Cigarette smoke affects keratinocytes SRB1 expression and localization via H2O2 production and HNE protein adducts formation

Scavenger Receptor B1 (SR-B1), also known as HDL receptor, is involved in cellular cholesterol uptake. Stratum corneum (SC), the outermost layer of the skin, is composed of more than 25% cholesterol. Several reports support the view that alteration of SC lipid composition may be the cause of impaired barrier function which gives rise to several skin diseases. For this reason the regulation of the genes involved in cholesterol uptake is of extreme significance for skin health. Being the first shield against external insults, the skin is exposed to several noxious substances and among these is cigarette smoke (CS), which has been recently associated with various skin pathologies. In this study we first have shown the presence of SR-B1 in murine and human skin tissue and then by using immunoblotting, immunoprecipitation, RT-PCR, and confocal microscopy we have demonstrated the translocation and the subsequent lost of SR-B1 in human keratinocytes (cell culture model) after CS exposure is driven by hydrogen peroxide (H(2)O(2)) that derives not only from the CS gas phase but mainly from the activation of cellular NADPH oxidase (NOX). This effect was reversed when the cells were pretreated with NOX inhibitors or catalase. Furthermore, CS caused the formation of SR-B1-aldheydes adducts (acrolein and 4-hydroxy-2-nonenal) and the increase of its ubiquitination, which could be one of the causes of SR-B1 loss. In conclusion, exposure to CS, through the production of H(2)O(2), induced post-translational modifications of SR-B1 with the consequence lost of the receptor and this may contribute to the skin physiology alteration as a consequence of the variation of cholesterol uptake.     

Dose-responsiveness and persistence of microRNA expression alterations induced by cigarette smoke in mouse lung

Our previous studies demonstrated that exposure to cigarette smoke (CS), either mainstream or environmental, results in a remarkable downregulation of microRNA expression in the lung of both mice and rats. The goals of the present study were to evaluate the dose responsiveness to CS and the persistence of microRNA alterations after smoking cessation. ICR (CD-1) neonatal mice were exposed whole-body to mainstream CS, at the doses of 119, 292, 438, and 631mg/m(3) of total particulate matter. Exposure started within 12h after birth and continued daily for 4 weeks. The levels of bulky DNA adducts and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were measured by (32)P postlabeling procedures, and the expression of 697 mouse microRNAs was analyzed by microarray. The highest CS dose was lethal. Exposure to CS caused a dose-dependent increase of DNA alterations. DNA adducts and, even more sharply, 8-oxodGuo were reverted 1 and 4 weeks after smoking cessation. Exposure to CS resulted in an evident dysregulation of microRNA expression profiles, mainly in the sense of downregulation. The two lowest doses were not particularly effective, while the highest nonlethal dose produced extensive microRNA alterations. The expression of most downregulated microRNAs, including among others 7 members of the let-7 family, was restored one week after smoking cessation. However, the recovery was incomplete for a limited array of microRNAs, including mir-34b, mir-345, mir-421, mir-450b, mir-466, and mir-469. Thus, it appears that microRNAs mainly behave as biomarkers of effect and that exposure to high-dose, lasting for an adequate period of time, is needed to trigger the CS-related carcinogenesis process in the experimental animal model used.     

Cholesterol sulfate, an activator of protein kinase C mediating squamous cell differentiation: a review

Activity of protein kinase C (PKC) depends on the interaction with polar head-groups of two membrane lipids, i.e., phosphatidylserine and diacylglycerol. We demonstrated that cholesterol metabolism is directly involved in activation of the eta isoform of protein kinase C (PKCeta), which is predominantly expressed in epithelial tissues in close association with epithelial differentiation. We found that PKCeta was activated by cholesterol sulfate (CS), a metabolite of cholesterol formed during squamous cell differentiation. In the presence of CS, phorbol ester only weakly enhanced the activity of PKCeta. CS also activated PKCeta, PKCdelta and PKCepsilon in a dose-dependent manner, when assayed using purified recombinant materials. However, when partially purified materials were used from overexpressing normal human keratinocytes, only PKCeta was activated by CS among the isoforms examined. All the existing lines of evidence, mainly supplied from our laboratory, suggest that CS is involved in a signal transduction of squamous cell differentiation and thereby modifying squamous cell carcinogenesis.     

Resveratrol and 4-hydroxynonenal act in concert to increase glutamate cysteine ligase expression and glutathione in human bronchial epithelial cells

Resveratrol has been shown to protect against oxidative stress through modulating antioxidant capacity. In this study, we investigated resveratrol-mediated induction of glutathione (GSH) and glutamate cysteine ligase (GCL), and the combined effect of resveratrol and 4-hydroxynonenal (HNE) on GSH synthesis in cultured HBE1 human bronchial epithelial cells. Resveratrol increased GSH and the mRNA contents of both the catalytic (GCLC) and modulatory subunit (GCLM) of GCL. Combined HNE and resveratrol treatment increased GSH content and GCL mRNAs to a greater extent than either compound did alone. Compared to individual agent, combining exposure to HNE and resveratrol also showed more protection against cell death caused by oxidative stress. These effects of combined exposure were additive rather than synergistic. In addition, Nrf2 silencing significantly decreased the combined effect of HNE and resveratrol on GCL induction. Our data suggest that resveratrol increases GSH and GCL gene expression and that there is an additive effect on GSH synthesis between resveratrol and HNE. The results also reveal that Nrf2-EpRE signaling was involved in the combined effects.     

Apoptosis signalling by 4-hydroxynonenal: a role for JNK-c-Jun/AP-1 pathway

4-Hydroxynonenal (HNE) is one of the most abundant aldehyde components of ox-LDL and it exerts various effects on intracellular and extracellular signaling cascades. In this mini-review, a brief synopsis of HNE-modulated signaling pathways will be presented mainly focused on cell death, including recent studies from our laboratory. The results of a number of studies demonstrate the ability of HNE to induce apoptosis and ROS formation in a dose-dependent manner. Several signaling pathways have been shown to be modulated by HNE, including MAP kinases, PKC isoforms, cell-cycle regulators, receptor tyrosine kinases and caspases. In order to get insight into the mechanisms of apoptotic response by HNE, MAP kinase and caspase activation pathways have been studied in 3T3 fibroblasts; HNE induced early activation of JNK and p38 proteins but down-regulated the basal activity of ERK-1/2. We have shown that HNE-induced release of cytochrome c from mitochondria, caspase-9 and caspase-3 activation. Activation of AP-1 along with increased c-Jun and phospho-c-Jun levels could be inhibited by pretreatment of cells with certain molecules such as resveratrol. Additionally, overexpression of dominant negative c-Jun and JNK1 in 3T3 fibroblasts prevented HNE-induced apoptosis, which indicated a role for JNK-c-Jun/AP-1 pathway. JNK-dependent induction of c-Jun/AP-1 activation data in the literature indicates a critical potential role for JNK in the cellular response against toxic products of lipid peroxidation.     

High-avidity monoclonal antibodies against the human scavenger class B type I receptor efficiently block hepatitis C virus infection in the presence of high-density lipoprotein

The human scavenger class B type 1 receptor (SR-B1/Cla1) was identified as a putative receptor for hepatitis C virus (HCV) because it binds to soluble recombinant HCV envelope glycoprotein E2 (sE2). High-density lipoprotein (HDL), a natural SR-B1 ligand, was shown to increase the in vitro infectivity of retroviral pseudoparticles bearing HCV envelope glycoproteins and of cell culture-derived HCV (HCVcc), suggesting that SR-B1 promotes viral entry in an HDL-dependent manner. To determine whether SR-B1 participates directly in HCV infection or facilitates HCV entry through lipoprotein uptake, we generated a panel of monoclonal antibodies (MAbs) against native human SR-B1. Two of them, 3D5 and C167, bound to conformation-dependent SR-B1 determinants and inhibited the interaction of sE2 with SR-B1. These antibodies efficiently blocked HCVcc infection of Huh-7.5 hepatoma cells in a dose-dependent manner. To examine the role of HDL in SR-B1-mediated HCVcc infection, we set up conditions for HCVcc production and infection in serum-free medium. HCVcc efficiently infected Huh-7.5 cells in the absence of serum lipoproteins, and addition of HDL led to a twofold increase in infectivity. However, the HDL-induced enhancement of infection had no impact on the neutralization potency of MAb C167, despite its ability to inhibit both HDL binding to cells and SR-B1-mediated lipid transfer. Of note, MAb C167 also potently blocked Huh-7.5 infection by an HCV strain recovered from HCVcc-infected chimpanzees. These results demonstrate that SR-B1 is essential for infection with HCV produced in vitro and in vivo and suggest the possible use of anti-SR-B1 antibodies as therapeutic agents.     

High molecular weight neurofilament proteins are physiological substrates of adduction by the lipid peroxidation product hydroxynonenal.

Protein adducts of the lipid peroxidation product trans-4-hydroxy-2-nonenal (HNE) are features of oxidative damage in neuronal cell bodies in Alzheimer's disease but are also seen in axons of normal as well as diseased individuals. In this study, focusing on the axons of the mouse sciatic nerve, we found that HNE adducts characterize axons of mice from birth to senility. Immunoblots of axonal proteins showed that HNE adducts are only detected in neurofilament heavy subunit (NFH) and, to a lesser extent, neurofilament medium subunit (NFM), both lysine-rich proteins, consistent with the adducts being limited to lysine residues. In vitro, HNE treatment of permeabilized sciatic nerve showed the same specificity, i.e. NFH and NFM are the only proteins that reacted with HNE, providing they are phosphorylated. Quantitative immunoblot analysis of two strains of mice ages 1-33 months showed that the levels of HNE adducts on NFH are consistent throughout life. Additionally, mice transgenic for human superoxide dismutase-1 with G85R mutation show no difference in HNE adduction to NFH compared with controls. Taken together, these studies indicate that HNE adduction to NFH is physiological, and its constancy from birth to senility as well as its dependence on phosphorylation argues that NFH and NFM modification may play a role in protecting the membrane-rich axon from toxic aldehydes resulting from oxidative damage.     

Resveratrol Prevents Oxidative Stress-Induced Senescence and Proliferative Dysfunction by Activating the AMPK-FOXO3 Cascade in Cultured Primary Human Keratinocytes

The aging process is perceived as resulting from a combination of intrinsic factors such as changes in intracellular signaling and extrinsic factors, most notably environmental stressors. In skin, the relationship between intrinsic changes and keratinocyte function is not clearly understood. Previously, we found that increasing the activity of AMP-activated protein kinase (AMPK) suppressed senescence in hydrogen peroxide (H₂O₂)-treated human primary keratinocytes, a model of oxidative stress-induced cellular aging. Using this model in the present study, we observed that resveratrol, an agent that increases the activities of both AMPK and sirtuins, ameliorated two age-associated phenotypes: cellular senescence and proliferative dysfunction. In addition, we found that treatment of keratinocytes with Ex527, a specific inhibitor of sirtuin 1 (SIRT1), attenuated the ability of resveratrol to suppress senescence. In keeping with the latter observation, we noted that compared to non-senescent keratinocytes, senescent cells lacked SIRT1. In addition to these effects on H₂O₂-induced senescence, resveratrol also prevented the H₂O₂-induced decrease in proliferation (as indicated by ³H-thymidine incorporation) in the presence of insulin. This effect was abrogated by inhibition of AMPK but not SIRT1. Compared to endothelium, we found that human keratinocytes expressed relatively high levels of Forkhead box O3 (FOXO3), a downstream target of both AMPK and SIRT1. Treatment of keratinocytes with resveratrol transactivated FOXO3 and increased the expression of its target genes including catalase. Resveratrol’s effects on both senescence and proliferation disappeared when FOXO3 was knocked down. Finally, we performed an exploratory study which showed that skin from humans over 50 years old had lower AMPK activity than skin from individuals under age 20. Collectively, these findings suggest that the effects of resveratrol on keratinocyte senescence and proliferation are regulated by the AMPK-FOXO3 pathway and in some situations, but not all, by SIRT1.     

Inactivation of human liver bile acid CoA:amino acid N-acyltransferase by the electrophilic lipid, 4-hydroxynonenal

The hepatic enzyme bile acid CoA:amino acid N-acyltransferase (BAT) catalyzes the formation of amino acid-conjugated bile acids. In the present study, protein carbonylation of BAT, consistent with modification by reactive oxygen species and their products, was increased in hepatic homogenates of apolipoprotein E knock-out mice. 4-Hydroxynonenal (4HNE), an electrophilic lipid generated by oxidation of polyunsaturated long-chain fatty acids, typically reacts with the amino acids Cys, His, Lys, and Arg to form adducts, some of which (Michael adducts) preserve the aldehyde (i.e., carbonyl) moiety. Because two of these amino acids (Cys and His) are members of the catalytic triad of human BAT, it was proposed that 4HNE would cause inactivation of this enzyme. As expected, human BAT (1.6 microM) was inactivated by 4HNE in a dose-dependent manner. To establish the sites of 4HNE's reaction with BAT, peptides from proteolysis of 4HNE-treated, recombinant human BAT were analyzed by peptide mass fingerprinting and by electrospray ionization-tandem mass spectrometry using a hybrid linear ion trap Fourier transform-ion cyclotron resonance mass spectrometer. The data revealed that the active-site His (His362) dose-dependently formed a 4HNE adduct, contributing to loss of activity, although 4HNE adducts on other residues may also contribute.     

Resveratrol affects undifferentiated and differentiated PC12 cells differently,particularly with respect to possible differences in mitochondrial and autophagic functions

Since resveratrol is considered to exert a unique dual effect, protective for normal cells but toxic to tumor cells, its action on undifferentiated (original) and differentiated PC12 cells was analyzed, because undifferentiated cells are tumorigenic and differentiated ones are neuronal in nature. Compared to resveratrol-untreated cells in both undifferentiated and differentiated cell groups, cells treated with different doses of resveratrol, at dosages of 1, 10 and 100 μM, showed the following alterations. Dying/dead cells were significantly increased in a dose-dependent manner in undifferentiated cells, but they were unchanged at doses of up to 10 μM resveratrol in differentiated cells. In living cells, neurites were short in undifferentiated cells, but drastically elongated with an increased number in differentiated cells. The expression of SIRT1 was drastically reduced in undifferentiated cells, but stable in differentiated cells. SIRT3 was significantly enhanced in a dose-dependent manner at resveratrol doses of up to 10 μM in both cells, with reduction and more enhanced at a dosage of 100 μM in undifferentiated and differentiated cells, respectively. Mitochondrial number and ATP synthase β subunit expression was unaltered at doses of up to 10 μM and were significantly reduced at doses of 100 μM in undifferentiated cells, but they were significantly increased in a dose-dependent manner, with a slight reduction in the ATP synthase at doses of 100 μM, in differentiated cells. In a dose-dependent manner, the number of autophagosomes and the LC3-II/LC3-I ratio were significantly less in undifferentiated cells and greater in differentiated cells. Also, in a dose-dependent manner, the expression of phosphorylated AMP-activated kinase (AMPK) was significantly less in undifferentiated cells and greater in differentiated cells. Resveratrol-induced AMPK suppression and activation, possibly through the modulation of SIRT protein activity, may thus be related to the inhibition and promotion of mitochondrial and autophagic functions, leading to cell death and survival in undifferentiated and differentiated cells, respectively.     

Corticotropin releasing hormone and related peptides can act as bioregulatory factors in human keratinocytes

Summary Following previous findings in human skin of the functional expression of genes for the corticotropin releasing hormone (CHR) receptor type 1 (CRH-R1) and CRH itself, we searched for local phenotypic effects for peptides related to CRH. We now report that CRH, sauvagine, and urocortin inhibit proliferation of human HaCaT keratinocytes in a dose-dependent manner. The peptides produced variable cyclic adenosine 3′∶5′-monophosphate stimulation with CRH having the highest potency. Binding of iodine 125 CRH to intact keratinocytes was inhibited by increasing doses of CRH, sauvagine, or urocortin, all showing equal inhibitory potency. Immunocytochemistry identified CRH-R1 immunoreactivity in HaCaT keratinocytes. In conclusion, CRH (exogenous or produced locally) and the related urocortin and sauvagine peptides can modify human keratinocyte phenotype through a receptor-mediated pathway.     

Antiatherogenic activity of extracts of Argania spinosa L. pericarp: beneficial effects on lipid peroxidation and cholesterol homeostasis

Prevention of lipoprotein oxidation by natural compounds may prevent atherosclerosis via reducing early atherogenesis. In this study, we investigated for the first time the beneficial properties of methanolic extract of argania pericarp (MEAP) towards atherogenesis by protecting human low-density lipoprotein (LDL) against oxidation while promoting high-density lipoprotein (HDL)-mediated cholesterol efflux. By measuring the formation of malondialdehyde (MDA) and conjugated diene as well as the lag phase and the progression rate of lipid peroxidation, the MEAP was found to possess an inhibitory effect. In addition, MEAP reduced the rate of disappearance of alpha-tocopherol as well as the apoB electrophoretic mobility in a dose-dependent manner. These effects are related to the free radical scavenging and copper-chelating effects of MEAP. In terms of cell viability, MEAP has shown a cytotoxic effect (0-40 microg/mL). Incubation of 3H-cholesterol-loaded J774 macrophages with HDL in the presence of increasing concentrations of MEAP enhanced HDL-mediated cholesterol efflux independently of ABCA1 receptor pathways. Our findings suggest that argania seed pericarp provides a source of natural antioxidants that inhibit LDL oxidation and enhance cholesterol efflux and thus can prevent development of cardiovascular diseases.     

Formation of diethylstilbestrol-DNA adducts in human breast epithelial cells and inhibition by resveratrol

Extensive evidence exists that the reaction of estrogen metabolites with DNA produces depurinating adducts that, in turn, induce mutations and cellular transformation. While it is clear that these estrogen metabolites result in a neoplastic phenotype in vitro, further evidence supporting the link between estrogen-DNA adduct formation and its role in neoplasia induction in vivo would strengthen the evidence for a genotoxic mechanism. Diethylstilbestrol (DES), an estrogen analogue known to increase the risk of breast cancer in women exposed in utero, is hypothesized to induce neoplasia through a similar genotoxic mechanism. Cultured MCF-10F human breast epithelial cells were treated with DES at varying concentrations and for various times to determine whether the addition of DES to MCF-10F cells resulted in the formation of depurinating adducts. This is the first demonstration of the formation of DES-DNA adducts in human breast cells. A dose-dependent increase in DES-DNA adducts was observed. Demonstrating that treatment of MCF-10F cells with DES, a known human carcinogen, yields depurinating adducts provides further support for the involvement of these adducts in the induction of breast neoplasia. Previous studies have demonstrated the ability of antioxidants such as resveratrol to prevent the formation of estrogen-DNA adducts, thus preventing a key carcinogenic event. In this study, when MCF-10F cells were treated with a combination of resveratrol and DES, a dose-dependent reduction in the level of DES-DNA adducts was also observed.     

Marrubium vulgare extract inhibits human-LDL oxidation and enhances HDL-mediated cholesterol efflux in THP-1 macrophage

The objective of the present study was to elucidate the beneficial properties of aqueous extracts of Marrubium vulgare (AEM) towards cardiovascular disease by protecting human-LDL against lipid peroxidation and promoting HDL-mediated cholesterol efflux. Human-LDL were oxidised by incubation with CuSO(4) in the presence of increased concentrations of AEM (0-100 microg/ml). LDL lipid peroxidation was evaluated by conjugated diene formation, vitamin E disappearance as well as LDL-electrophoretic mobility. HDL-mediated cholesterol efflux assay was carried out in human THP-1 macrophages. Incubation of LDL with AEM significantly prolonged the lag phase (P=0.014), lowered the progression rate of lipid peroxidation (P=0.004), reduced the disappearance of vitamin E and the electrophoretic mobility in a dose-dependent manner. Also, incubation of HDL with AEM significantly increased HDL-mediated cholesterol efflux from THP-1 macrophages implicating an independent ATP binding cassette A1 (ABCA1) pathways. Our findings suggest that M. vulgare provides a source of natural antioxidants, which inhibit LDL oxidation and enhance reverse cholesterol transport and thus can prevent cardiovascular diseases development. These antioxidant properties increase the anti-atherogenic potential of HDL.     

Ozone mediators effect on “in vitro” scratch wound closure

The beneficial effect of low doses of ozone on wound healing has been well documented and attributed mainly to its bactericidal and pro-oxidant properties. Because ozone itself does not penetrate the cells but immediately reacts with polyunsaturated fatty acids, its effects are the results of oxidative mediators. Among the molecule produces by the interaction of ozone with biological systems, there are HNE and H₂O₂. At today, the cellular mechanisms accounting for the positive effects of mild ozonization on wound closure are still largely unexplored. The aim of the present study was to evaluate the effect of different non-toxic doses of ozonated saline ranging from 2 to 300?μM, in an in vitro wound scratch model by the use of human keratinocytes. The results showed that ozonated saline is able to improve in vitro wound healing by stimulating cell proliferation as measured by BrdU assay and PCNA protein levels. In order to better elucidate the molecules that play the main role in the beneficial effect of ozonated saline in wound healing, HNE and H₂O₂ were used alone or in combination to mimic ozonated saline effect. Surprisingly, keratinocytes treated with different doses of HNE and H₂O₂ did not significantly improve the wound closure, while the combination of the two compounds was able to improve wound closure. In addition, Nrf2 pathways were also activated as determined by its translocation to the nucleus and the increased HO1 gene expression. The present work suggests that ozonated saline effect on wound closure is the results of the combination of more molecules among which HNE and H₂O₂ play a key role.     

Effects of 4-hydroxynonenal on adenylate cyclase and 5'-nucleotidase activities in rat liver plasma membranes

Adenylate cyclase and 5'-nucleotidase activities in rat liver plasma membranes were assayed in vitro in the presence of 4-hydroxy-2,3-trans-nonenal (HNE), a major end-product of microsomal lipid peroxidation. Both basal and glucagon-stimulated adenylate cyclase were inhibited in a dose-dependent manner, even at micromolar HNE concentrations, whereas fluoride-stimulated activity increased. A biphasic, dose- and time-dependent effect was noted when the basal activity was monitored at increasing doses. 5'-Nucleotidase activity was also decreased by HNE, but only at millimolar concentrations. These findings are related to the view that aldehydes, especially HNE, may act as diffusible cytotoxic compounds when lipid peroxidative derangement of membrane lipids is provoked by toxic conditions.     

UCP1 and defense against oxidative stress. 4-Hydroxy-2-nonenal effects on brown fat mitochondria are uncoupling protein 1-independent

Uncoupling proteins have been ascribed a role in defense against oxidative stress, particularly by being activated by products of oxidative stress such as 4-hydroxy-2-nonenal (HNE). We have investigated here the ability of HNE to activate UCP1. Using brown fat mitochondria from UCP1+/+ and UCP1-/- mice to allow for identification of UCP1-dependent effects, we found that HNE could neither (re)activate purine nucleotide-inhibited UCP1, nor induce additional activation of innately active UCP1. The aldehyde nonenal had a (re)activating effect only if converted to the corresponding fatty acid by aldehyde dehydrogenase; the presence of a carboxyl group was thus an absolute requirement for (re)activation. The UCP1-dependent proton leak was not increased by HNE but HNE changed basal proton leak characteristics in a UCP1-independent manner. In agreement with the in vitro results, we found, as compared with UCP1+/+ mice, no increase in HNE/protein adducts in brown fat mitochondria isolated from UCP1-/- mice, irrespective of whether they were adapted to thermoneutral temperature (30 degrees C) or to the cold (4 degrees C). The absence of oxidative damage in UCP1-/- mitochondria was not due to enhanced activity of antioxidant enzymes. Thus, HNE did not affect UCP1 activity, and UCP1 would appear not to be physiologically involved in defense against oxidative stress. Additionally, it was concluded that at least in brown adipose tissue, conditions of high mitochondrial membrane potential, high oxygen tension, and high substrate supply do not necessarily lead to increased oxidative damage.     

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics.