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Abstract Annexins are cytosolic proteins with conserved three-dimensional structures that bind acidic phospholipids in cellular membranes at elevated Ca2+ levels. Through this they act as Ca2+-regulated membrane binding modules that organize membrane lipids, facilitating cellular membrane transport but also displaying extracellular activities. Recent discoveries highlight annexins as sensors and regulators of cellular and organismal stress, controlling inflammatory reactions in mammals, environmental stress in plants, and cellular responses to plasma membrane rupture. Here, we describe the role of annexins as Ca2+-regulated membrane binding modules that sense and respond to cellular stress and share our view on future research directions in the field.

Infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause coronavirus disease 2019 (COVID-19), a disease with very heterogeneous symptoms. Dyslipidaemia is prevalent in at least 20% of Europeans, and dyslipidaemia before SARS-CoV-2 infection increases the risk for severe COVID-19 and mortality by 139%. Many reports described reduced serum cholesterol levels in virus-infected patients, in particular in those with severe disease. The liver is the major organ for lipid homeostasis and hepatic dysfunction appears to occur in one in five patients infected with SARS-CoV-2. Thus, SARS-CoV-2 infection, COVID-19 disease severity and liver injury may be related to impaired cholesterol homeostasis. These observations prompted efforts to assess the therapeutic opportunities of cholesterol-lowering medications to reduce COVID-19 severity. The majority of studies implicate statins to have beneficial effects on disease severity and outcome in COVID-19. Proprotein convertase subtilisin/kexin type 9 (PCSK9) antibodies have also shown potential to protect against COVID-19. This review describes the relationship between systemic cholesterol levels, liver injury and COVID-19 disease severity. The potential effects of statins and PCSK9 in COVID-19 are summarised. Finally, the relationship between cholesterol and lung function, the first organ to be affected by SARS-CoV-2, is described.

Abstract Background Acquired treatment resistance is a significant problem in breast cancer management, and alterations in lipid metabolism have been proposed to contribute to the development of drug resistance as well as other aspects of tumor progression. The present study aimed to identify the role of cholesterol metabolism in MCF-7 and MDA-MB-231 breast cancer cell response to cisplatin (CDDP) treatment in the acute setting and in a model of CDDP resistance. Methods MCF-7 (luminal A), MDA-MB-231 (triple-negative) and CDDP-resistant MDA-MB-231 (MDACR) cell lines were grown in the presence or absence of CDDP in combination with atorvastatin (ATV), lipid depletion or low-density lipoprotein loading and were analyzed by a variety of biochemical and radiometric techniques. Results Co-administration of CDDP and ATV strongly reduced cell proliferation and viability to a greater extent than CDDP alone, especially in MDA-MB-231 cells. These findings were associated with reduced cholesteryl ester synthesis and storage in MDA-MB-231 cells. In MDACR cells, acetyl-CoA acetyltransferase 1 (ACAT-1) was upregulated compared to naïve MDA-MB-231 cells and ATV treatment restored CDDP sensitivity, suggesting that aberrant ACAT-1 expression and associated changes in cholesterol metabolism contribute to CDDP resistance in MDA-MB-231 cells. Conclusion These findings indicate that the elevated susceptibility of MDA-MB-231 cells to co-administration of CDDP and ATV, is associated with an increased reliance on cholesteryl ester availability. Our data from these cell culture-based studies identifies altered cholesterol homeostasis as an adaptive response to CDDP treatment that contributes to aggressiveness and chemotherapy resistance.

Abstract Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.

Abstract Background Prostate cancer growth is driven by androgen receptor signaling, and advanced disease is initially treatable by depleting circulating androgens. However, prostate cancer cells inevitably adapt, resulting in disease relapse with incurable castrate-resistant prostate cancer. Androgen deprivation therapy has many side effects, including hypercholesterolemia, and more aggressive and castrate-resistant prostate cancers typically feature cellular accumulation of cholesterol stored in the form of cholesteryl esters. As cholesterol is a key substrate for de novo steroidogenesis in prostate cells, this study hypothesized that castrate-resistant/advanced prostate cancer cell growth is influenced by the availability of extracellular, low-density lipoprotein (LDL)-derived, cholesterol, which is coupled to intracellular cholesteryl ester homeostasis. Methods C4-2B and PC3 prostate cancer cells were cultured in media supplemented with fetal calf serum (FCS), charcoal-stripped FCS (CS-FCS), lipoprotein-deficient FCS (LPDS), or charcoal-stripped LPDS (CS-LPDS) and analyzed by a variety of biochemical techniques. Cell viability and proliferation were measured by MTT assay and Incucyte, respectively. Results Reducing lipoprotein availability led to a reduction in cholesteryl ester levels and cell growth in C4-2B and PC3 cells, with concomitant reductions in PI3K/mTOR and p38MAPK signaling. This reduced growth in LPDS-containing media was fully recovered by supplementation of exogenous low-density lipoprotein (LDL), but LDL only partially rescued growth of cells cultured with CS-LPDS. This growth pattern was not associated with changes in androgen receptor signaling but rather increased p38MAPK and MEK1/ERK/MSK1 activation. The ability of LDL supplementation to rescue cell growth required cholesterol esterification as well as cholesteryl ester hydrolysis activity. Further, growth of cells cultured in low androgen levels (CS-FCS) was suppressed when cholesteryl ester hydrolysis was inhibited. Conclusions Overall, these studies demonstrate that androgen-independent prostate cancer cell growth can be influenced by extracellular lipid levels and LDL-cholesterol availability and that uptake of extracellular cholesterol, through endocytosis of LDL-derived cholesterol and subsequent delivery and storage in the lipid droplet as cholesteryl esters, is required to support prostate cancer cell growth. This provides new insights into the relationship between extracellular cholesterol, intracellular cholesterol metabolism, and prostate cancer cell growth and the potential mechanisms linking hypercholesterolemia and more aggressive prostate cancer.

Accumulating evidence implicates obesity as a risk factor for increased severity of disease outcomes in patients infected with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Obesity is associated with adipose tissue dysfunction, which not only predisposes individuals to metabolic complications, but also substantially contributes to low-grade systemic inflammation, altered immune cell composition, and compromised immune function. This seems to impact the susceptibility and outcome of diseases caused by viruses, as obese people appear more vulnerable to developing infections and they recover later from infectious diseases than normal-weight individuals. Based on these findings, increased efforts to identify suitable diagnostic and prognostic markers in obese Coronavirus disease 2019 (COVID-19) patients to predict disease outcomes have been made. This includes the analysis of cytokines secreted from adipose tissues (adipokines), which have multiple regulatory functions in the body; for instance, modulating insulin sensitivity, blood pressure, lipid metabolism, appetite, and fertility. Most relevant in the context of viral infections, adipokines also influence the immune cell number, with consequences for overall immune cell activity and function. Hence, the analysis of the circulating levels of diverse adipokines in patients infected with SARS-CoV-2 have been considered to reveal diagnostic and prognostic COVID-19 markers. This review article summarizes the findings aimed to correlate the circulating levels of adipokines with progression and disease outcomes of COVID-19. Several studies provided insights on chemerin, adiponectin, leptin, resistin, and galectin-3 levels in SARS-CoV-2-infected patients, while limited information is yet available on the adipokines apelin and visfatin in COVID-19. Altogether, current evidence points at circulating galectin-3 and resistin levels being of diagnostic and prognostic value in COVID-19 disease.

The use of biomarkers is of great clinical value for the diagnosis and prognosis of disease and the assessment of treatment efficacy. In this context, adipokines secreted from adipose tissue are of interest, as their elevated circulating levels are associated with a range of metabolic dysfunctions, inflammation, renal and hepatic diseases and cancers. In addition to serum, adipokines can also be detected in the urine and feces, and current experimental evidence on the analysis of fecal and urinary adipokine levels points to their potential as disease biomarkers. This includes increased urinary adiponectin, lipocalin-2, leptin and interleukin-6 (IL-6) levels in renal diseases and an association of elevated urinary chemerin as well as urinary and fecal lipocalin-2 levels with active inflammatory bowel diseases. Urinary IL-6 levels are also upregulated in rheumatoid arthritis and may become an early marker for kidney transplant rejection, while fecal IL-6 levels are increased in decompensated liver cirrhosis and acute gastroenteritis. In addition, galectin-3 levels in urine and stool may emerge as a biomarker for several cancers. With the analysis of urine and feces from patients being cost-efficient and non-invasive, the identification and utilization of adipokine levels as urinary and fecal biomarkers could become a great advantage for disease diagnosis and predicting treatment outcomes. This review article highlights data on the abundance of selected adipokines in urine and feces, underscoring their potential to serve as diagnostic and prognostic biomarkers.

Membrane contact sites (MCS) are specialized small areas of close apposition between two different organelles that have led researchers to reconsider the dogma of intercellular communication via vesicular trafficking. The latter is now being challenged by the discovery of lipid and ion transfer across MCS connecting adjacent organelles. These findings gave rise to a new concept that implicates cell compartments not to function as individual and isolated entities, but as a dynamic and regulated ensemble facilitating the trafficking of lipids, including cholesterol, and ions. Hence, MCS are now envisaged as metabolic platforms, crucial for cellular homeostasis. In this context, well-known as well as novel proteins were ascribed functions such as tethers, transporters, and scaffolds in MCS, or transient MCS companions with yet unknown functions. Intriguingly, we and others uncovered metabolic alterations in cell-based disease models that perturbed MCS size and numbers between coupled organelles such as endolysosomes, the endoplasmic reticulum, mitochondria, or lipid droplets. On the other hand, overexpression or deficiency of certain proteins in this narrow 10–30 nm membrane contact zone can enable MCS formation to either rescue compromised MCS function, or in certain disease settings trigger undesired metabolite transport. In this “Mini Review” we summarize recent findings regarding a subset of annexins and discuss their multiple roles to regulate MCS dynamics and functioning. Their contribution to novel pathways related to MCS biology will provide new insights relevant for a number of human diseases and offer opportunities to design innovative treatments in the future.

Cancer cells undergo drastic metabolic adaptions to cover increased bioenergetic needs, contributing to resistance to therapies. This includes a higher demand for cholesterol, which often coincides with elevated cholesterol uptake from low-density lipoproteins (LDL) and overexpression of the LDL receptor in many cancers. This implies the need for cancer cells to accommodate an increased delivery of LDL along the endocytic pathway to late endosomes/lysosomes (LE/Lys), providing a rapid and effective distribution of LDL-derived cholesterol from LE/Lys to other organelles for cholesterol to foster cancer growth and spread. LDL-cholesterol exported from LE/Lys is facilitated by Niemann–Pick Type C1/2 (NPC1/2) proteins, members of the steroidogenic acute regulatory-related lipid transfer domain (StARD) and oxysterol-binding protein (OSBP) families. In addition, lysosomal membrane proteins, small Rab GTPases as well as scaffolding proteins, including annexin A6 (AnxA6), contribute to regulating cholesterol egress from LE/Lys. Here, we summarize current knowledge that links upregulated activity and expression of cholesterol transporters and related proteins in LE/Lys with cancer growth, progression and treatment outcomes. Several mechanisms on how cellular distribution of LDL-derived cholesterol from LE/Lys influences cancer cell behavior are reviewed, some of those providing opportunities for treatment strategies to reduce cancer progression and anticancer drug resistance.

Chronic liver diseases are commonly associated with dysregulated cholesterol metabolism. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease of the proprotein convertase family that is mainly synthetized and secreted by the liver, and represents one of the key regulators of circulating low-density lipoprotein (LDL) cholesterol levels. Its ability to bind and induce LDL-receptor degradation, in particular in the liver, increases circulating LDL-cholesterol levels in the blood. Hence, inhibition of PCSK9 has become a very potent tool for the treatment of hypercholesterolemia. Besides PCSK9 limiting entry of LDL-derived cholesterol, affecting multiple cholesterol-related functions in cells, more recent studies have associated PCSK9 with various other cellular processes, including inflammation, fatty acid metabolism, cancerogenesis and visceral adiposity. It is increasingly becoming evident that additional roles for PCSK9 beyond cholesterol homeostasis are crucial for liver physiology in health and disease, often contributing to pathophysiology. This review will summarize studies analyzing circulating and hepatic PCSK9 levels in patients with chronic liver diseases. The factors affecting PCSK9 levels in the circulation and in hepatocytes, clinically relevant studies and the pathophysiological role of PCSK9 in chronic liver injury are discussed.

Breast cancer (BrCa) metabolism is geared toward biomass synthesis and maintenance of reductive capacity. Changes in glucose and glutamine metabolism in BrCa have been widely reported, yet the contribution of fatty acids (FAs) in BrCa biology remains to be determined. We recently reported that adipocyte coculture alters MCF‐7 and MDA‐MB‐231 cell metabolism and promotes proliferation and migration. Since adipocytes are FA‐rich, and these FAs are transferred to BrCa cells, we sought to elucidate the FA metabolism of BrCa cells and their response to FA‐rich environments. MCF‐7 and MDA‐MB‐231 cells incubated in serum‐containing media supplemented with FAs accumulate extracellular FAs as intracellular triacylglycerols (TAG) in a dose‐dependent manner, with MDA‐MB‐231 cells accumulating more TAG. The differences in TAG levels were a consequence of distinct differences in intracellular partitioning of FAs, and not due to differences in the rate of FA uptake. Specifically, MCF‐7 cells preferentially partition FAs into mitochondrial oxidation, whereas MDA‐MB‐231 cells partition FAs into TAG synthesis. These differences in intracellular FA handling underpin differences in the sensitivity to palmitate‐induced lipotoxicity, with MDA‐MB‐231 cells being highly sensitive, whereas MCF‐7 cells are partially protected. The attenuation of palmitate‐induced lipotoxicity in MCF‐7 cells was reversed by inhibition of FA oxidation. Pretreatment of MDA‐MB‐231 cells with FAs increased TAG synthesis and reduced palmitate‐induced apoptosis. Our results provide novel insight into the potential influences of obesity on BrCa biology, highlighting distinct differences in FA metabolism in MCF‐7 and MDA‐MB‐231 cells and how lipid‐rich environments modulate these effects.

Delivery of low-density lipoprotein-derived cholesterol to the endoplasmic reticulum (ER) is essential for cholesterol homeostasis, yet the mechanism of this transport has largely remained elusive. Two recent reports shed some light on this process, uncovering a role for Niemann Pick type-C1 protein (NPC1) in the formation of membrane contact sites (MCS) between late endosomes (LE)/lysosomes (Lys) and the ER. Both studies identified a loss of MCS in cells lacking functional NPC1, where cholesterol accumulates in late endocytic organelles. Remarkably, and taking different approaches, both studies have made a striking observation that expansion of LE/Lys-ER MCS can rescue the cholesterol accumulation phenotype in NPC1 mutant or deficient cells. In both cases, the cholesterol was shown to be transported to the ER, demonstrating the importance of ER-LE/Lys contact sites in the direct transport of low-density lipoprotein-derived cholesterol to the ER.

Routine manipulation of the mouse genome has become a landmark in biomedical research. Traits that are only associated with advanced developmental stages can now be investigated within a living organism, and the in vivo analysis of corresponding phenotypes and functions advances the translation into the clinical setting. The annexins, a family of closely related calcium (Ca2+)- and lipid-binding proteins, are found at various intra- and extracellular locations, and interact with a broad range of membrane lipids and proteins. Their impacts on cellular functions has been extensively assessed in vitro, yet annexin-deficient mouse models generally develop normally and do not display obvious phenotypes. Only in recent years, studies examining genetically modified annexin mouse models which were exposed to stress conditions mimicking human disease often revealed striking phenotypes. This review is the first comprehensive overview of annexin-related research using animal models and their exciting future use for relevant issues in biology and experimental medicine.

The calcium (Ca2+)-dependent membrane-binding Annexin A6 (AnxA6), is a multifunctional, predominantly intracellular scaffolding protein, now known to play relevant roles in different cancer types through diverse, often cell-type-specific mechanisms. AnxA6 is differentially expressed in various stages/subtypes of several cancers, and its expression in certain tumor cells is also induced by a variety of pharmacological drugs. Together with the secretion of AnxA6 as a component of extracellular vesicles, this suggests that AnxA6 mediates distinct tumor progression patterns via extracellular and/or intracellular activities. Although it lacks enzymatic activity, some of the AnxA6-mediated functions involving membrane, nucleotide and cholesterol binding as well as the scaffolding of specific proteins or multifactorial protein complexes, suggest its potential utility in the diagnosis, prognosis and therapeutic strategies for various cancers. In breast cancer, the low AnxA6 expression levels in the more aggressive basal-like triple-negative breast cancer (TNBC) subtype correlate with its tumor suppressor activity and the poor overall survival of basal-like TNBC patients. In this review, we highlight the potential tumor suppressor function of AnxA6 in TNBC progression and metastasis, the relevance of AnxA6 in the diagnosis and prognosis of several cancers and discuss the concept of therapy-induced expression of AnxA6 as a novel mechanism for acquired resistance of TNBC to tyrosine kinase inhibitors.

We recently identified elevated annexin A6 (AnxA6) protein levels in Niemann–Pick-type C1 (NPC1) mutant cells. In these cells, AnxA6 depletion rescued the cholesterol accumulation associated with NPC1 deficiency. Here, we demonstrate that elevated AnxA6 protein levels in NPC1 mutants or upon pharmacological NPC1 inhibition, using U18666A, were not due to upregulated AnxA6 mRNA expression, but caused by defects in AnxA6 protein degradation. Two KFERQ-motifs are believed to target AnxA6 to lysosomes for chaperone-mediated autophagy (CMA), and we hypothesized that the cholesterol accumulation in endolysosomes (LE/Lys) triggered by the NPC1 inhibition could interfere with the CMA pathway. Therefore, AnxA6 protein amounts and cholesterol levels in the LE/Lys (LE-Chol) compartment were analyzed in NPC1 mutant cells ectopically expressing lysosome-associated membrane protein 2A (Lamp2A), which is well known to induce the CMA pathway. Strikingly, AnxA6 protein amounts were strongly decreased and coincided with significantly reduced LE-Chol levels in NPC1 mutant cells upon Lamp2A overexpression. Therefore, these findings suggest Lamp2A-mediated restoration of CMA in NPC1 mutant cells to lower LE-Chol levels with concomitant lysosomal AnxA6 degradation. Collectively, we propose CMA to permit a feedback loop between AnxA6 and cholesterol levels in LE/Lys, encompassing a novel mechanism for regulating cholesterol homeostasis in NPC1 disease.

Calmodulin is a ubiquitous signalling protein that controls many biological processes due to its capacity to interact and/or regulate a large number of cellular proteins and pathways, mostly in a Ca2+-dependent manner. This complex interactome of calmodulin can have pleiotropic molecular consequences, which over the years has made it often difficult to clearly define the contribution of calmodulin in the signal output of specific pathways and overall biological response. Most relevant for this review, the ability of calmodulin to influence the spatiotemporal signalling of several small GTPases, in particular KRas and Rac1, can modulate fundamental biological outcomes such as proliferation and migration. First, direct interaction of calmodulin with these GTPases can alter their subcellular localization and activation state, induce post-translational modifications as well as their ability to interact with effectors. Second, through interaction with a set of calmodulin binding proteins (CaMBPs), calmodulin can control the capacity of several guanine nucleotide exchange factors (GEFs) to promote the switch of inactive KRas and Rac1 to an active conformation. Moreover, Rac1 is also an effector of KRas and both proteins are interconnected as highlighted by the requirement for Rac1 activation in KRas-driven tumourigenesis. In this review, we attempt to summarize the multiple layers how calmodulin can regulate KRas and Rac1 GTPases in a variety of cellular events, with biological consequences and potential for therapeutic opportunities in disease settings, such as cancer.

ABSTRACT To transfer the viral genome into the host cell cytoplasm, internalized influenza A virus (IAV) particles depend on the fusion of the IAV envelope with host endosomal membranes. The antiviral host interferon (IFN) response includes the upregulation of interferon-induced transmembrane protein 3 (IFITM3), which inhibits the release of the viral content into the cytosol. Although IFITM3 induction occurs concomitantly with late endosomal/lysosomal (LE/L) cholesterol accumulation, the functional significance of this process is not well understood. Here we report that LE/L cholesterol accumulation itself plays a pivotal role in the early antiviral defense. We demonstrate that inducing LE/L cholesterol accumulation is antiviral in non-IFN-primed cells, restricting incoming IAV particles and impairing mixing of IAV/endosomal membrane lipids. Our results establish a protective function of LE/L cholesterol accumulation and suggest endosomal cholesterol balance as a possible antiviral target. IMPORTANCE With annual epidemics occurring in all parts of the world and the risk of global outbreaks, influenza A virus (IAV) infections remain a major threat to public health. Infected host cells detect viral components and mount an interferon (IFN)-mediated response to restrict virus propagation and spread of infection. Identification of cellular factors and underlying mechanisms that establish such an antiviral state can provide novel strategies for the development of antiviral drugs. The contribution of LE/L cholesterol levels, especially in the context of the IFN-induced antiviral response, has remained controversial so far. Here, we report that accumulation of cholesterol in the LE/L compartment contributes to the IFN-induced host cell defense against incoming IAV. Our results establish cholesterol accumulation in LE/L per se as a novel antiviral barrier and suggest the endosomal cholesterol balance as a putative druggable host cell factor in IAV infection.

Annexin A6 (AnxA6) controls cholesterol and membrane transport in endo- and exocytosis, and modulates triglyceride accumulation and storage. In addition, AnxA6 acts as a scaffolding protein for negative regulators of growth factor receptors and their effector pathways in many different cell types. Here we investigated the role of AnxA6 in the regulation of whole body lipid metabolism and insulin-regulated glucose homeostasis. Therefore, wildtype (WT) and AnxA6-knockout (KO) mice were fed a high-fat diet (HFD) for 17 weeks. During the course of HFD feeding, AnxA6-KO mice gained less weight compared to controls, which correlated with reduced adiposity. Systemic triglyceride and cholesterol levels of HFD-fed control and AnxA6-KO mice were comparable, with slightly elevated high density lipoprotein (HDL) and reduced triglyceride-rich lipoprotein (TRL) levels in AnxA6-KO mice. AnxA6-KO mice displayed a trend towards improved insulin sensitivity in oral glucose and insulin tolerance tests (OGTT, ITT), which correlated with increased insulin-inducible phosphorylation of protein kinase B (Akt) and ribosomal protein S6 kinase (S6) in liver extracts. However, HFD-fed AnxA6-KO mice failed to downregulate hepatic gluconeogenesis, despite similar insulin levels and insulin signaling activity, as well as expression profiles of insulin-sensitive transcription factors to controls. In addition, increased glycogen storage in livers of HFD- and chow-fed AnxA6-KO animals was observed. Together with an inability to reduce glucose production upon insulin exposure in AnxA6-depleted HuH7 hepatocytes, this implicates AnxA6 contributing to the fine-tuning of hepatic glucose metabolism with potential consequences for the systemic control of glucose in health and disease.

Inhibition of cholesterol export from late endosomes causes cellular cholesterol imbalance, including cholesterol depletion in the trans-Golgi network (TGN). Here, using Chinese hamster ovary (CHO) Niemann-Pick type C1 (NPC1) mutant cell lines and human NPC1 mutant fibroblasts, we show that altered cholesterol levels at the TGN/endosome boundaries trigger Syntaxin 6 (Stx6) accumulation into VAMP3, transferrin, and Rab11-positive recycling endosomes (REs). This increases Stx6/VAMP3 interaction and interferes with the recycling of αVβ3 and α5β1 integrins and cell migration, possibly in a Stx6-dependent manner. In NPC1 mutant cells, restoration of cholesterol levels in the TGN, but not inhibition of VAMP3, restores the steady-state localization of Stx6 in the TGN. Furthermore, elevation of RE cholesterol is associated with increased amounts of Stx6 in RE. Hence, the fine-tuning of cholesterol levels at the TGN-RE boundaries together with a subset of cholesterol-sensitive SNARE proteins may play a regulatory role in cell migration and invasion.

Obesity and the associated comorbidities are a growing health threat worldwide. Adipose tissue dysfunction, impaired adipokine activity, and inflammation are central to metabolic diseases related to obesity. In particular, the excess storage of lipids in adipose tissues disturbs cellular homeostasis. Amongst others, organelle function and cell signaling, often related to the altered composition of specialized membrane microdomains (lipid rafts), are affected. Within this context, the conserved family of annexins are well known to associate with membranes in a calcium (Ca2+)- and phospholipid-dependent manner in order to regulate membrane-related events, such as trafficking in endo- and exocytosis and membrane microdomain organization. These multiple activities of annexins are facilitated through their diverse interactions with a plethora of lipids and proteins, often in different cellular locations and with consequences for the activity of receptors, transporters, metabolic enzymes, and signaling complexes. While increasing evidence points at the function of annexins in lipid homeostasis and cell metabolism in various cells and organs, their role in adipose tissue, obesity and related metabolic diseases is still not well understood. Annexin A1 (AnxA1) is a potent pro-resolving mediator affecting the regulation of body weight and metabolic health. Relevant for glucose metabolism and fatty acid uptake in adipose tissue, several studies suggest AnxA2 to contribute to coordinate glucose transporter type 4 (GLUT4) translocation and to associate with the fatty acid transporter CD36. On the other hand, AnxA6 has been linked to the control of adipocyte lipolysis and adiponectin release. In addition, several other annexins are expressed in fat tissues, yet their roles in adipocytes are less well examined. The current review article summarizes studies on the expression of annexins in adipocytes and in obesity. Research efforts investigating the potential role of annexins in fat tissue relevant to health and metabolic disease are discussed.

Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS) and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC) with cholesterol and the oxysterol 7-ketocholesterol (7KC). Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1) colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL)-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF)-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

ABSTRACT Influenza is caused by influenza A virus (IAV), an enveloped, negative-stranded RNA virus that derives its envelope lipids from the host cell plasma membrane. Here, we examined the functional role of cellular cholesterol in the IAV infection cycle. We show that shifting of cellular cholesterol pools via the Ca2+-regulated membrane-binding protein annexin A6 (AnxA6) affects the infectivity of progeny virus particles. Elevated levels of cellular AnxA6, which decrease plasma membrane and increase late endosomal cholesterol levels, impaired IAV replication and propagation, whereas RNA interference-mediated AnxA6 ablation increased viral progeny titers. Pharmacological accumulation of late endosomal cholesterol also diminished IAV virus propagation. Decreased IAV replication caused by upregulated AnxA6 expression could be restored either by exogenous replenishment of host cell cholesterol or by ectopic expression of the late endosomal cholesterol transporter Niemann-Pick C1 (NPC1). Virus released from AnxA6-overexpressing cells displayed significantly reduced cholesterol levels. Our results show that IAV replication depends on maintenance of the cellular cholesterol balance and identify AnxA6 as a critical factor in linking IAV to cellular cholesterol homeostasis. IMPORTANCE Influenza A virus (IAV) is a major public health concern, and yet, major host-pathogen interactions regulating IAV replication still remain poorly understood. It is known that host cell cholesterol is a critical factor in the influenza virus life cycle. The viral envelope is derived from the host cell membrane during the process of budding and, hence, equips the virus with a special lipid-protein mixture which is high in cholesterol. However, the influence of host cell cholesterol homeostasis on IAV infection is largely unknown. We show that IAV infection success critically depends on host cell cholesterol distribution. Cholesterol sequestration in the endosomal compartment impairs progeny titer and infectivity and is associated with reduced cholesterol content in the viral envelope.

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

BACKGROUND: Lipoprotein receptors from the low density lipoprotein (LDL) receptor family are multifunctional membrane proteins which can efficiently mediate endocytosis and thereby facilitate lipoprotein clearance from the plasma. The biggest member of this family, the LDL receptor-related protein 1 (LRP1), facilitates the hepatic uptake of triglyceride-rich lipoproteins (TRL) via interaction with apolipoprotein E (apoE). In contrast to the classical LDL degradation pathway, TRL disintegrate in peripheral endosomes, and core lipids and apoB are targeted along the endocytic pathway for lysosomal degradation. Notably, TRL-derived apoE remains within recycling endosomes and is then mobilized by high density lipoproteins (HDL) for re-secretion. The aim of this study is to investigate the involvement of LRP1 in the regulation of apoE recycling. PRINCIPAL FINDINGS: Immunofluorescence studies indicate the LRP1-dependent trapping of apoE in EEA1-positive endosomes in human hepatoma cells. This processing is distinct from other LRP1 ligands such as RAP which is efficiently targeted to lysosomal compartments. Upon stimulation of HDL-induced recycling, apoE is released from LRP1-positive endosomes but is targeted to another, distinct population of early endosomes that contain HDL, but not LRP1. For subsequent analysis of the recycling capacity, we expressed the full-length human LRP1 and used an RNA interference approach to manipulate the expression levels of LRP1. In support of LRP1 determining the intracellular fate of apoE, overexpression of LRP1 significantly stimulated HDL-induced apoE recycling. Vice versa LRP1 knockdown in HEK293 cells and primary hepatocytes strongly reduced the efficiency of HDL to stimulate apoE secretion. CONCLUSION: We conclude that LRP1 enables apoE to accumulate in an early endosomal recycling compartment that serves as a pool for the intracellular formation and subsequent re-secretion of apoE-enriched HDL particles.

Caveolin-1 (Cav1), a structural protein required for the formation of invaginated membrane domains known as caveolae, has been implicated in cholesterol trafficking and homeostasis. Here we investigated the contribution of Cav1 to apolipoprotein A-I (apoA-I) cell surface binding and intracellular processing using mouse embryonic fibroblasts (MEFs) derived from wild type (WT) or Cav1-deficient (Cav1(-/-)) animals. We found that cells expressing Cav1 have 2.6-fold more apoA-I binding sites than Cav1(-/-) cells although these additional binding sites are not associated with detergent-free lipid rafts. Further, Cav1-mediated binding targets apoA-I for internalization and degradation and these processes are not correlated to cholesterol efflux. Despite lower apoA-I binding, cholesterol efflux from Cav1(-/-) MEFs is 1.7-fold higher than from WT MEFs. Stimulation of ABCA1 expression with an LXR agonist enhances cholesterol efflux from both WT and Cav1(-/-) cells without increasing apoA-I surface binding or affecting apoA-I processing. Our results indicate that there are at least two independent lipid binding sites for apoA-I; Cav1-mediated apoA-I surface binding and uptake is not linked to cholesterol efflux, indicating that membrane domains other than caveolae regulate ABCA1-mediated cholesterol efflux.

The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process.