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Schippers, IJ; Moshage, Han; Roelofsen, Han; Müller, Michael; Heymans, H. S. A.; Ruiters, Marcel H. J.; Kuipers, Folkert

doi:10.1023/a:1007404028681

Cited by 92 publications

(37 citation statements)

References 18 publications

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“…This also resulted in increased expression of a panel of studied SREBP2 targets in HepG2 cells (Fig. 2), more prominently evident in IHH cells, which is consistent with them being more metabolically responsive (20). Notably, the increase was apparent when cells were cultured in both complete medium and under sterol depletion conditions.…”

Section: Resultssupporting

confidence: 70%

“…Cells were cultured at 37°C and 5% CO 2 in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). IHH cells were a kind gift from Geesje Dallinga-Thie (Amsterdam, The Netherlands) and cultured in William's E medium supplemented with 2 mM glutamine, 10% FBS, 20 mU/ml bovine insulin, and 50 nM dexamethasone, as previously reported (20). Where indicated in the figures and figure legends, cells were sterol depleted by culture in sterol depletion medium (DMEM supplemented with 10% lipoprotein-deficient serum [LPDS], 2.5 g/ml simvastatin, and 100 M mevalonate) for 24 h. For detection of PCSK9 in culture medium, cells were washed with phosphate-buffered saline (PBS) and cultured in Opti-MEM (Invitrogen) for 24 h prior to cell lysis.…”

Section: Methodsmentioning

confidence: 99%

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A MARCH6 and IDOL E3 Ubiquitin Ligase Circuit Uncouples Cholesterol Synthesis from Lipoprotein Uptake in Hepatocytes

Loregger

Cook

Nelson

et al. 2016

Molecular and Cellular Biology

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Cholesterol synthesis and lipoprotein uptake are tightly coordinated to ensure that the cellular level of cholesterol is adequately maintained. Hepatic dysregulation of these processes is associated with pathological conditions, most notably cardiovascular disease. Using a genetic approach, we have recently identified the E3 ubiquitin ligase MARCH6 as a regulator of cholesterol biosynthesis, owing to its ability to promote degradation of the rate-limiting enzymes 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) and squalene epoxidase (SQLE). Here, we present evidence for MARCH6 playing a multifaceted role in the control of cholesterol homeostasis in hepatocytes. We identify MARCH6 as an endogenous inhibitor of the sterol regulatory element binding protein (SREBP) transcriptional program. Accordingly, loss of MARCH6 increases expression of SREBP-regulated genes involved in cholesterol biosynthesis and lipoprotein uptake. Unexpectedly, this is associated with a decrease in cellular lipoprotein uptake, induced by enhanced lysosomal degradation of the low-density lipoprotein receptor (LDLR). Finally, we provide evidence that induction of the E3 ubiquitin ligase IDOL represents the molecular mechanism underlying this MARCH6-induced phenotype. Our study thus highlights a MARCH6-dependent mechanism to direct cellular cholesterol accretion that relies on uncoupling of cholesterol synthesis from lipoprotein uptake. Cholesterol is an essential constituent of cellular membranes and signaling pathways and is a precursor of sterol-derived molecules (1). Yet elevated levels of cholesterol are toxic to cells, and dysregulated cholesterol metabolism is associated, most evidently, with development of cardiovascular disease. As such, multiple transcriptional networks and posttranscriptional processes regulate the synthesis, uptake, and efflux of cholesterol. Transcriptionally, these processes are largely governed by the opposing actions of the transcription factors sterol regulatory element binding proteins (SREBPs) and the liver X receptors (LXRs) (2-6). Upon sensing low cholesterol levels in the endoplasmic reticulum (ER), SREBPs are processed into their mature, transcriptionally active form. This results in induction of the full set of genes required for de novo biosynthesis of cholesterol via the mevalonate pathway and of the low-density lipoprotein receptor (LDLR) that is required for uptake of LDL-derived cholesterol (7, 8). In contrast, LXRs, members of the nuclear receptor family, are activated when cellular cholesterol levels are elevated. Once activated by their cognate oxysterol ligands, LXRs induce cholesterol efflux pathways (e.g., via the transporters ABCA1 and ABCG1) and limit LDL uptake by inducing expression of the E3 ubiquitin ligase (E3)-inducible degrader of the LDLR (IDOL) (6, 9, 10). The coordinated action of these two transcription factor families ensures that cellular cholesterol is adequately maintained at an appropriate level.Next to transcriptional regulation, posttranscriptional mechanisms are ...

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Section: Resultssupporting

confidence: 70%

Section: Methodsmentioning

confidence: 99%

A MARCH6 and IDOL E3 Ubiquitin Ligase Circuit Uncouples Cholesterol Synthesis from Lipoprotein Uptake in Hepatocytes

Loregger

Cook

Nelson

et al. 2016

Molecular and Cellular Biology

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“…One day prior to experiments, immortalized human hepatocyte (IHH) cells [21] were inoculated in 6 well plates containing 3 ml medium per well. On the day of the experiment, cells were placed in medium, which did not contain insulin and dexamethasone and were pre-incubated with a vanadium complex that is a specific inhibitor of PTEN (VO-OHpic, 50nM) for 15 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Role of PTEN in Oxidative Stress and DNA Damage in the Liver of Whole-Body Pten Haplodeficient Mice

Bankoglu

Tschopp²,

Schmitt

et al. 2016

PLoS ONE

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Type 2 diabetes (T2DM) and obesity are frequently associated with non-alcoholic fatty liver disease (NAFLD) and with an elevated cancer incidence. The molecular mechanisms of carcinogenesis in this context are only partially understood. High blood insulin levels are typical in early T2DM and excessive insulin can cause elevated reactive oxygen species (ROS) production and genomic instability. ROS are important for various cellular functions in signaling and host defense. However, elevated ROS formation is thought to be involved in cancer induction. In the molecular events from insulin receptor binding to genomic damage, some signaling steps have been identified, pointing at the PI3K/AKT pathway. For further elucidation Phosphatase and Tensin homolog (Pten), a tumour suppressor phosphatase that plays a role in insulin signaling by negative regulation of PI3K/AKT and its downstream targets, was investigated here. Dihydroethidium (DHE) staining was used to detect ROS formation in immortalized human hepatocytes. Comet assay and micronucleus test were performed to investigate genomic damage in vitro. In liver samples, DHE staining and western blot detection of HSP70 and HO-1 were performed to evaluate oxidative stress response. DNA double strand breaks (DSBs) were detected by immunohistostaining. Inhibition of PTEN with the pharmacologic inhibitor VO-OHpic resulted in increased ROS production and genomic damage in a liver cell line. Knockdown of Pten in a mouse model yielded increased oxidative stress levels, detected by ROS levels and expression of the two stress-proteins HSP70 and HO-1 and elevated genomic damage in the liver, which was significant in mice fed with a high fat diet. We conclude that PTEN is involved in oxidative stress and genomic damage induction in vitro and that this may also explain the in vivo observations. This further supports the hypothesis that the PI3K/AKT pathway is responsible for damaging effects of high levels of insulin.

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“…Cell Culture-Human immortalized hepatocytes are described elsewhere (30). They were cultured on collagencoated flasks in William's E medium in the presence of a 10% fetal calf serum.…”

Section: Methodsmentioning

confidence: 99%

Dual Mechanisms for the Fibrate-mediated Repression of Proprotein Convertase Subtilisin/Kexin Type 9

Kourimate

May

Langhi

et al. 2008

Journal of Biological Chemistry

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Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with familial autosomal dominant hypercholesterolemia and is a natural inhibitor of the LDL receptor (LDLr). PCSK9 is degraded by other proprotein convertases: PC5/6A and furin. Both PCSK9 and the LDLr are up-regulated by the hypocholesterolemic statins. Thus, inhibitors or repressors of PCSK9 should amplify their beneficial effects. In the present study, we showed that PPAR␣ activation counteracts PCSK9 induction by statins by repressing PCSK9 promoter activity and by increasing PC5/6A and furin expression. Quantification of mRNA and protein levels showed that various fibrates decreased PCSK9 and increased PC5/6A and furin expression. Fenofibric acid (FA) reduced PCSK9 protein content in immortalized human hepatocytes (IHH) as well as its cellular secretion. FA suppressed PCSK9 induction by statins or by the liver X receptor agonist TO901317. PCSK9 repression is occurring at the promoter level. We showed that PC5/6A and furin fibrate-mediated up-regulation is PPAR␣-dependent. As a functional test, we observed that FA increased by 30% the effect of pravastatin on the LDLr activity in vitro. In conclusion, fibrates simultaneously decreased PCSK9 expression while increasing PC5/6A and furin expression, indicating a broad action of PPAR␣ activation in proprotein convertase-mediated lipid homeostasis. Moreover, this study validates the functional relevance of a combined therapy associating PCSK9 repressors and statins.Proprotein convertase subtilisin/kexin type 9 (PCSK9) 3 is associated with autosomal dominant hypercholesterolemia (1) and is a natural inhibitor of the LDL receptor (LDLr) (2). Its autocatalytic activity is necessary to its processing and for the mature form to reach the LDLr and induce its degradation (3). This catalytic activity is not involved in LDLr degradation per se. Indeed, cellular or secreted mature PCSK9 probably acts as a chaperone and prevents the LDLr from being recycled by binding to its EGFA domain (4 -10). Accordingly, hepatic transient overexpression of wild-type PCSK9 in mice (11)(12)(13)(14) or elevated circulating levels in parabiotic mice (15) decrease the LDLr expression and lead to hypercholesterolemia. It has been shown that the proprotein convertase furin and to a lesser extent PC5/6A cleave PCSK9 into a secreted, truncated, inactive form (16). Interestingly, some "gain of function" mutations do not necessarily induce a higher affinity of PCSK9 for the LDLr, but decrease PCSK9 sensitivity to this degradation (4). In contrast, PCSK9 deficiency lowers plasmatic cholesterol concentration and confers protection against cardiovascular disease (17-20). Thus, treatments inhibiting PCSK9 synthesis, processing, or binding to the LDLr could be useful for hypercholesterolemic patients who do not reach therapeutic goals. In particular, these inhibitors could be added to statins to amplify their effect on the LDLr activity. Surprisingly, statins also increase PCSK9 expression via SREBP-2, a pathway that exerts a break on ...

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Untitled

Cited by 92 publications

References 18 publications

A MARCH6 and IDOL E3 Ubiquitin Ligase Circuit Uncouples Cholesterol Synthesis from Lipoprotein Uptake in Hepatocytes

A MARCH6 and IDOL E3 Ubiquitin Ligase Circuit Uncouples Cholesterol Synthesis from Lipoprotein Uptake in Hepatocytes

Role of PTEN in Oxidative Stress and DNA Damage in the Liver of Whole-Body Pten Haplodeficient Mice

Dual Mechanisms for the Fibrate-mediated Repression of Proprotein Convertase Subtilisin/Kexin Type 9

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