sq-23377 and 25-hydroxycholesterol

The in vivo turnover rate of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the mevalonate (MVA) pathway, is accelerated when excess MVA or sterols are added to the growth medium of cells. As we have shown recently (Roitelman, J., Bar-Nun, S., Inoue, S., and Simoni, R. D. (1991) J. Biol. Chem. 266, 16085-16091), perturbation of cellular Ca2+ homeostasis abrogates the MVA-accelerated degradation of HMG-CoA reductase and HMGal. Here we show that, in contrast, the sterol-accelerated degradation of HMG-CoA reductase is unaffected by Ca2+ perturbation achieved either by Ca2+ ionophore or by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. The differential effects of Ca2+ perturbation can be attributed neither to global alteration in protein synthesis nor to inhibition of MVA conversion to sterols. Yet, such manipulations markedly reduce the incorporation of MVA into cellular macromolecules, including prenylated proteins. Furthermore, we directly demonstrate that MVA gives rise to at least two distinct signals, one that is essential to support the effect of sterols and another that operates independently of sterols. Our results indicate that the cellular signals operating in the MVA-accelerated turnover of HMG-CoA reductase are distinct from those involved in the sterol-regulated degradation. A working model for the degradation pathway is proposed.

Topics: Animals; Antioxidants; beta-Galactosidase; Calcimycin; Calcium; CHO Cells; Cholesterol; Cricetinae; Endoplasmic Reticulum; Homeostasis; Hydroquinones; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Ionomycin; Kinetics; Mevalonic Acid; Models, Biological; Recombinant Fusion Proteins; Signal Transduction; Sterols; Terpenes; Thapsigargin; Time Factors; Transfection

Upon stimulation with antigen or antibodies directed at the CD3.T cell receptor complex, T lymphocytes undergo a series of biochemical events that result in DNA synthesis and cellular proliferation. The purpose of the current study was to explore the role of mevalonic acid and its metabolites in this process. Stimulation of freshly isolated human T cells with immobilized anti-CD3 monoclonal antibody (mAb) results in the induction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase message, with maximum induction occurring at 24 h of culture, approximately 12 h before the onset of DNA synthesis. Protein kinase C (PKC) probably mediates this induction, as H7, which inhibits PKC and cyclic nucleotide-dependent protein kinases, but not HA1004, which inhibits all of these protein kinases except PKC, completely abrogates the appearance of HMG-CoA reductase message. The importance of HMG-CoA reductase induction and mevalonate production in cell cycle progression was demonstrated by the observation that either 25-hydroxycholesterol, which inhibits this induction, or lovastatin, a competitive inhibitor of HMG-CoA reductase, inhibited anti-CD3-induced T cell mitogenesis in a dose-dependent manner. The presence of lovastatin during the first 24-36 h of culture results in a progressive delay of cell cycle progression, whereas this agent, when present only for the first 12 h of culture, had no effect on T cell proliferation. These results suggest that mevalonate is required for cell cycle progression from mid-G1 into late G1. Exogenous mevalonate overcomes the antiproliferative effect of lovastatin but not of 25-hydroxycholesterol. Since 25-hydroxycholesterol suppresses the metabolism of mevalonic acid at multiple points, this result suggests that one or more metabolites of mevalonate, rather than mevalonate itself, plays an essential role in cell cycle progression. One metabolite of mevalonate, farnesol pyrophosphate, may play such a role, since free farnesol suppresses anti-CD3 mAb-induced T cell proliferation in a concentration-dependent manner. In mAb is associated with PKC-dependent induction of HMG-CoA reductase which, in turn, leads to the generation of mevalonic acid and its metabolites, one or more of which play a requisite role in cell cycle progression.

Topics: Antibodies, Monoclonal; Antigens, CD; Blotting, Northern; Cell Division; Humans; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Ionomycin; Lovastatin; Mevalonic Acid; Mitogens; RNA; T-Lymphocytes; Tetradecanoylphorbol Acetate

Granulosa cells, isolated by collagenase digestion from the mature ovarian follicle of laying hens, were incubated in the presence of two ionophores, lasalocid (X537A) and ionomycin, to determine their effects on basal and stimulated steroidogenesis, as well as their effects on various cell parameters including DNA, RNA, and protein synthesis. Both ionophores caused a dose-dependent inhibition of agonist-promoted progesterone production and, in the presence of calcium, a small but significant increase in basal output of progesterone. Whereas the conversion of pregnenolone to progesterone was unaffected by the ionophores, the activity of cholesterol side-chain cleavage enzyme was inhibited in a dose-related manner. Both ionophores decreased cellular levels of ATP and inhibited the incorporation of radioactively-labeled precursors into DNA, RNA, and proteins. Morphologically, ionophore-treated cells showed swelling of the rough endoplasmic reticulum. Similar morphological changes were also observed in cells treated with oligomycin, a known metabolic inhibitor. These results suggest that the ionophores lasalocid and ionomycin impair release of energy and thereby exert the principal cause of the inhibited steroidogenic response by granulosa cells to a variety of agonists.

Topics: Animals; Chickens; Ethers; Female; Granulosa Cells; Hydroxycholesterols; In Vitro Techniques; Ionomycin; Kinetics; Lasalocid; Microscopy, Electron; Mitochondria; Progesterone