ABSTACT: The cyclic fluctuations of HMG-CoA reductase activ- ity and mRNA are reportedly related to feeding the cells in cul- ture or to variations in food consumption by the animals over a 24-h cycle. In this work, we demonstrate cyclic increments in HMG-CoA reductase activity in smooth muscle cells (SMC) not associated with the culture feeding. Since reductase activity also shows a marked rise preceding the S phase, one of the major goals of the present work was to evaluate this dual role of reduc- tase activity and mRNA fluctuations related to the cell cycle and to food intake in the SMC-C/SMC-Ch cultures derived from con- trol-fed (SMC-C) and cholesterol-fed (SMC-Ch) chicks. The pe- riod and amplitude oscillations in HMG-CoA reductase activity varied depending on culture conditions: lipoprotein-deficient serum vs. FBS, young vs. senescent cells, or confluent vs. non- confluent cultures. The HMG-CoA reductase mRNA concentra- tion showed a marked rise after feeding not correlated to the fluc- tuation activity, suggesting posttranscriptional modulation. Re- ductase activity and mRNA were down-regulated in SMC-Ch. Since the nutritional culture conditions were the same in both cell lines, these findings indicate that consumption of a high-cho- lesterol diet by the animals prior to the establishment of the SMC cultures induced changes in the HMG-CoA reductase gene ex- pression in aortic SMC. Paper no. L9929 in Lipids 41, 1089–1099 (December 2006). The enzyme HMG-CoA reductase (EC 1.1.1.34), from the en- doplasmic reticulum, catalyzes the conversion of HMG-CoA to mevalonate, the rate-limiting step in cholesterol biosynthesis (1). The rate of cholesterol biosynthesis from different tissues was shown to have a diurnal rhythm many years ago (2–4). These rhythmic changes are associated with changes in HMG- CoA reductase activity (1,2,5,6). In rodent liver, diurnal varia- tion of hepatic cholesterol synthesis is driven primarily by varying the steady-state mRNA levels for HMG-CoA reduc- tase (6–11). The diurnal rhythm of HMG-CoA reductase from chick liver and intestine has also been observed in our labora- tory. In this animal, both reductase activities were maximal dur- ing the light period, in agreement with chick feeding habits. However, chick liver and intestine reductase do not show diur- nal rhythm at hatching (5) or during the first week of life, when the chick is still reabsorbing the yolk (12) and thereby devel- oping a transient hypercholesterolemia, which becomes almost three times higher in cholesterol-fed chicks (13). Indeed, the rise in reductase activity is depressed in hypercholesterolemic chicks (13) and following cholesterol or mevalonate feeding in cell cultures (14). Moreover, the diurnal rhythm of mevalonate metabolism by sterol and nonsterol pathways and of meval- onate-activating enzymes has been demonstrated (15). The multivalent feedback regulation of HMG-CoA reductase in- volves cholesterol and one or more of the other products that are synthesized from mevalonate (16). Both cholesterol and mevalonate are required for the depression of reductase activ- ity at the transcriptional, posttranscriptional, and posttransla- tional levels (17–20). The diurnal variation in activity is a good example of short-term physiological regulation of hepatic HMG-CoA reductase (1,2) and is due to changes in the state of phosphorylation of the enzyme; however it has been reported that the diurnal variation in hepatic HMG-CoA reductase is due to changes in enzyme protein levels rather than changes in the phosphorylation state of the enzyme (21). It has also been demonstrated in vivo that the short-term changes in enzyme ac- tivity are due to changes in the quantity of enzyme rather than to a modulation of the catalytic activity (22). On the other hand, HMG-CoA reductase activity regulation participates in cell division (23–26), as reflected by a marked rise in HMG-CoA reductase activity just preceding the S phase of the cell cycle (25). However, parallel changes in HMG-CoA reductase activity have been observed in synchronized and un- synchronized cells (14). In fact, HMG-CoA reductase activity was low at the time of the medium change and increased 10- to 20-fold 5–10 h after medium change, returning to basal levels by 24 h in both synchronized and unsynchronized cells. Thus, other factors besides the cell cycle may also play a role. If the rise in HMG-CoA reductase activity is blocked by a competi- tive inhibitor such as compactin, the cells will not enter the S phase (23). The arrest in the cell cycle cannot be overcome by the addition of cholesterol, but rather by the addition of meval- onate (24,25). Studies in a rat embryo fibroblast cell line, syn- chronized by double thymidine (dThd) block and cultured in cholesterol-containing medium, have demonstrated cyclic vari- ations of HMG-CoA reductase activity with two maxima in the S and G 2 M phases (26). In this culture model, the cyclic varia- Copyright © 2006 by AOCS Press 1089 Lipids, Vol. 41, no. 12 (2006) *To whom correspondence should be addressed at Dept. of Biochemistry and Molecular Biology, Faculty of Sciences, C/ Fuentenueva s/n, University of Granada, 18071 Granada, Spain. E-mail: analinar@ugr.es The first two authors listed contributed equally to this work. Abbreviations: dNTP, deoxy nucleotide 5′-triphosphate; dThd, double thymi- dine; LPDS, lipoprotein-deficient serum; PI, propidium iodide; RT-PCR, re- verse transcription-polymerase chain reaction; SMC, smooth muscle cell; SMC-C, smooth muscle cell–control fed chicks; SMC-Ch, smooth muscle cell–cholesterol fed chicks; SRE, sterol regulatory element; SREBP sterol regulatory element-binding protein. ARTICLES Cyclic Fluctuations of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Aortic Smooth Muscle Cell Cultures Mª José Alejandre, Sonia Perales, Ángel Carazo, Rogelio Palomino-Morales, and Ana Linares* Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, 18071 Granada, Spain