Alberto Canfrán-Duque

MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels.

The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL-cholesterol (LDL-C). While the transcriptional regulation of LDLR is well-characterized, the post-transcriptional mechanisms which govern LDLR expression are just beginning to emerge. Here, we developed a high-throughput genome-wide screening assay to systematically identify microRNAs (miRNAs) that regulate LDLR activity in human hepatic cells. From this screen, we characterize miR-148a as a negative regulator of LDLR expression and activity, and define a novel SREBP1-mediated pathway by which miR-148a regulates LDL-C uptake. Importantly, inhibition of miR-148a increases hepatic LDLR expression and decreases plasma LDL-C in vivo. We also provide evidence that miR-148a regulates hepatic ABCA1 expression and circulating HDL-C levels. Collectively, these studies uncover miR-148a as an important regulator of hepatic LDL-C clearance through direct regulation of LDLR expression, and demonstrate the therapeutic potential of inhibiting miR-148a to ameliorate the elevated LDL-C/HDL-C ratio, a prominent risk factor for cardiovascular disease.

Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro

Haloperidol, a typical antipsychotic, has been shown to inhibit cholesterol biosynthesis by affecting Δ7-reductase, Δ8,7-isomerase, and Δ14-reductase activities, which results in the accumulation of different sterol intermediates. In the present work, we investigated the effects of atypical or second-generation antipsychotics (SGA), such as clozapine, risperidone, and ziprasidone, on intracellular lipid metabolism in different cell lines. All the SGAs tested inhibited cholesterol biosynthesis. Ziprasidone and risperidone had the same targets as haloperidol at inhibiting cholesterol biosynthesis, although with different relative activities (ziprasidone > haloperidol > risperidone). In contrast, clozapine mainly affected Δ24-reductase and Δ8,7-isomerase activities. These amphiphilic drugs also interfered with the LDL-derived cholesterol egress from the endosome/lysosome compartment, thus further reducing the cholesterol content in the endoplasmic reticulum. This triggered a homeostatic response with the stimulation of sterol regulatory element-binding protein (SREBP)-regulated gene expression. Treatment with SGAs also increased the synthesis of complex lipids (phospholipids and triacylglycerides). Once the antipsychotics were removed from the medium, a rebound in the cholesterol biosynthesis rate was detected, and the complex-lipid synthesis further increased. In this condition, apolipoprotein B secretion was also stimulated as demonstrated in HepG2 cells. These effects of SGAs on lipid homeostasis may be relevant in the metabolic side effects of antipsychotics, especially hypertriglyceridemia.

Haloperidol disrupts lipid rafts and impairs insulin signaling in SH-SY5Y cells

Haloperidol exerts its therapeutic effects basically by acting on dopamine receptors. We previously reported that haloperidol inhibits cholesterol biosynthesis in cultured cells. In the present work we investigated its effects on lipid-raft composition and functionality. In both neuroblastoma SH-SY5Y and promyelocytic HL-60 human cell lines, haloperidol inhibited cholesterol biosynthesis resulting in a decrease of the cell cholesterol content and the accumulation of different sterol intermediates (7-dehydrocholesterol, zymostenol and cholesta-8,14-dien-3beta-ol) depending on the dose of the drug. As a consequence, the cholesterol content in lipid rafts was greatly reduced, and several pre-cholesterol sterols, particularly cholesta-8,14-dien-3beta-ol, were incorporated into the cell membrane. This was accompanied by the disruption of lipid rafts, with redistribution of flotillin-1 and Fyn and the impairment of insulin-Akt signaling. Supplementing the medium with free cholesterol abrogated the effects of haloperidol on lipid-raft composition and functionality. LDL (low-density lipoprotein), a physiological vehicle of cholesterol in plasma, was much less effective in preventing the effects of haloperidol, which is attributed to the drugs inhibition of intracellular vesicular trafficking. These effects on cellular cholesterol homeostasis that ultimately result in the alteration of lipid-raft-dependent insulin signaling action may underlie some of the metabolic effects of this widely used antipsychotic.

microRNAs and HDL life cycle

miRNAs have emerged as important regulators of lipoprotein metabolism. Work over the past few years has demonstrated that miRNAs control the expression of most of the genes associated with high-density lipoprotein (HDL) metabolism, including the ATP transporters, ABCA1 and ABCG1, and the scavenger receptor SRB1. These findings strongly suggest that miRNAs regulate HDL biogenesis, cellular cholesterol efflux, and HDL cholesterol (HDL-C) uptake in the liver, thereby controlling all of the steps of reverse cholesterol transport. Recent work in animal models has demonstrated that manipulating miRNA levels including miR-33 can increase circulating HDL-C. Importantly, antagonizing miR-33 in vivo enhances the regression and reduces the progression of atherosclerosis. These findings support the idea of developing miRNA inhibitors for the treatment of dyslipidaemia and related cardiovascular disorders such as atherosclerosis. This review article focuses on how HDL metabolism is regulated by miRNAs and how antagonizing miRNA expression could be a potential therapy for treating cardiometabolic diseases.

Macrophage deficiency of miR‐21 promotes apoptosis, plaque necrosis, and vascular inflammation during atherogenesis

Atherosclerosis, the major cause of cardiovascular disease, is a chronic inflammatory disease characterized by the accumulation of lipids and inflammatory cells in the artery wall. Aberrant expression of microRNAs has been implicated in the pathophysiological processes underlying the progression of atherosclerosis. Here, we define the contribution of miR‐21 in hematopoietic cells during atherogenesis. Interestingly, we found that miR‐21 is the most abundant miRNA in macrophages and its absence results in accelerated atherosclerosis, plaque necrosis, and vascular inflammation. miR‐21 expression influences foam cell formation, sensitivity to ER‐stress‐induced apoptosis, and phagocytic clearance capacity. Mechanistically, we discovered that the absence of miR‐21 in macrophages increases the expression of the miR‐21 target gene, MKK3, promoting the induction of p38‐CHOP and JNK signaling. Both pathways enhance macrophage apoptosis and promote the post‐translational degradation of ABCG1, a transporter that regulates cholesterol efflux in macrophages. Altogether, these findings reveal a major role for hematopoietic miR‐21 in atherogenesis.

Post-lanosterol biosynthesis of cholesterol and cancer

Mammalian cells require cholesterol for proliferation. Cholesterol contributes not only to the physicochemical properties of membranes but also to the organization of lipid rafts involved in signal transduction. Inhibition of cholesterol biosynthesis from lanosterol results in the inhibition of cell cycle progression and, in certain cell types, also in the induction of cell differentiation. Cholesterol metabolism, thus, appears to play a relevant role in the decision making between cell proliferation and differentiation. Several regulators of cholesterol metabolism, including certain microRNAs, are also involved in cell cycle regulation. The relevance of these processes in cancer underscores the interest for studying the role of cholesterol in tumorigenesis and exploring the possibility of interfering with the growth of malignant cells by manipulation of cholesterol metabolism.

The miR-199–dynamin regulatory axis controls receptor-mediated endocytosis

ABSTRACT Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate many physiological processes; however, their role in regulating intracellular transport remains largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a and miR-199b family of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a and miR-199b regulate endocytic transport by controlling the expression of important mediators of endocytosis such as clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). Importantly, miR-199a-5p and miR-199b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa). Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated endocytosis. Taken together, our work identifies a new mechanism by which microRNAs regulate intracellular trafficking. In particular, we demonstrate that the DNM, miR-199a-5p and miR-199b-5p genes act as a bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the regulation of intracellular trafficking. Summary: A characterization of how an intronic miRNA encoded in the dynamin genes controls cellular endocytosis by targeting multiple components of the endocytic machinery.

Curcumin promotes exosomes/microvesicles secretion that attenuates lysosomal cholesterol traffic impairment

SCOPE Exosomes/microvesicles are originated from multivesicular bodies that allow the secretion of endolysosome components out of the cell. In the present work, we investigated the effects of curcumin, a polyphenol, on exosomes/microvesicles secretion in different cells lines, using U18666A as a model of intracellular cholesterol trafficking impairment. METHODS AND RESULTS In both HepG2 hepatocarcinoma cells and THP-1 differentiated macrophages, treatment with curcumin affected the size and the localization of endosome/lysosomes accumulated by U18666A, and reduced the cholesterol cell content. To ascertain the mechanism, we analyzed the incubation medium. Curcumin stimulated the release of cholesterol and the lysosomal β-hexosaminidase enzyme, as well as the exosome markers, flotillin-2 and CD63. Electron microscopy studies demonstrated the presence of small vesicles similar to exosomes/microvesicles in the secretion fluid. These vesicles harbored CD63 on their surface, indicative of their endolysosomal origin. These effects of curcumin were particularly intense in cells treated with U18666A. CONCLUSION These findings indicate that curcumin ameliorates the U18666A-induced endolysosomal cholesterol accumulation by shuttling cholesterol and presumably other lipids out of the cell via exosomes/microvesicles secretion. This action may contribute to the potential of curcumin in the treatment of lysosomal storage diseases.

Micro-RNAs and High-Density Lipoprotein Metabolism

Improved prevention and treatment of cardiovascular diseases (CVD) 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 disease (CVD). The cardioprotective effect of HDL is due to its ability to remove cholesterol from plaques in the artery wall to the liver for excretion by a process known as reverse cholesterol transport (RCT). Numerous studies have reported the role that microRNAs (miRNAs) play in the regulation of the different steps in RCT, including HDL biogenesis, cholesterol efflux, cholesterol uptake in the liver and bile acid synthesis and secretion. Due to their ability to control different aspects of HDL metabolism and function, miRNAs have emerged as potential therapeutic targets to combat CVDs. 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 CVD biomarkers.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.

Neuregulin-activated ERBB4 induces the SREBP-2 cholesterol biosynthetic pathway and increases low-density lipoprotein uptake.

The epidermal growth factor receptor family member ERBB4 stimulates cholesterol biosynthesis in breast epithelial cells. Providing cholesterol for proliferation Although cholesterol has received a lot of bad press, this lipid molecule is actually an essential component of cellular membranes. Growing or dividing cells need more cholesterol than quiescent cells. The activity of epidermal growth factor receptor (EGFR) family members stimulates cell proliferation in physiological and pathophysiological contexts, such as cancer. Haskins et al. found that neuregulin 1 (NRG1)–mediated activation of the EGFR member ERBB4 stimulated the transcription factor SREBP-2, which enhanced the expression of the receptor needed to uptake cholesterol-rich low-density lipoproteins and genes involved in cholesterol biosynthesis in cultured breast epithelial cells. Pharmacological inhibition of EGFR activity or SREBP-2 activity suppressed the NRG1-mediated induction of cholesterol synthesis–related genes. Thus, EGFR signaling alters cellular lipid metabolism, enabling cells to acquire or synthesize molecules necessary for proliferation. Cholesterol is a lipid that is critical for steroid hormone production and the integrity of cellular membranes, and, as such, it is essential for cell growth. The epidermal growth factor receptor (EGFR) family member ERBB4, which forms signaling complexes with other EGFR family members, can undergo ligand-induced proteolytic cleavage to release a soluble intracellular domain (ICD) that enters the nucleus to modify transcription. We found that ERBB4 activates sterol regulatory element binding protein-2 (SREBP-2) to enhance low-density lipoprotein (LDL) uptake and cholesterol biosynthesis. Expression of the ERBB4 ICD in mammary epithelial cells or activation of ERBB4 with the ligand neuregulin 1 (NRG1) induced the expression of SREBP target genes involved in cholesterol biosynthesis, including HMGCR and HMGCS1, and lipid uptake, LDLR, which encodes the LDL receptor. Addition of NRG1 increased the abundance of the cleaved, mature form of SREBP-2 through a pathway that was blocked by addition of inhibitors of PI3K (phosphatidylinositol 3-kinase) or dual inhibition of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2, but not by inhibition of AKT or mTORC1. Pharmacological inhibition of the activity of SREBP site 1 protease or of all EGFR family members (with lapatinib), but not EGFR alone (with erlotinib), impaired NRG1-induced expression of cholesterol biosynthesis genes. Collectively, our findings indicated that activation of ERBB4 promotes SREBP-2–regulated cholesterol metabolism. The connections of EGFR and ERBB4 signaling with SREBP-2–regulated cholesterol metabolism are likely to be important in ERBB-regulated developmental processes and may contribute to metabolic remodeling in ERBB-driven cancers.