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While therapeutic modulation of miRNAs provides a promising approach for numerous diseases, the promiscuous nature of miRNAs raises concern over detrimental off-target effects. miR-33 has emerged as a likely target for treatment of cardiovascular diseases. However, the deleterious effects of long-term anti-miR-33 therapies and predisposition of miR-33−/−mice to obesity and metabolic dysfunction exemplify the possible pitfalls of miRNA-based therapies. Our work provides an in-depth characterization of miR-33−/−mice and explores the mechanisms by which loss of miR-33 promotes insulin resistance in key metabolic tissues. Contrary to previous reports, our data do not support a direct role for SREBP-1-mediated lipid synthesis in promoting these effects. Alternatively, in adipose tissue of miR-33−/−mice, we observe increased pre-adipocyte proliferation, enhanced lipid uptake, and impaired lipolysis. Moreover, we demonstrate that the driving force behind these abnormalities is increased food intake, which can be prevented by pair feeding with wild-type animals.

As an important regulator of macrophage cholesterol efflux and HDL biogenesis, miR-33 is a promising target for treatment of atherosclerosis, and numerous studies demonstrate that inhibition of miR-33 increases HDL levels and reduces plaque burden. However, important questions remain about how miR-33 impacts atherogenesis, including whether this protection is primarily due to direct effects on plaque macrophages or regulation of lipid metabolism in the liver. We demonstrate that miR-33 deficiency in Ldlr−/−mice promotes obesity, insulin resistance, and hyperlipidemia but does not impact plaque development. We further assess how loss of miR-33 or addition of miR-33b in macrophages and other hematopoietic cells impact atherogenesis. Macrophage-specific loss of miR-33 decreases lipid accumulation and inflammation under hyperlipidemic conditions, leading to reduced plaque burden. Therefore, the pro-atherogenic effects observed in miR-33-deficient mice are likely counterbalanced by protective effects in macrophages, which may be the primary mechanism through which anti-miR-33 therapies reduce atherosclerosis.

Macrophages perform critical functions in both innate immunity and cholesterol metabolism. Here, we report that activation of Toll-like receptor 4 (TLR4) in macrophages causes lanosterol, the first sterol intermediate in the cholesterol biosynthetic pathway, to accumulate. This effect is due to type I interferon (IFN)-dependent histone deacetylase 1 (HDAC1) transcriptional repression of lanosterol-14α-demethylase, the gene product of Cyp51A1. Lanosterol accumulation in macrophages, because of either treatment with ketoconazole or induced conditional disruption of Cyp51A1in mouse macrophages in vitro, decreases IFNβ-mediated signal transducer and activator of transcription (STAT)1-STAT2 activation and IFNβ-stimulated gene expression. These effects translate into increased survival to endotoxemic shock by reducing cytokine secretion. In addition, lanosterol accumulation increases membrane fluidity and ROS production, thus potentiating phagocytosis and the ability to kill bacteria. This improves resistance of mice to Listeria monocytogenesinfection by increasing bacterial clearance in the spleen and liver. Overall, our data indicate that lanosterol is an endogenous selective regulator of macrophage immunity.

Improved prevention and treatment of cardiovascular diseases is one of the challenges in Western societies, where ischemic heart disease and stroke are the leading cause of death. Early epidemiological studies have shown an inverse correlation between circulating high-density lipoprotein-cholesterol (HDL-C) and cardiovascular diseases. The cardioprotective effect of HDL is because of its ability to remove cholesterol from plaques in the artery wall to the liver for excretion by a process known as reverse cholesterol transport. Numerous studies have reported the role that micro-RNAs (miRNA) play in the regulation of the different steps in reverse cholesterol transport, including HDL biogenesis, cholesterol efflux, and cholesterol uptake in the liver and bile acid synthesis and secretion. Because of their ability to control different aspects of HDL metabolism and function, miRNAs have emerged as potential therapeutic targets to combat cardiovascular diseases. In this review, we summarize the recent advances in the miRNA–mediated control of HDL metabolism. We also discuss how HDL particles serve as carriers of miRNAs and the potential use of HDL-containing miRNAs as cardiovascular diseases biomarkers.

Although the role for microRNAs (miRNAs) in regulating multiple physiological processes including apoptosis, cell differentiation, and cancer is well established, the importance of these tiny RNAs in regulating lipid metabolism has only recently been uncovered. This review summarizes the evidence for a critical role of miRNAs in regulating lipid metabolism.

Plitidepsin (aplidin) is a marine cyclic depsipeptide in phase II clinical development against several neoplasias. Plitidepsin is a potent inducer of apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We have reported that this activation depends on the early induction of oxidative stress, activation of Rac1 small GTPase, and the later down-regulation of MKP-1 phosphatase. Using Scatchard and saturation binding analyses, we have found that 14C-labeled plitidepsin binds to a moderately high-affinity receptor (Kdof 44.8 ± 3.1 and 35.5 ± 4.8 nM, respectively) in MDA-MB-231 breast cancer cells. Two minutes after addition to cells, half of the drug was membrane-bound and was subsequently found in the cytosolic fraction. At 4°C, plitidepsin cellular binding was around 10-fold lower than at 37°C but sufficed to induce cell death, suggesting that this process is triggered from the membrane. Depletion of plasma membrane cholesterol by short treatment with methyl-β-cyclodextrin diminished plitidepsin binding and Rac1 and JNK activation. Rac1 is targeted to the plasma membrane by plitidepsin as shown by subcellular fractioning and immunofluorescence analysis followed by confocal microscopy. Methyl-β-cyclodextrin blocked this effect. A subline of HeLa cells (HeLa-R), partially resistant to plitidepsin, showed similar affinity (Kdof 79.5 ± 2.5 versus 37.7 ± 8.2 nM) but 7.5-fold lower binding capacity than wild-type HeLa cells. Moreover, HeLa-R cells had lower total (71%) and membrane (67%) cholesterol content and membrane-bound Rac1, and showed no Rac1 activation upon plitidepsin treatment. In conclusion, cellular plitidepsin uptake and induction of apoptosis via activation of the Rac1-JNK pathway is membrane-cholesterol dependent.

Plitidepsin (aplidin) is a marine cyclic depsipeptide in phase II clinical development against several neoplasias. Plitidepsin is a potent inducer of apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We have reported that this activation depends on the early induction of oxidative stress, activation of Rac1 small GTPase, and the later down-regulation of MKP-1 phosphatase. Using Scatchard and saturation binding analyses, we have found that 14C-labeled plitidepsin binds to a moderately high-affinity receptor (Kd of 44.8 ± 3.1 and 35.5 ± 4.8 nM, respectively) in MDA-MB-231 breast cancer cells. Two minutes after addition to cells, half of the drug was membrane-bound and was subsequently found in the cytosolic fraction. At 4°C, plitidepsin cellular binding was around 10-fold lower than at 37°C but sufficed to induce cell death, suggesting that this process is triggered from the membrane. Depletion of plasma membrane cholesterol by short treatment with methyl-β-cyclodextrin diminished plitidepsin binding and Rac1 and JNK activation. Rac1 is targeted to the plasma membrane by plitidepsin as shown by subcellular fractioning and immunofluorescence analysis followed by confocal microscopy. Methyl-β-cyclodextrin blocked this effect. A subline of HeLa cells (HeLa-R), partially resistant to plitidepsin, showed similar affinity (Kd of 79.5 ± 2.5 versus 37.7 ± 8.2 nM) but 7.5-fold lower binding capacity than wild-type HeLa cells. Moreover, HeLa-R cells had lower total (71%) and membrane (67%) cholesterol content and membrane-bound Rac1, and showed no Rac1 activation upon plitidepsin treatment. In conclusion, cellular plitidepsin uptake and induction of apoptosis via activation of the Rac1-JNK pathway is membrane-cholesterol dependent.

Dietary phytosterols are cholesterol-lowering agents that interfere with the intestinal absorption of cholesterol. In the present study, we have studied their effects on cholesterol biosynthesis in human cells, particularly in the sterol-conversion pathway. For this, both Caco-2 (intestinal mucosa) and HL-60 (promyelocytic) human cell lines were incubated with [14C]acetate, and the incorporation of radioactivity into sterols was determined using HPLC and radioactivity detection online. Sterols containing a double bond at C-22 in the side chain (stigmasterol, brassicasterol and ergosterol) dramatically inhibited the activity of sterol Δ24-reductase, as indicated by the decrease in radioactivity incorporation into cholesterol and the accumulation of its precursors (mainly desmosterol). Phytosterols with the saturated side chain (β-sitosterol and campesterol) were inactive in this regard. The inhibition of sterol 24-reductase was confirmed in rat liver microsomes by using 14C-labelled desmosterol as the substrate. The 22-unsaturated phytosterols acted as competitive inhibitors of sterol 24-reductase, with Ki values (41.1, 42.7 and 36.8μM for stigmasterol, brassicasterol and ergosterol respectively) similar to the estimated Km for desmosterol (26.3μM). The sterol 5,22-cholestedien-3β-ol, an unusual desmosterol isomer that lacks the alkyl groups characteristic of phytosterols, acted as a much stronger inhibitor of 24-reductase (Ki = 3.34μM). The usually low intracellular concentrations of the physiological substrates of 24-reductase explains the strong inhibition of cholesterol biosynthesis that these compounds exert in cells. Given that inhibition of sterol 24-reductase was achieved at physiologically relevant concentrations, it may represent an additional mechanism for the cholesterol-lowering action of phytosterols, and opens up the possibility of using certain 22-unsaturated sterols as effective hypocholesterolaemic agents.

As a major component of mammalian cell plasma membranes, cholesterol is essential for cell growth. Accordingly, the restriction of cholesterol provision has been shown to result in cell proliferation inhibition. We explored the potential regulatory role of cholesterol on cell cycle progression. MOLT‐4 and HL‐60 cell lines were cultured in a cholesterol‐deficient medium and simultaneously exposed to SKF 104976, which is a specific inhibitor of lanosterol 14‐α demethylase. Through HPLC analyses with on‐line radioactivity detection, we found that SKF 104976 efficiently blocked the [14C]‐acetate incorporation into cholesterol, resulting in an accumulation of lanosterol and dihydrolanosterol, without affecting the synthesis of mevalonic acid. The inhibitor also produced a rapid and intense inhibition of cell proliferation (IC50= 0.1 µM), as assessed by both [3H]‐thymidine incorporation into DNA and cell counting. Flow cytometry and morphological examination showed that treatment with SKF 104976 for 48 h or longer resulted in the accumulation of cells specifically at G2 phase, whereas both the G1 traversal and the transition through S were unaffected. The G2 arrest was accompanied by an increase in the hyperphosphorylated form of p34cdc2and a reduction of its activity, as determined by assaying the H1 histone phosphorylating activity of p34cdc2immunoprecipitates. The persistent deficiency of cholesterol induced apoptosis. However, supplementing the medium with cholesterol, either in the form of LDL or free cholesterol dissolved in ethanol, completely abolished these effects, whereas mevalonate was ineffective. Caffeine, which abrogates the G2 checkpoint by preventing p34cdc2phosphorylation, reduced the accumulation in G2 when added to cultures containing cells on transit to G2, but was ineffective in cells arrested at G2 by sustained cholesterol starvation. Cells arrested in G2, however, were still viable and responded to cholesterol provision by activating p34cdc2and resuming the cell cycle. We conclude that in both lymphoblastoid and promyelocytic cells, cholesterol availability governs the G2 traversal, probably by affecting p34cdc2activity.—Martínez‐Botas, J., Suárez, Y., Ferruelo, A. J., Gómez‐Coronado, D., Lasunción, M.A. Cholesterol starvation decreases P34cdc2kinase activity and arrests the cell cycle at G2. FASEB J.13, 1359–1370 (1999)

The epidermal growth factor receptor family member ERBB4 stimulates cholesterol biosynthesis in breast epithelial cells.