Role of mevalonate in regulation of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase in cultured cells and their cytoplasts

H4-II-E-C3 hepatoma cells in culture respond to lipid-depleted media and to mevinolin with increased sterol synthesis from [14C]acetate and rise of 3-hydroxy-3-methylglutaryl coenzyme A reductase levels. Mevalonate at 4 mM concentration represses sterol synthesis and the reductase, and completely abolishes the effects of mevinolin. Mevalonate has little or no effect on sterol synthesis or reductase in enucleated hepatoma cells (cytoplasts) or on reductase in cytoplasts of cultured Chinese hamster ovary (CHO) cells. The sterol-synthesizing system of hepatoma cell cytoplasts and the reductase in the cytoplasts of CHO cells were completely stable for at least 4 hr. While reductase levels and sterol synthesis from acetate followed parallel courses, the effects on sterol synthesis--both increases and decreases--exceeded those on reductase. In vitro translation of hepatoma cell poly(A)+RNAs under various culture conditions gave an immunoprecipitable polypeptide with a mass of 97,000 daltons. The poly(A)+RNA from cells exposed for 24 hr to lipid-depleted media plus mevinolin (1 microgram/ml) contained 2.8 to 3.6 times more reductase-specific mRNA than that of cells kept in full-growth medium, or cells exposed to lipid-depleted media plus mevinolin plus mevalonate. Northern blot hybridization of H4 cell poly(A)+RNAs with [32P]cDNA to the reductase of CHO cells gave two 32P-labeled bands of 4.6 and 4.2 K-bases of relative intensities 1.0, 0.61-1.1, 2.56, and 1.79 from cells kept, respectively, in full-growth medium, lipid-depleted medium plus mevinolin plus mevalonate, lipid-depleted medium plus mevinolin, and lipid-depleted medium. These values approximate the reductase levels of these cells. We conclude that mevalonate suppresses cholesterol biosynthesis in part by being a source of a product that decreases the level of reductase-specific mRNA.     

Effects of mevinolin in rat liver: evidence for a lack of coupling between synthesis of hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase activity

The effect of treatment of rats with the hydroxymethylglutaryl-CoA reductase inhibitor, mevinolin, on 7 alpha-hydroxylation of cholesterol was studied. Treatment with 0.1% mevinolin in diet for 3 days was found to have an inhibitory effect on 7 alpha-hydroxylation of cholesterol (about 35%). Treatment with cholestyramine increased 7 alpha-hydroxylation of both exogenously added and endogenous microsomal cholesterol 3-4-fold. Combined treatment with both cholestyramine and mevinolin decreased this stimulation to 2-2.5-fold. Treatment with 2% cholesterol in diet increased 7 alpha-hydroxylation of exogenous cholesterol about 2-fold and 7 alpha-hydroxylation of endogenous cholesterol about 3.5-fold. The stimulatory effect of cholesterol was reduced or abolished when 0.1% mevinolin was added to the cholesterol-containing diet. With the exception of the experiments with cholesterol in the diet, all experiments including mevinolin gave a marked stimulation (up to 60-fold) of the hydroxymethylglutaryl-CoA reductase activity under the in vitro conditions employed. The concentration of free cholesterol in the liver microsomes was not significantly changed in any of these experiments. It is concluded that there is no coupling between induction of synthesis of hydroxymethylglutaryl-CoA reductase protein and cholesterol 7 alpha-hydroxylase activity. The inhibitory effect of mevinolin on cholesterol 7 alpha-hydroxylase activity under experimental conditions where most of the effect of mevinolin on hydroxymethylglutaryl-CoA reductase was abolished by treatment with cholesterol suggest that the effect of mevinolin on the cholesterol 7 alpha-hydroxylase may be independent of its effect on cholesterol synthesis. The over-all results do not favour the hypothesis that cholesterol synthesis and cholesterol availability are the most important determinants for the regulation of the cholesterol 7 alpha-hydroxylase.     

Effect of cordycepin (3''-deoxyadenosine) on virus-specific RNA species synthesized in Newcastle disease virus-infected cells.

Cordycepin (3'-deoxyadenosine) has no effect on the size or relative proportions of Newcastle disease virus-specific 18-22S mRNA species nor on the amount or size of the polyadenylic acid associated with them. Cordycepin does, however, cause an inhibition of incorporation of [3H]uridine into 50S virus-specific RNA relative to 18-22S RNA. This inhibition is probably not a direct effect of the drug on the synthesis of 50S viral RNA. Like cycloheximide, another drug which inhibits 50S RNA accumulation in paramyxovirus-infected cells, cordycepin inhibits protein synthesis as measured by amino acid incorporation. It is likely that the inhibition of 50S RNA accumulation is a secondary effect of protein synthesis inhibition. This is supported by the finding that concentrations of cordycepin and cycloheximide, which inhibit protein synthesis to the same extent, have the same effect on the ratio of 50 to 18-22S virus-specific RNA.     

INCREASED RATE OF ACETYLCHOLINESTERASE SYNTHESIS IN DIFFERENTIATING NEUROBLASTOMA CELLS

When neuroblastoma cells (N18) in vitro are maintained in the absence of serum, the specific activity of AChE begins to rise rapidly after an initial lag period of about 2–3 days, reaching a maximum level (10–20-fold increase) by 7 days after induction. In order to clarify the mechanism of induction, it was necessary to measure the rate of AChE synthesis and its sensitivity to metabolic inhibitors. Return of enzymatic activity after irreversible inhibition of AChE in "differentiated" cells was blocked by cycloheximide, but not by cordycepin or actinomycin D, suggesting that protein but not mRNA synthesis was required for replacement. By using the initial rate of this replacement as a measure of the rate of synthesis of the enzyme, it was shown that cells which had differentiated in the absence of serum synthesized AChE 50-fold faster on a specific activity basis than their undifferentiated counterparts. In contrast, cordycepin effectively blocked the increase in the rate of AChE synthesis that occurs as a result of serum deprivation, indicating that the induction process itself requires the synthesis of new mRNA. Axonation, another index of differentiation, was not completely blocked by inhibition of RNA or protein synthesis and presumably utilizes only pools of pre-existing structural proteins.     

Effect of Ammonium, Sucrose and Light on the Regulation of Nitrate Reductase Level in Pisum sativum

In shoot apices of 7-day-old dark-grown peas the addition of ammonium along with the inducer nitrate resulted in a more than two-fold increase in nitrate reductase activity. Individual amino acids, amides and amino-acid mixture could not replace the ammonium effect. Ammonium also stimulated NADH-glutamate dehydrogenase but not glucose-6-phosphate dehydrogenase. Sucrose caused a marked stimulation of nitrate reductase induction and showed synergistic effect with light. In presence of cordycepin and cycloheximide, induction of nitrate reductase was inhibited more if ammonium or sucrose was supplied along with the inducer. With actinomycin D, α-amanitin or chloramphenicol, no differential inhibition took place in presence of ammonium. The inhibition of enzyme activity by chloramphenicol and 3-(3,4-dichlorophenyl)-l,dimethyl urea was completely relieved by sucrose. Incorporation of ¹⁴C-lysine was markedly stimulated by sucrose, but was not affected by ammonium. The effect of sucrose and light on ¹⁴C-lysine incorporation was additive. Cordycepin and cycloheximide did not have any differential effect on ¹⁴C-lysine incorporation in the presence of ammonium as well as sucrose. The inhibition of ¹⁴C-lysine incorporation caused by chloramphenicol was relieved by sucrose. Sucrose also caused a marked increase in ³H-uridine incorporation but ammonium had no effect. Actinomycin D and cordycepin blocked the sucrose dependent increase in ³H-uridine incorporation. The results suggest that ammonium mediated stimulation may depend on a regulatory protein(s) synthesized in response to ammonium, whereas sucrose acts mainly by an overall increase in RNA and protein synthesis. The effect of light does not seem to be dependent on photosynthetic light reactions.     

THE EFFECT OF CORDYCEPIN ON THE APPEARANCE OF [3H]RNA IN THE GOLDFISH OPTIC TECTUM FOLLOWING INTRAOCULAR INJECTION OF [3H]URIDINE

Abstract— Although biochemical and electron microscopic evidence has shown that RNA molecules may be found within axons, the origin of this RNA is not known. In order to determine if the RNA found in axons is synthesized in the nerve cell body and axonally transported, we have studied the effect of the RNA inhibitor cordycepin (3′-deoxyadenosine) on the retinal synthesis and axonal migration of radioactive RNA. Ten μg of cordycepin was injected into the right eye of 11 fish and 3 h later [³H]uridine was injected into the same eye. Twelve control fish were injected with [³H]uridine only and all fish were sacrificed 6 days later. Results of RNA extraction of retina and tecta showed that cordycepin decreased retinal RNA synthesis by approx 24%, while inhibiting the amount of [³H]RNA appearing in the contralateral tectum by 74%. Since the transport of RNA precursors was depressed by only 50%, (significantly different from the effect on RNA, P < 0.01) it seems unlikely that the action of cordycepin in decreasing tectal [³H]RNA levels was due solely to a decrease in the availability of labeled precursors for tectal RNA synthesis. For the purpose of blocking tectal RNA synthesis, 200 μg of cordycepin was injected intracranially several days after the intraocular injection of [³H]uridine. This route of cordycepin administration failed to significantly block the appearance of [³H]RNA in the tectum, suggesting that at least some of the [³H]RNA in the tectum was synthesized before arrival in the tectum itself. To be sure that cordycepin itself was not being transported, we injected cordycepin into the right eye of fish and 5 days later, injected fish intracranially with [³H]uridine. Autoradiograms were prepared and grains were counted over the fiber layers of left (experimental) and right (control) tecta. No significant difference was observed in the number of grains of left vs right tecta indicating that cordycepin itself is not axonally transported. These experiments support earlier findings from our laboratory which suggest that RNA may be axonally transported in goldfish optic fibers.     

Regulation of rat liver phosphoenolpyruvate carboxykinase (GTP) messenger ribonucleic acid activity by N6, O2'-dibutyryladenosine 3',5'-phosphate

N6,O2'-Dibutyryladenosine 3',5'-phosphate (Bt2cAMP) induces the synthesis of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.32), in rat liver by increasing the activity of messenger ribonucleic acid (mRNA) coding for this enzyme (mRNAPEPCK) more than 20-fold (from less than 0.01% to greater than 0.20% of total mRNA activity) as determined by using in vitro translation systems which measure only active mRNAPEPCK. The increase in mRNAPEPCK activity could result from increased synthesis, increased processing, or decreased inactivation rates. Actinomycin D and cordycepin inhibit mRNAPEPCK induction by 89% and 70%, respectively, a result that indicates a requirement for ongoing RNA synthesis but that does not distinguish which of these steps is regulated by cAMP. We have employed a kinetic approach, not involving RNA synthesis inhibitors, to determine the half-life of mRNAPEPCK both during a period of deinduction following glucose feeding and during a subsequent induction by Bt2cAMP. An estimated half-life of 20 +/-5 min during both of these periods indicates that Bt2cAMP has no effect on the rate of inactivation of mRNAPEPCK. We conclude that Bt2cAMP effects the increase in activity of mRNAPEPCK by promoting its synthesis or processing.     

THE EFFECT OF INTRAOCULAR INJECTION OF CORDYCEPIN ON RETINAL RNA SYNTHESIS AND ON RNA AXONALLY TRANSPORTED DURING REGENERATION OF THE OPTIC NERVES OF GOLDFISH

Abstract— The presence of relatively large amounts of RNA has been demonstrated in regenerating axons of the goldfish optic nerve. Previous experiments have suggested that this R NA may be composed of only small molecular weight 4S RNA. The present experiments were performed in order to see if inhibiting RNA transport by intraocular injections of cordycepin causes a selective depletion of 4S RNA arriving in the contralateral optic tectum, and thus add further evidence that 4S RNA is axonally transported. Optic nerves were crushed in a group of goldfish and 18 days later 10.0 /tg of cordycepin was injected into the right eye followed 3 h later by injections of [³H]uridine into the same eye. Six days later the amount of axonally transported [³H]RNA was decreased by 89% compared with non-cordycepin treated controls. The effect of cordycepin on retinal RNA synthesis was shown by autoradiography to be primarily on retinal ganglion cell RNA synthesis with lesser effects on other cellular elements of the retina. SDS polyacrylamide gel electrophoresis at both 1 and 6 days after intraocular injections of cordycepin and [³H]uridine, showed that cordycepin blocks the retinal synthesis of ribosomal RNAs but appeared to have little effect on the synthesis of 4S RNA. When transported RNA in the tectum was fractionated by gel electrophoresis 6 days after injection, it was found that the amount of ribosomal RNA was decreased by approx 70% as a result of cordycepin pretreatment. This correlated well with the effect of cordycepin on the transport of available RNA precursors (also decreased by approx 70%) and is consistent with the contention that in these experiments ribosomal RNA is synthesized in the tectum itself and is not axonal. The amount of [³H] 4S RNA arriving in the tectum, however, was decreased by greater than 90% suggesting that its presence in the tectum was not entirely dependent on the availability of ³H precursors for local synthesis in the tectum. These results are consistent with data suggesting that 4S RNA is the predominant, if not the only, RNA species axonally transported during regeneration of goldfish optic nerves.     

Rapid Inhibition of Auxin-induced Elongation of Avena Coleoptile Segments by Cordycepin

The effects of cordycepin (3'-deoxyadenosine), an RNA synthesis inhibitor, on auxin-induced elongation in Avena coleoptile segments were studied with a position-sensing transducer. Cordycepin rapidly inhibited auxin-stimulated growth in the coleoptile segments whether added before, at the same time as, or after, the 2 mum auxin treatment. Midcourse additions of 100, 50, and 25 mug/ml cordycepin inhibited auxin-promoted elongation in an average of 18, 22, and 35 minutes, respectively. Additions of cordycepin before or at the same time as the auxin treatment partially inhibited the magnitude of the subsequent auxin-promoted growth but did not appreciably alter the latent period of the auxin response. It was concluded that if cordycepin is inhibiting the synthesis of RNA required for growth, the decay time for this RNA may be considerably shorter than that suggested in the literature from actinomycin D experiments. Preliminary kinetic evidence indicated that cordycepin does not inhibit auxin-induced elongation by acting as a respiratory inhibitor. Studies in mung bean shoot mitochondria demonstrated that cordycepin has no effect on respiration, respiratory control, or ADP/oxygen ratios.     

Linkage of the isoprenoid biosynthetic pathway with induction of DNA synthesis in mouse lymphocytes. Effects of compactin on mitogen-induced lymphocytes in serum-free medium

Concanavalin A induction of DNA synthesis in mouse spleen lymphocytes cultured in serum-free medium was shown to be very sensitive to inhibition by compactin (ML-236B), a specific competitive inhibitor of hydroxymethylglutaryl-CoA reductase. As low as 0.1 microM compactin could give 98% inhibition of mitogen induction of a 5.10(6) cells/ml culture. This inhibition could be reversed completely by addition of exogenous mevalonate, but could not be reversed by either exogenous cholesterol or isopentenyladenine. Oxygenated sterol inhibition of mitogen-induced DNA synthesis could be reversed by cholesterol or by mevalonate, whereas cyclic AMP inhibition could not be reversed by either compound. These results suggest that endogenous cholesterol production is a necessary but not sufficient factor co-ordinated with mitogen-induced DNA synthesis, and that the presence of some additional product of mevalonate metabolism is involved also. Isopentenyladenine, though, did not have as significant effect of alleviating any of the above inhibitions. Since mevalonate could not relieve cyclic AMP inhibition, but could overcome compactin inhibition, cyclic AMP inhibition cannot be explained as due only to blockage of mevalonate production.     

Polyadenylate sequences of human rhinovirus and poliovirus RNA and cordycepin sensitivity of virus replication.

The polyadenylate [poly(A)] content of the genome RNA of human rhinovirus type 14 (HRV-14) is nearly twice as large as that of the genome RNA of poliovirus type 2. The poly(A) content of viral RNA was determined to be the RNase-resistant fraction of 32P-labeled viral RNA extracted from purified virions. Polyacrylamide gel electrophoresis indicated that the poly(A) sequences of HRV-14 are more heterogenous and on an average larger than those of poliovirus RNA. On the basis of susceptibility to micrococcal polynucleotide phosphorylase the rhinovirus genome terminates in poly(A). Replication of both viruses is almost totally inhibited by cordycepin at 50 mug/ml. At lower concentrations, rhinovirus replication is more sensitive to cordycepin than poliovirus replication. Addition of cordycepin (75 mug/ml) to infected culture prior to or during viral RNA replication results in more or less complete inhibition of virus-specific RNA synthesis. The results do not indicate that cordycepin sensitivity of either virus is due to preferential inhibition of viral poly(A) synthesis by this antibiotic.     

Selective inhibition of initial polyadenylation in isolated nuclei by low levels of cordycepin 5"-triphosphate.

The effect of cordycepin 5'-triphosphate on poly(A) synthesis was investigated in isolated rat hepatic nuclei. Nuclei were incubated in the absence and presence of exogenous primer in order to distinguish the chromatin-associated poly(A) polymerase from the "free" enzyme (Jacob, S.T., Roe, F.J. and Rose, K.M. (1976) Biochem. J. 153, 733--735). The chromatin-bound enzyme, which adds adenylate residues onto the endogenous RNA, was selectively inhibited at low concentrations of cordycepin 5'-triphosphate, 50% inhibition being achieved at 2microng/ml. At least 80 times more inhibitor was required for 50% reduction in the "free" nuclear poly(A) polymerase activity. Inhibition of DNA-dependent RNA synthesis also required higher concentrations of the nucleotide analogue. These data not only offer a mechanism for the selective inhibition of initial polyadenylation of heterogeneous nuclear RNA in vivo by cordycepin, but also provide a satisfactory explanation for the indiscriminate effect of the inhibitor on partially purified or "free" poly(A) and RNA polymerases.     

The effect of hydrocortisone on cholesterol metabolism of cultured human skin fibroblasts

Hydrocortisone in physiologic concentrations resulted in a reduction in sterol synthesis by cultured normal human skin fibroblasts. These changes were observed when [14C]acetate, [14C]octanoic acid and 3H2O were used as precursors. However, the incorporation of [3H]mevalonic acid lactone into digitonin-precipitable sterols was not affected by hydrocortisone, suggesting that hydrocortisone inhibits sterol synthesis at a site prior to the formation of mevalonic acid. In contrast, the activity of hydroxymethylglutaryl-CoA reductase was stimulated several-fold by the hormone. Thus, the inhibitory effect of hydrocortisone on the cholesterol synthetic pathway may be on hydroxymethylglutaryl-CoA synthase.     

Cholesterol biosynthesis in human lymphocytes,monocytes, and granulocytes

Lymphocytes, monocytes and granulocytes were separated by counter-flow centrifugation from the blood of normal individuals and were incubated in full serum medium or lipid-depleted medium. The monocytes incorporated about five times more [2-¹⁴C]acetate into sterols than did the lymphocytes in full serum medium and approximately twenty times more than the lymphocytes in lipid-depleted medium. The granulocytes were unable to synthesize sterols from either [2-¹⁴C]acetate or [2-¹⁴C]mevalonate, but they were able to use these substrates for the synthesis of squalene and demonstrated approximately a two fold increase in the incorporation of [2-¹⁴C]acetate (but not [2-¹⁴C]mevalonate) into squalene when incubated in the lipid-depleted medium as compared to the full serum medium.     

Apparent coordination of the biosynthesis of lipids in cultured cells: its relationship to the regulation of the membrane sterol:phospholipid ratio and cell cycling

The coordination of the syntheses of the several cellular lipid classes with one another and with cell cycle control were investigated in proliferating L6 myoblasts and fibroblasts (WI-38 and CEF). Cells cultured in lipid-depleted medium containing one of two inhibitors of hydroxymethylglutaryl-CoA reductase, 25-hydroxycholesterol or compactin, display a rapid, dose-dependent inhibition of cholesterol synthesis. Inhibition of the syntheses of each of the other lipid classes is first apparent after the rate of sterol synthesis is depressed severalfold. 24 h after the addition of the inhibitor, the syntheses of DNA, RNA, and protein also decline. The inhibition of sterol synthesis leads to a threefold reduction in the sterol:phospholipid ratio that parallels the development of proliferative and G1 cell cycle arrests and alterations in cellular morphology. All of these responses are reversed upon reinitiation of cholesterol synthesis or addition of exogenous cholesterol. A comparison of the timing of these responses with respect to the development of the G1 arrest indicates that the primary factor limiting cell cycling is the availability of cholesterol provided either from an exogenous source or by de novo synthesis. The G1 arrest appears to be responsible for the general inhibition of macromolecular synthesis in proliferating cells treated with 25-hydroxycholesterol. In contrast, the apparent coordinated inhibition of lipid synthesis is not a consequence of the G1 arrest but may in fact give rise to it. Sequential inhibition of lipid syntheses is also observed in cycling cells when the synthesis of choline-containing lipids is blocked by choline deprivation and is observed in association with G1 arrests caused by confluence or differentiation. In the nonproliferating cells, the syntheses of lipid and protein do not appear coupled.     

Cordycepin. An inhibitor of newly synthesized globin messenger RNA.

The effect of cordycepin (3'-deoxyadenosine) on newly synthesized globin mRNA in cultured mouse fetal liver erythroid cells is investigated. At cordycepin concentrations that do not inhibit amino acid incorporation into acid-precipitable material, the quantity of pulse-labeled (radioactive) globin mRNA nucleotide sequences is reduced by 90%, as compared to adenosine-treated controls. The reduction of radioactivity in globin-specific RNA sequences is greater than the inhibition of total RNA synthesis in experiments in which the labeling times range from 6 to 60 min. Control experiments demonstrate that cordycepin does not reduce the recovery of total cell RNA or steady state (unlabeled) globin mRNA. The hybridization assay used to detect radioactive globin mRNA sequences is independent of the cellular location or the number of 3'-terminal adenylate residues in the mRNA-containing molecules. These data thus indicate that cordycepin inhibits newly synthesized mRNA as effectively as it inhibits ribosomal and transfer RNA synthesis.