1973) J. Biol. Chem. 248. 6928-6929. Brown. M.S.. Dana. S.E. and Goldstein. J.L. (1973) J. Biol. Chem. 70. 2162-2166. Golds&, J.‘L. and’Brown, M.S. (1976) Cur: Top..CeIL Regal. 11,147-181. Boy;m, A. (1968) Stand. J. Clin. Lab. Invest. 97. Suppl. 21. 77-89. McFarlane, A.S. (1942) Nature 149, 439. Perrv. R.P. (1963) Exu. Cell Res. 29.400-406. Zylbei, E.. cesco; C. and Penman, S.. (1969) J. MoL Biol. 44. 195-204. Penman, S.. Rosbach. M. and Penman, M. (1970) Proc. Natl. Acad. Sci U.S.A. 67, 1878-1885. RudIand. P.S., We& S. and Hunter, A.R. (1975) J. Mol. Biol. 96. 745-766. Bligh. E.G. and Dyer. W.J. (1959) Can. J. Biochem. Phvsiol. 37. 911-917. Singer, R.H. and tie&an, Sl (19i2) Nature 240. IOO-i02. Maale, G.. Stein, G. and Mans, R. (1975) Nature 255, 80-82. Horowitz, B., Goldfinger. B.A. and Marmur, J. (1976) Arch. Biochem. Biophys 172. 143-148. Mendecki, J., Lee, S.Y. and Brawerman, G. (1972) Biochemistry 11, 792-798. Cholou. J.J. and Stud&&i, G.P. (1974) Science 184, 160-161. Lewin. B. (1975) Cell 4. 11-20.
