Cholesterol biosynthetic pathway induces cellular senescence through ERRa

Cellular senescence is a cell program induced by various stresses that leads to a stable proliferation arrest and to a senescence-associated secretory phenotype. Accumulation of senescent cells during age-related diseases participates in these pathologies and regulates healthy lifespan. Recent evidences point out a global dysregulated intracellular metabolism associated to senescence phenotype. Nonetheless, the functional contribution of metabolic homeostasis in regulating senescence is barely understood. In this work, we describe how the mevalonate pathway, an anabolic pathway leading to the endogenous biosynthesis of poly-isoprenoids, such as cholesterol, acts as a positive regulator of cellular senescence in normal human cells. Mechanistically, this mevalonate-induced senescence is partly mediated by the downstream cholesterol biosynthetic pathway. This pathway promotes transcriptional activity of ERRα leading to dysfunctional mitochondria, ROS production, DNA damage and a p53-dependent senescence. Supporting the relevance of these observations, increase of senescence in liver due to a high-fat diet regimen is abrogated in ERRα knockout mouse. Overall, this work unravels the role of cholesterol biosynthesis in the induction of an ERRα-dependent mitochondrial program leading to cellular senescence and related pathological alterations.

AG-205 Upregulates Enzymes Involved in Cholesterol Biosynthesis and Steroidogenesis in Human Endometrial Cells Independently of PGRMC1 and Related MAPR Proteins

An inappropriate response to progestogens in the human endometrium can result in fertility issues and jeopardize progestin-based treatments against pathologies such as endometriosis. PGRMC1 can mediate progesterone response in the breast and ovaries but its endometrial functions remain unknown. AG-205 is an alleged PGRMC1 inhibitor but its specificity was recently questioned. We added AG-205 in the cultures of two endometrial cell lines and performed a transcriptomic comparison. AG-205 significantly increased expression of genes coding enzymes of the cholesterol biosynthetic pathway or of steroidogenesis. However, these observations were not reproduced with cells transfected with siRNA against PGRMC1 or its related proteins (MAPRs). Furthermore, AG-205 retained its ability to increase expression of selected target genes even when expression of PGRMC1 or all MAPRs was concomitantly downregulated, indicating that neither PGRMC1 nor any MAPR is required to mediate AG-205 effect. In conclusion, although AG-205 has attractive effects encouraging its use to develop therapeutic strategies, for instance against breast cancer, our study delivers two important warning messages. First, AG-205 is not specific for PGRMC1 or other MAPRs and its mechanisms of action remain unclear. Second, due to its effects on genes involved in steroidogenesis, its use may increase the risk for endometrial pathologies resulting from imbalanced hormones concentrations.

HMGCR inhibition stabilizes the glycolytic enzyme PKM2 to support the growth of renal cell carcinoma

Renal cell carcinoma (RCC) is responsible for most cases of the kidney cancer. Previous research showed that low serum levels of cholesterol level positively correlate with poorer RCC-specific survival outcomes. However, the underlying mechanisms and functional significance of the role of cholesterol in the development of RCC remain obscure. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) plays a pivotal role in RCC development as it is the key rate-limiting enzyme of the cholesterol biosynthetic pathway. In this study, we demonstrated that the inhibition of HMGCR could accelerate the development of RCC tumors by lactate accumulation and angiogenesis in animal models. We identified that the inhibition of HMGCR led to an increase in glycolysis via the regulated HSP90 expression levels, thus maintaining the levels of a glycolysis rate-limiting enzyme, pyruvate kinase M2 (PKM2). Based on these findings, we reversed the HMGCR inhibition-induced tumor growth acceleration in RCC xenograft mice by suppressing glycolysis. Furthermore, the coadministration of Shikonin, a potent PKM2 inhibitor, reverted the tumor development induced by the HMGCR signaling pathway.

Warburg’s Ghost—Cancer’s Self-Sustaining Phenotype: The Aberrant Carbon Flux in Cholesterol-Enriched Tumor Mitochondria via Deregulated Cholesterogenesis

Interpreting connections between the multiple networks of cell metabolism is indispensable for understanding how cells maintain homeostasis or transform into the decontrolled proliferation phenotype of cancer. Situated at a critical metabolic intersection, citrate, derived via glycolysis, serves as either a combustible fuel for aerobic mitochondrial bioenergetics or as a continuously replenished cytosolic carbon source for lipid biosynthesis, an essentially anaerobic process. Therein lies the paradox: under what conditions do cells control the metabolic route by which they process citrate? The Warburg effect exposes essentially the same dilemma—why do cancer cells, despite an abundance of oxygen needed for energy-generating mitochondrial respiration with citrate as fuel, avoid catabolizing mitochondrial citrate and instead rely upon accelerated glycolysis to support their energy requirements? This review details the genesis and consequences of the metabolic paradigm of a “truncated” Krebs/TCA cycle. Abundant data are presented for substrate utilization and membrane cholesterol enrichment in tumors that are consistent with criteria of the Warburg effect. From healthy cellular homeostasis to the uncontrolled proliferation of tumors, metabolic alterations center upon the loss of regulation of the cholesterol biosynthetic pathway. Deregulated tumor cholesterogenesis at the HMGR locus, generating enhanced carbon flux through the cholesterol synthesis pathway, is an absolute prerequisite for DNA synthesis and cell division. Therefore, expedited citrate efflux from cholesterol-enriched tumor mitochondria via the CTP/SLC25A1 citrate transporter is fundamental for sustaining the constant demand for cytosolic citrate that fuels the elevated flow of carbons from acetyl-CoA through the deregulated pathway of cholesterol biosynthesis.

A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate

Cholesterol biosynthesis is a high-cost process and, therefore, tightly regulated by both transcriptional and posttranslational negative feedback mechanisms in response to the level of cellular cholesterol. Squalene monooxygenase (SM, also known as squalene epoxidase or SQLE) is a rate-limiting enzyme in the cholesterol biosynthetic pathway and catalyzes epoxidation of squalene. The stability of SM is negatively regulated by cholesterol via its N-terminal regulatory domain (SM-N100). In this study, using a SM-luciferase fusion reporter cell line, we performed a chemical genetics screen that identified inhibitors of SM itself as up-regulators of SM. This effect was mediated through the SM-N100 region, competed with cholesterol-accelerated degradation, and required the E3 ubiquitin ligase MARCH6. However, up-regulation was not observed with statins, well-established cholesterol biosynthesis inhibitors, and this pointed to the presence of another mechanism other than reduced cholesterol synthesis. Further analyses revealed that squalene accumulation upon treatment with the SM inhibitor was responsible for the up-regulatory effect. Using photoaffinity labeling, we demonstrated that squalene directly bound to the N100 region, thereby reducing interaction with and ubiquitination by MARCH6. Our findings suggest that SM senses squalene via its N100 domain to increase its metabolic capacity, highlighting squalene as a feedforward factor for the cholesterol biosynthetic pathway.

Kinetic study of the expression of genes related to hepatic steatosis, glucose and lipid metabolism, and cellular stress during overfeeding in mule ducks

Induced by overfeeding, hepatic steatosis is a process exploited for the “foie gras” production in mule ducks. To better understand the mechanisms underlying its development, the physiological responses of mule ducks overfed with corn for a duration of 11 days were analyzed. A kinetic analysis of glucose and lipid metabolism and cell protection mechanisms was performed on 96 male mule ducks during overfeeding with three sampling times (after the 4th, the 12th, and the 22nd meal). Gene expression and protein analysis realized on the liver, muscle, and abdominal fat showed an activation of a cholesterol biosynthetic pathway during the complete overfeeding period mainly in livers with significant correlations between its weight and its cholesterolemia ( r = 0.88; P < 0.0001) and between the liver weight and the hmgcr and soat1 expression ( r = 0.4, P < 0.0001 and r = 0.67; P < 0.0001, respectively). Results also revealed an activation of insulin and amino acid cells signaling a pathway suggesting that ducks boost insulin sensitivity to raise glucose uptake and use via glycolysis and lipogenesis. Cellular stress analysis revealed an upregulation of key autophagy-related gene expression atg8 and sqstm1( P < 0.0001) during the complete overfeeding period, mainly in the liver, in contrast to an induction of cyp2e1( P < 0.0001), suggesting that autophagy could be suppressed during steatosis development. This study has highlighted different mechanisms enabling mule ducks to efficiently handle the starch overload by keeping its liver in a nonpathological state. Moreover, it has revealed potential biomarker candidates of hepatic steatosis as plasma cholesterol for the liver weight.

Kinetic study of the expression of genes related to hepatic steatosis, global intermediate metabolism and cellular stress during overfeeding in mule ducks

ABSTRACTInduced by overfeeding, hepatic steatosis is a reversible process exploited for “foie gras” production. To better understand the mechanisms underlying this non-pathological phenomenon, we analysed the physiological responses of the mule duck to cope with 22 carbohydrate meals. A kinetic analysis of intermediate metabolism and cell protection mechanisms was performed during overfeeding. As expected, dietary carbohydrates are up taken mainly by the liver (chrebp, glut1/2/8) and converted into lipids (acox, scd1, acsl1, fas, dgat2). Our study showed an activation of cholesterol biosynthetic pathway with significant correlations between plasma cholesterol, expression of key genes (hmgcr, soat1) and liver weight. Results revealed an activation of insulin and amino acid cell signalling pathway suggesting that ducks boost insulin sensitivity to raise glucose uptake and useviaglycolysis and lipogenesis. Expression ofcpt1a, acad, hadhsuggested an induction of beta-oxidation probably to remove part of newly synthesized lipids and avoid lipotoxicity. Cellular stress analysis revealed an upregulation of autophagy-related gene expression (atg8, atg9, sqstm1) in contrast with an induction ofcyp2e1suggesting that autophagy could be suppressed.Lamp2aandplin2enhanced, conflicting with the idea of an inhibition of lipophagy.Hsbp1overexpression indicated that mechanisms are carried out during overfeeding to limit cellular stress and apoptosis to prevent the switch to pathological state.Atf4andasnsoverexpression reflects the nutritional imbalance during overfeeding. These results permitted to highlight the mechanisms enabling mule ducks to efficiently handle the huge starch overload and reveal potential biomarker candidates of hepatic steatosis as plasma cholesterol for liver weight.

The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1

Mammalian HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-accelerated degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that RNF145 and gp78 independently co-ordinate HMGCR ubiquitination and degradation. RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR via Insigs, promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of both RNF145 and gp78, Hrd1, a third UBE2G2-dependent E3 ligase, partially regulates HMGCR activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR regulation and elucidate the complexity of sterol-accelerated HMGCR degradation.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

Cholesterol metabolism in innate and adaptive response

It has been long recognized that cholesterol is a critical molecule in mammalian cell biology, primarily for its contribution to the plasma membrane’s composition and its role in assuring proper transmembrane receptor signaling as part of lipid rafts. Efforts have also been made to characterize the cholesterol biosynthetic pathway, cholesterol homeostasis, and cholesterol-derived metabolites in order to gain insights into their dysregulation during metabolic diseases. Despite the central role cholesterol metabolism plays in shaping human health, its regulation during immune activation, such as immune response to pathogens or autoimmune/autoinflammatory diseases, is poorly understood. The immune system is composed of several type of cells with distinct developmental origin, life span, molecular requirements, and gene expressions. It is unclear whether the same array of cholesterol metabolism regulators are equally employed by different immune cells and whether distinct cholesterol metabolites have similar biological consequences in different immune cells. In this review, we will describe how cholesterol metabolism is controlled during the adaptive and the innate immune response and the role for intracellular and extracellular receptors for cholesterol and its derivatives.