Role In Cholesterol Trafficking Research Articles

Hydroxypropyl-beta and -gamma cyclodextrins rescue cholesterol accumulation in Niemann\u2013Pick C1 mutant cell via lysosome-associated membrane protein 1

Niemann–Pick type C (NPC) disease is a fatal hereditary neurodegenerative disorder characterized by a massive accumulation of cholesterol in lysosomes and late endosomes due to a defect in intracellular cholesterol trafficking. Dysfunction in intracellular cholesterol trafficking is responsible for about 50 rare inherited lysosomal storage disorders including NPC. The lysosomal proteins NPC1 and NPC2 play a crucial role in trafficking of cholesterol from late endosomes and lysosomes to other cellular compartments. However, the detailed mechanisms of cholesterol trafficking at the late endosomes/lysosomes (LE/LY) are poorly understood. Studies showed that 2-hydroxypropyl-β-cyclodextrin (HPβCD) alleviates the cholesterol accumulation defect in animal model and has been approved for a phase 2b/3 clinical trial for NPC. HPβCD is known to bind cholesterol; however, the mechanisms how HPβCD mediates the exit of cholesterol from the LE/LY compartments are still unknown. Further, another cyclodextrin (CD) derivative, 2-hydroxypropyl-γ-cyclodextrin (HPγCD), was shown to reduce intracellular cholesterol accumulation in NPC patient cells and NPC mice model. Herein, we identified a number of candidate proteins differentially expressed in NPC patient-derived cells compared to cells derived from a healthy donor using a proteomic approach. Interestingly, both HPβCD and HPγCD treatments modulated the expression of most of these NPC-specific proteins. Data showed that treatment with both CDs induces the expression of the lysosome-associated membrane protein 1 (LAMP-1) in NPC patient-derived cells. Remarkably, LAMP-1 overexpression in HeLa cells rescued U18666A-induced cholesterol accumulation suggesting a role of LAMP-1 in cholesterol trafficking. We propose that HPβCD and HPγCD facilitate cholesterol export from the LE/LY compartments via the LAMP-1 protein, which may play a crucial role in cholesterol trafficking at the LE/LY compartments when there is no functional NPC1 protein. Together, this study uncovers new cellular mechanisms for cholesterol trafficking, which will contribute to development of novel therapeutic approaches for lysosomal storage diseases.

Abstract 045: SR-BI and PCPE2 Modulate Lipid Trafficking in Adipocytes

Adipose tissue undergoes distinct structural remodeling in response to increases in fat mass, as in obesity. During this process, the extracellular matrix (ECM) plays a pivotal role with respect to both adipocyte receptor signaling and structural remodeling. One ECM protein, procollagen endopeptidase enhancer 2 (PCPE2), has been shown by our lab to work in partnership with the scavenger receptor class B type I (SR-BI) to regulate adipocyte cholesterol homeostasis. To investigate the functional impact of the partnership between SR-BI and PCPE2, we studied mature adipocytes differentiated from murine embryonic fibroblasts (MEF) and compared to our adipose specific PCPE2 knockout mice. Our studies showed the presence of an SR-BI-PCPE2 protein complex based on the co-immunoprecipitation of PCPE2 with SR-BI in both differentiated MEFs and mouse adipose tissue. Furthermore, stimulated emission depletion (STED) microscopy showed SR-BI and PCPE2 localized on the lipid droplet surface separated by distances ranging from 24 – 90 nm, suggesting that the SR-BI-PCPE2 complex may contain other proteins. Most significantly, a loss of SR-BI function was shown in PCPE2-deficient adipocytes by the SR-BI mediated HDL binding and cholesteryl ester (CE) uptake assays. Compared to differentiated MEFs from Ldlr -/- mice, binding of HDL to differentiated MEFs from Ldlr -/- Pcpe2 -/- mice was reduced by 75%, and HDL cholesteryl oleyl ether uptake was reduced by 50%. Disruption of adipocyte SR-BI function in the absence of PCPE2 also reduced free cholesterol efflux indicating substantial disruption to intracellular cholesterol homeostasis. Moreover, analysis of RNAseq from several human clinical studies show that PCPE2 mRNA expression is positively correlated with fat mass in specific adipose depots, while negatively correlated with plasma insulin, glucose and triglyceride levels, with the latter two more robust in type 2 diabetic populations (FDR < 0.004). Overall, these results suggest that the partnership between SR-BI and PCPE2 in adipocytes plays an important role in cholesterol trafficking and homeostasis which influences fat mass expansion, glucose tolerance and insulin resistance.

Increased bile lithogenicity by SCP2 via HMGCR and CYP7A1 regulation in human hepatocytes

Lithogenic bile is a major cause of cholesterol gallstones, and hypersecretion of biliary cholesterol is believed to be an important cause of lithogenic bile. Sterol carrier protein 2 plays a key role in cholesterol trafficking and may regulate lipid metabolism in hepatocytes. Human sterol carrier protein 2 cDNA was cloned and shRNAs against sterol carrier protein 2 were synthesized. Sterol carrier protein 2-modified hepatocyte models were then set up using adenoviral vectors to study its regulatory function in lipid metabolism. Following overexpression and knockdown of sterol carrier protein 2, HMGCR and CYP7A1 expression as well as the secretion of bile lipids were studied in hepatocytes. The expression of HMGCR was enhanced by sterol carrier protein 2 overexpression and downregulated by sterol carrier protein 2 knockdown. The concentration of cholesterol in the supernatant was elevated under sterol carrier protein 2 overexpression and decreased under sterol carrier protein 2 downregulation. Although sterol carrier protein 2 overexpression could repress the expression of CYP7A1, no changes were noted in total bile acid concentration. Thus, the expression of sterol carrier protein 2 could influence bile lithogenicity in the sterol carrier protein 2-modified hepatocyte models. Sterol carrier protein 2 may function as a moderator of HMGCR in human hepatocytes. Likely influences cholesterol metabolism and bile lithogenicity of human liver cells by regulating the expression of HMGCR and CYP7A1.

Involvement of cathepsins B and L in inflammation and cholesterol trafficking protein NPC2 secretion in macrophages

Obesity and its related chronic inflammation are the major risk factors for developing metabolic disturbances. The roles of cathepsin cysteine proteases have been tied to inflammation and atherosclerosis. Cathepsins are important functional links between inflammation, cholesterol metabolism, and atherosclerosis in obesity. NPC2, a lysosomal protein, plays an important role in cholesterol trafficking. The objective of this study was to examine the regulation of cathepsins and NPC2 in adipose tissue and macrophages in obesity, and the effect of modifying cathepsin activity in cholesterol metabolism and trafficking in macrophages. We found that high fat diet (HFD) feeding altered the mRNA and protein expression levels of cathepsin B and L and NPC2 in adipose tissue in mice; the differential regulation of these proteins was observed between adipose depots. In vitro studies showed that TNF-α reduces intracellular protein levels of CtB, CtL, and NPC2, but increases their secretion in 3T3-L1 adipocytes. Likewise, LPS stimulated the secretion of CtB and NPC2 in Raw264.7 macrophages. Using the inhibitors of cathepsin enzymatic activity, we found that cathepsin B and L regulate TNFα production, the expression and secretion of NPC2 protein, and the mRNA levels of the genes involved in cholesterol trafficking in macrophages. Our findings suggest that cathepsin B and L have a significant involvement in mediating the inflammatory response, in cholesterol trafficking, and in regulating NPC2 secretion.

Sterol carrier protein-2 (SCP-2) involvement in cholesterol hydroperoxide cytotoxicity as revealed by SCP-2 inhibitor effects

Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells. The purpose of this study was to determine whether SCP-2, under oxidative stress conditions, might also traffic hydroperoxides of cholesterol, thereby disseminating their cytotoxic effects. Two inhibitors, SCPI-1 and SCPI-3, known to block cholesterol binding by an insect SCP-2, were used to investigate this. A mouse fibroblast transfectant clone (SC2F) overexpressing SCP-2 was found to be substantially more sensitive to apoptotic killing induced by liposomal 7α-hydroperoxycholesterol (7α-OOH) than a wild-type control. 7α-OOH uptake by SC2F cells and resulting apoptosis were both inhibited by SCPI-1 or SCPI-3 at a subtoxic concentration. Preceding cell death, reactive oxidant accumulation and loss of mitochondrial membrane potential were also strongly inhibited. Similar SCPI protection against 7α-OOH was observed with two other types of SCP-2-expressing mammalian cells. In striking contrast, neither inhibitor had any effect on H(2)O(2)-induced cell killing. To learn whether 7α-OOH cytotoxicity is due to uptake/transport by SCP-2, we used a fluorescence-based competitive binding assay involving recombinant SCP-2, NBD-cholesterol, and SCPI-1/SCPI-3 or 7α-OOH. The results clearly showed that 7α-OOH binds to SCP-2 in SCPI-inhibitable fashion. Our findings suggest that cellular SCP-2 not only binds and translocates cholesterol but also cholesterol hydroperoxides, thus expanding their redox toxicity and signaling ranges under oxidative stress conditions.

Use of dansyl-cholestanol as a probe of cholesterol behavior in membranes of living cells

While plasma membrane cholesterol-rich microdomains play a role in cholesterol trafficking, little is known about the appearance and dynamics of cholesterol through these domains in living cells. The fluorescent cholesterol analog 6-dansyl-cholestanol (DChol), its biochemical fractionation, and confocal imaging of L-cell fibroblasts contributed the following new insights: i) fluorescence properties of DChol were sensitive to microenvironment polarity and mobility; (ii) DChol taken up by L-cell fibroblasts was distributed similarly as cholesterol and preferentially into cholesterol-rich vs. -poor microdomains resolved by affinity chromatography of purified plasma membranes; iii) DChol reported similar polarity (dielectric constant near 18) but higher mobility near phospholipid polar head group region for cholesterol in purified cholesterol-rich versus -poor microdomains; and iv) real-time confocal imaging, quantitative colocalization analysis, and fluorescence resonance energy transfer with cholesterol-rich and -poor microdomain markers confirmed that DChol preferentially localized in plasma membrane cholesterol-rich microdomains of living cells. Thus, DChol sensed a unique, relatively more mobile microenvironment for cholesterol in plasma membrane cholesterol-rich microdomains, consistent with the known, more rapid exchange dynamics of cholesterol from cholesterol-rich than -poor microdomains.

Amyloid β-protein stimulates trafficking of cholesterol and caveolin-1 from the plasma membrane to the Golgi complex in mouse primary astrocytes

The Golgi complex plays a key role in cholesterol trafficking in cells. Our earlier study demonstrated amyloid β-protein (Aβ) alters cholesterol distribution and abundance in the Golgi complex of astrocytes. We now test the hypothesis that the Aβ-induced increase in Golgi complex cholesterol is due to retrograde movement of the cholesterol carrier protein caveolin-1 from the cell plasma membrane to the Golgi complex in astrocytes. Results with mouse primary astrocytes indicated that Aβ 1-42-induced increase in cholesterol and caveolin abundance in the Golgi complex was accompanied by a reduction in cholesterol and caveolin levels in the plasma membrane. Transfected rat astrocytes (DITNC1) with siRNA directed at caveolin-1 mRNA inhibited the Aβ 1-42-induced redistribution of both cholesterol and caveolin from the plasma membrane to the Golgi complex. In astrocytes not treated with Aβ 1-42, suppression of caveolin-1 expression also significantly reduced cholesterol abundance in the Golgi complex, further demonstrating the role for caveolin in retrograde transport of cholesterol from the plasma membrane to the Golgi complex. Perturbation of this process by Aβ 1-42 could have consequences on membrane structure and cellular functions requiring optimal levels of cholesterol.

Translocator protein 2 (TSPO2), a new family of proteins involved in cholesterol redistribution in erythropoiesis

Translocator protein (18‐kDa; TSPO) is a highly conserved cholesterol‐ and drug‐binding protein. Analysis of Tspo‐like sequences from different organisms revealed the presence of evolutionary conserved genes which arose by gene duplications before the divergence of avians and mammals. This new family of genes was named Tspo2 and found to be under strong purifying selection, suggesting that it serves a conserved biological function. The predicted cholesterol‐binding motif at the C‐terminus of TSPO was present in TSPO2, suggesting a role in cholesterol trafficking and/or targeting. In contrast to the mitochondrial TSPO, TSPO2 was found in nuclear and ER membranes. Heterologous expression of TSPO2 in Saccharomyces cerevisiae demonstrated that TSPO2 indeed binds cholesterol with high affinity. However, TSPO2 lost the TSPO drug binding ability. Spatial and temporal distribution of Tspo2 mRNA in the mouse demonstrated its presence in hematopoetic tissues and expression in an erythroid cell type‐specific manner. Overexpression of Tspo2 in erythroid cells resulted in the redistribution of the intracellular free cholesterol which has been linked to the expulsion of the nucleus from erythroblasts leading to erythrocyte maturation. These data suggest that the identified gene expansion in Tspo family supports a specialized function of the TSPO2 in erythropoiesis of endothermic animals, including humans.

Niemann-Pick C1 Protein Facilitates the Efflux of the Anticancer Drug Daunorubicin from Cells According to a Novel Vesicle-Mediated Pathway

Niemann-Pick C1 (NPC1) is a late endosomal/lysosomal membrane protein originally reported on for its role in cholesterol trafficking in mammalian cells. NPC1 has been shown recently to share significant structural homology with a family of prokaryotic permeases and was proposed to play a role in intracellular drug transport; however, the mechanism for this has not been fully understood. We provide evidence here that is consistent with NPC1's involvement in a vesicle-mediated clearance of the anticancer agent daunorubicin from cells. In experiments with human fibroblasts, we demonstrate that lysosomal efflux of daunorubicin, as well as dextran molecules, are significantly reduced in cells with mutated and dysfunctional NPC1 compared with wild-type fibroblasts. Furthermore, we show that NPC1 is implicated in a lysosomal drug sequestration phenotype exhibited by the multidrug-resistant (MDR) human leukemic HL-60 cancer cell line. Evaluations of cholesterol trafficking, NPC1 mRNA levels, and protein expression are all consistent with a loss of NPC1 activity that is associated with the emergence of the MDR phenotype in this cell line. Collectively, this work proposes a novel role for NPC1 in a vesicle-mediated pathway responsible for the clearance of drugs from cells and provides an explanation for a drug sequestration phenotype exhibited by the MDR HL-60 cell line.

Cholesterol Distribution in the Golgi Complex of DITNC1 Astrocytes Is Differentially Altered by Fresh and Aged Amyloid औ-Peptide-(1–42)

The Golgi complex plays an important role in cholesterol trafficking in cells, and amyloid beta-peptides (Abetas) alter cholesterol trafficking. The hypothesis was tested that fresh and aged Abeta-(1-42) would differentially modify Golgi cholesterol content in DINTC1 astrocytes and that the effects of Abeta-(1-42) would be associated with the region of the Golgi complex. Two different methods were used to determine the effects of Abeta-(1-42) on Golgi complex cholesterol. Confocal microscopy showed that fresh Abeta-(1-42) significantly increased cholesterol and that aged Abeta-(1-42) significantly reduced cholesterol content in the Golgi complex. Isolation of the Golgi complex into two fractions using density gradient centrifugation showed effects of aged Abeta-(1-42) similar to those observed with confocal microscopy but revealed the novel finding that fresh Abeta-(1-42) had opposite effects on the two Golgi fractions suggesting a specificity of Abeta-(1-42) perturbation of the Golgi complex. Phosphatidylcholine-phospholipase D activity, cell membrane cholesterol, and apolipoprotein E levels were associated with effects of fresh Abeta-(1-42) on cholesterol distribution but not with effects of aged Abeta-(1-42), arguing against a common mechanism. Extracellular Abeta-(1-42) targets the Golgi complex and disrupts cell cholesterol homeostasis, and this action of Abeta-(1-42) could alter cell functions requiring optimal levels of cholesterol.

P21-activated Kinase-1 (PAK1) Inhibition of the Human Scavenger Receptor Class B, Type I Promoter in Macrophages Is Independent of PAK1 Kinase Activity, but Requires the GTPase-binding Domain

Scavenger receptor class B, type I (SR-BI), is a high density lipoprotein receptor that mediates the flux of cholesterol between high density lipoprotein and cells. Recent evidence suggests that SR-BI plays a role in atherosclerosis and that inflammatory mediators down-regulate SR-BI in the macrophage. The purpose of this study was to evaluate the ability of lipopolysaccharide (LPS) to down-regulate the activity of the human SR-BI promoter in the macrophage and to delineate the mechanisms involved. Experiments with cultured cells and in vivo derived macrophages showed that LPS has a powerful suppressive effect on SR-BI expression both in vitro and in vivo. Transient transfection studies demonstrated that LPS represses SR-BI promoter activity in the macrophage cell line RAW 264.7. Cotransfection with either a constitutively active p21-activated protein kinase-1 (PAK1) construct (T423E) or a kinase-deficient PAK1 construct (K299R) resulted in repression of the SR-BI promoter, similar to LPS. These results demonstrate that PAK1-mediated down-regulation of the SR-BI promoter is independent of PAK1 kinase activity and suggest that PAK1 mediates the LPS-induced decrease in promoter activity. Cotransfection with constitutively active Cdc42 or Rac expression constructs also resulted in down-regulation of the promoter; whereas the dominant-negative Cdc42 and Rac constructs elevated basal promoter activity and blunted the LPS response. Cotransfection of PAK1 constructs containing mutations in both the kinase domain and the Cdc42/Rac-binding domain attenuated the PAK1-mediated down-regulation of the promoter, suggesting that Rac and Cdc42 are required for PAK1-mediated decreases in SR-BI promoter activity. 5'-Deletion analysis and gel shift data suggest that LPS inhibits binding of a novel transcription factor to a myeloid zing finger protein-1-like element (-476 to -456) in the human SR-BI promoter. These results demonstrate that the PAK1 pathway down-regulates the SR-BI promoter and suggest that activation of this pathway may play an important role in cholesterol trafficking in the vessel wall.

Caveolin is present in intestinal cells: role in cholesterol trafficking?

It was postulated that specialized microdomains of the plasma membrane, consistent with caveolae, might play a role in cholesterol trafficking in intestinal cells. The existence, therefore, of caveolin and the role of detergent-resistant microdomains of the plasma membrane in cholesterol trafficking were investigated in human small intestine and CaCo-2 cells. Caveolin mRNA was detected by RT-PCR in small intestinal brushings and biopsies and in CaCo-2 cells. Northern hybridization of caveolin mRNA detected 3 kb and 0.8 kb transcripts in CaCo-2 cells. From brushings of distal duodenum and in CaCo-2 cells, Western analysis for detection of caveolin protein demonstrated a 21 kDa-sized protein and a 600 kDa homooligomer. In CaCo-2 cells, caveolin was demonstrated by immunoflourescence in apical membranes as well as within cells. Using sucrose-density gradients, caveolin was localized to detergent-resistant microdomains of the plasma membrane. As determined by cholesterol oxidase-accessible cholesterol, 3–5% of plasma membrane cholesterol in CaCo-2 cells was estimated to be in these detergent-resistant microdomains. After the absorption of cholesterol from bile-salt micelles, more plasma membrane cholesterol moved to these specialized microdomains within the plasma membrane and was esterified. In CaCo-2 cells, filipin, N-ethyl maleimide, and cholesterol depletion, treatments that disrupt caveolar function, interfered with the transport of plasma membrane cholesterol to the endoplasmic reticulum, whereas okadaic acid, sphingomyelinase, and cholesterol oxidase did not. Changes in cholesterol flux at the apical membrane of the cell did not alter mRNA levels or mass of caveolin. The results suggest that caveolin is present in intestinal and CaCo-2 cells and is associated with detergent-resistant microdomains of cellular membranes. With the influx of micellar cholesterol from the lumen, plasma membrane cholesterol moves or “clusters” to these microdomains and is transported to the endoplasmic reticulum for esterification and eventual transport. Caveolin/caveolae may play a role in cholesterol trafficking in intestinal cells.—Field, F. J., E. Born, S. Murthy, and S. N. Mathur. Caveolin is present in intestinal cells: role in cholesterol trafficking? J. Lipid Res. 1998. 39: 1938–1950.

Loss of phosphoserine polar group asymmetry and inhibition of cholesterol transport in Jurkat cells treated with cholesterylphosphoserine

Cholesterylphosphoserine (CPHS) is a synthetic ester of cholesterol showing immunosuppressive activity. In the present study, we have used the T cell line Jurkat to investigate its mechanism of action. CPHS incorporates into cells reaching a molar ratio of 0.23 and 3.9 with the total phospholipid and cholesterol content, without inducing necrosis or apoptosis. CPHS incorporation elicits a dose-dependent binding of fluorescein isothiocyanate-labeled annexin V, suggesting that the steroid distributes in the external leaflet of plasma membrane exposing the phosphoserine group to the external cell environment and inserting the steroid ring into the phospholipid bilayer. In agreement with a preferential steroid association with sphingolipids, CPHS is included in a Triton X-100-insoluble complex when mixed with sphingomyelin and cholesterol. CPHS incorporation inhibits the esterification of low density lipoprotein (LDL)-derived cholesterol, producing a minor influence on the endogenous synthesis of cholesterol and on the acyl-CoA:cholesterol acyltransferase activity. In this effect, CPHS is as potent as progesterone (IC50 of 3.5 μm).▪ It is concluded that the insertion of cholesterylphosphoserine (CPHS) in the Jurkat plasma membrane neutralizes the asymmetric distribution of the phosphoserine group and inhibits the movement of cholesterol to the endoplasmic reticulum. As CPHS is a negatively charged steroid, this last effect may be linked to the perturbation of sphingolipid/cholesterol-based microdomains, proposed to play a role in cholesterol trafficking.—Cusinato, F., W. Habeler, F. Calderazzo, F. Nardi, and A. Bruni. Loss of phosphoserine polar group asymmetry and inhibition of cholesterol transport in Jurkat cells treated with cholesterylphosphoserine.

Effects of bile salts on rat hepatic acyl CoA:cholesterol acyltransferase.

Acyl CoA:cholesterol acyltransferase (EC2.3.1.26, ACAT), responsible for intracellular esterification of cholesterol, may play an important role in cholesterol trafficking within the cell, and thus, in maintenance of cellular cholesterol homeostasis. Bile acids are potential regulators of cholesterol trafficking in the liver. Therefore, the effect of bile salts on hepatic ACAT activity was studied in the perfused rat liver. ACAT activity was increased after liver perfusion with either taurocholate or taurochenodeoxycholate. However, addition of these bile salts at physiological concentrations in vitro had little effect on microsomal ACAT activity. The increase in hepatic ACAT activity due to perfusion with bile salts was accompanied by reduced accumulation of very low density lipoprotein cholesterol in the perfusate, but there was no effect on 3-hydroxy-3-methylglutaryl-CoA reductase activity. Hepatic ACAT activity was decreased after bile diversion for four hours in the intact animal. This treatment had no statistically significant effect on 3-hydroxy-3-methylglutaryl-CoA reductase activity. These data suggest that bile salts induce changes in hepatic compartmentation and traffic of cholesterol within the hepatocyte accompanied by response of ACAT activity to maintain cellular cholesterol homeostasis.