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Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1.

Cianciola NL, Greene DJ, Morton RE, Carlin CR - Mol. Biol. Cell (2013)

Bottom Line: Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7.The molecular identity of putative alternative pathways, however, is poorly characterized.We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195 Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106.

ABSTRACT
Niemann-Pick disease type C (NPC) is caused by mutations in NPC1 or NPC2, which coordinate egress of low-density-lipoprotein (LDL)-cholesterol from late endosomes. We previously reported that the adenovirus-encoded protein RIDα rescues the cholesterol storage phenotype in NPC1-mutant fibroblasts. We show here that RIDα reconstitutes deficient endosome-to-endoplasmic reticulum (ER) transport, allowing excess LDL-cholesterol to be esterified by acyl-CoA:cholesterol acyltransferase and stored in lipid droplets (LDs) in NPC1-deficient cells. Furthermore, the RIDα pathway is regulated by the oxysterol-binding protein ORP1L. Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7. Our data, however, suggest that ORP1L may play a role in transport of LDL-cholesterol to a specific ER pool designated for LD formation. In contrast to NPC1, which is dispensable, the RIDα/ORP1L-dependent route requires functional NPC2. Although NPC1/NPC2 constitutes the major pathway, therapies that amplify minor egress routes for LDL-cholesterol could significantly improve clinical management of patients with loss-of-function NPC1 mutations. The molecular identity of putative alternative pathways, however, is poorly characterized. We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

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Related in: MedlinePlus

RIDα does not modulate sterol-regulated gene expression in NPC1-deficient cells with stable RIDα expression. (A) Schematic summarizing homeostatic responses to increased free cholesterol (Ch). High levels of ER Ch become esterified by ACAT and also inhibit the SREBP pathway by facilitating SCAP–SREBP interactions and ER retention. Increased mitochondrial Ch stimulates 27-hydroxycholesterol (27-HC) biosynthesis, which binds Insig, contributing to SREBP ER retention, and activates nuclear LXR transcription factors. (B–E) HMGCR (B), LDLR (C), ABCA1 (D), and CYP7B (E) mRNA levels quantified by real-time PCR. Values are expressed as relative units after internal normalization to glyceraldehyde 3-phosphate dehydrogenase mRNA levels and compared with control samples from the same cell lines cultured in 10% FBS, which was set to 1 (dashed line) from three independent experiments. Data are presented as mean ± SEM. (F, G) Quantification of esterified cholesterol in Chinese hamster ovary, CT43, and CT43-RIDα cells (F) or shControl, shNPC1, and shNPC1-RIDα cells (G) with and without 24 h treatment with LDL using the Amplex Red Cholesterol Assay kit as described in Materials and Methods. Values were normalized to total cellular protein and are displayed as mean ± SEM. (H) Equal aliquots of total cellular protein from CT43 and CT43-RIDα cells or shNPC1 and shNPC1-RIDα cells were immunoblotted with antibodies to FLAG or actin for loading control.
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Figure 4: RIDα does not modulate sterol-regulated gene expression in NPC1-deficient cells with stable RIDα expression. (A) Schematic summarizing homeostatic responses to increased free cholesterol (Ch). High levels of ER Ch become esterified by ACAT and also inhibit the SREBP pathway by facilitating SCAP–SREBP interactions and ER retention. Increased mitochondrial Ch stimulates 27-hydroxycholesterol (27-HC) biosynthesis, which binds Insig, contributing to SREBP ER retention, and activates nuclear LXR transcription factors. (B–E) HMGCR (B), LDLR (C), ABCA1 (D), and CYP7B (E) mRNA levels quantified by real-time PCR. Values are expressed as relative units after internal normalization to glyceraldehyde 3-phosphate dehydrogenase mRNA levels and compared with control samples from the same cell lines cultured in 10% FBS, which was set to 1 (dashed line) from three independent experiments. Data are presented as mean ± SEM. (F, G) Quantification of esterified cholesterol in Chinese hamster ovary, CT43, and CT43-RIDα cells (F) or shControl, shNPC1, and shNPC1-RIDα cells (G) with and without 24 h treatment with LDL using the Amplex Red Cholesterol Assay kit as described in Materials and Methods. Values were normalized to total cellular protein and are displayed as mean ± SEM. (H) Equal aliquots of total cellular protein from CT43 and CT43-RIDα cells or shNPC1 and shNPC1-RIDα cells were immunoblotted with antibodies to FLAG or actin for loading control.

Mentions: RIDα increases LD formation in NPC1-deficient cells. (A) Confocal images of normal and NPC1- and NPC2-mutant fibroblasts transfected with RIDα and stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI to visualize LDs and nuclei, respectively. Mock-transfected cells lacking FLAG-RIDα expression are shown in the same field and designated with an asterisk. (B) Confocal images of shControl, shNPC1, and shNPC1-RIDα cells stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. (C) Confocal images of CT43 and CT43-RIDα cells stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. (D, E) Quantification of average LD area (D) and average LD number (E) per cell in cells treated similarly to cells in C as described in Materials and Methods. Data are presented as mean ± SEM (*p < 0.001). (F) Quantification of esterified cholesterol in Chinese hamster ovary, CT43, and CT43-RIDα cells using the Amplex Red Cholesterol Assay kit as described in Materials and Methods. Values were normalized to total cellular protein and are displayed as mean ± SEM (*p < 0.001). (G) ACAT mRNA levels quantified by real-time PCR similarly to cells in Figure 4. Data are presented as mean ± SEM. (H) Experimental setup of cholesterol transport assay. Purified human LDL was labeled with [3H]cholesteryl palmitate, and cells were incubated with the labeled LDL and excess oleate. The labeled LDL was transported to Ly (step 1) and deesterified by lysosomal acid lipase (LAL; step 2). The liberated [3H]cholesterol can then be transported to the ER (step 3), where it can be reesterified by ACAT along with the excess oleate to form [3H]cholesteryl oleate and stored in LDs (step 4). (I) shControl, shNPC1, and shNPC1-RIDα cells were incubated with [3H]cholesteryl palmitate along with excess oleate as described in Materials and Methods. The [3H]cholesteryl oleate production was quantified, and values were normalized to total cellular protein and are displayed as mean ± SD (*p < 0.0001) from three independent experiments. Bars, 10 μm.


Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1.

Cianciola NL, Greene DJ, Morton RE, Carlin CR - Mol. Biol. Cell (2013)

RIDα does not modulate sterol-regulated gene expression in NPC1-deficient cells with stable RIDα expression. (A) Schematic summarizing homeostatic responses to increased free cholesterol (Ch). High levels of ER Ch become esterified by ACAT and also inhibit the SREBP pathway by facilitating SCAP–SREBP interactions and ER retention. Increased mitochondrial Ch stimulates 27-hydroxycholesterol (27-HC) biosynthesis, which binds Insig, contributing to SREBP ER retention, and activates nuclear LXR transcription factors. (B–E) HMGCR (B), LDLR (C), ABCA1 (D), and CYP7B (E) mRNA levels quantified by real-time PCR. Values are expressed as relative units after internal normalization to glyceraldehyde 3-phosphate dehydrogenase mRNA levels and compared with control samples from the same cell lines cultured in 10% FBS, which was set to 1 (dashed line) from three independent experiments. Data are presented as mean ± SEM. (F, G) Quantification of esterified cholesterol in Chinese hamster ovary, CT43, and CT43-RIDα cells (F) or shControl, shNPC1, and shNPC1-RIDα cells (G) with and without 24 h treatment with LDL using the Amplex Red Cholesterol Assay kit as described in Materials and Methods. Values were normalized to total cellular protein and are displayed as mean ± SEM. (H) Equal aliquots of total cellular protein from CT43 and CT43-RIDα cells or shNPC1 and shNPC1-RIDα cells were immunoblotted with antibodies to FLAG or actin for loading control.
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Related In: Results  -  Collection

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Figure 4: RIDα does not modulate sterol-regulated gene expression in NPC1-deficient cells with stable RIDα expression. (A) Schematic summarizing homeostatic responses to increased free cholesterol (Ch). High levels of ER Ch become esterified by ACAT and also inhibit the SREBP pathway by facilitating SCAP–SREBP interactions and ER retention. Increased mitochondrial Ch stimulates 27-hydroxycholesterol (27-HC) biosynthesis, which binds Insig, contributing to SREBP ER retention, and activates nuclear LXR transcription factors. (B–E) HMGCR (B), LDLR (C), ABCA1 (D), and CYP7B (E) mRNA levels quantified by real-time PCR. Values are expressed as relative units after internal normalization to glyceraldehyde 3-phosphate dehydrogenase mRNA levels and compared with control samples from the same cell lines cultured in 10% FBS, which was set to 1 (dashed line) from three independent experiments. Data are presented as mean ± SEM. (F, G) Quantification of esterified cholesterol in Chinese hamster ovary, CT43, and CT43-RIDα cells (F) or shControl, shNPC1, and shNPC1-RIDα cells (G) with and without 24 h treatment with LDL using the Amplex Red Cholesterol Assay kit as described in Materials and Methods. Values were normalized to total cellular protein and are displayed as mean ± SEM. (H) Equal aliquots of total cellular protein from CT43 and CT43-RIDα cells or shNPC1 and shNPC1-RIDα cells were immunoblotted with antibodies to FLAG or actin for loading control.
Mentions: RIDα increases LD formation in NPC1-deficient cells. (A) Confocal images of normal and NPC1- and NPC2-mutant fibroblasts transfected with RIDα and stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI to visualize LDs and nuclei, respectively. Mock-transfected cells lacking FLAG-RIDα expression are shown in the same field and designated with an asterisk. (B) Confocal images of shControl, shNPC1, and shNPC1-RIDα cells stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. (C) Confocal images of CT43 and CT43-RIDα cells stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. (D, E) Quantification of average LD area (D) and average LD number (E) per cell in cells treated similarly to cells in C as described in Materials and Methods. Data are presented as mean ± SEM (*p < 0.001). (F) Quantification of esterified cholesterol in Chinese hamster ovary, CT43, and CT43-RIDα cells using the Amplex Red Cholesterol Assay kit as described in Materials and Methods. Values were normalized to total cellular protein and are displayed as mean ± SEM (*p < 0.001). (G) ACAT mRNA levels quantified by real-time PCR similarly to cells in Figure 4. Data are presented as mean ± SEM. (H) Experimental setup of cholesterol transport assay. Purified human LDL was labeled with [3H]cholesteryl palmitate, and cells were incubated with the labeled LDL and excess oleate. The labeled LDL was transported to Ly (step 1) and deesterified by lysosomal acid lipase (LAL; step 2). The liberated [3H]cholesterol can then be transported to the ER (step 3), where it can be reesterified by ACAT along with the excess oleate to form [3H]cholesteryl oleate and stored in LDs (step 4). (I) shControl, shNPC1, and shNPC1-RIDα cells were incubated with [3H]cholesteryl palmitate along with excess oleate as described in Materials and Methods. The [3H]cholesteryl oleate production was quantified, and values were normalized to total cellular protein and are displayed as mean ± SD (*p < 0.0001) from three independent experiments. Bars, 10 μm.

Bottom Line: Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7.The molecular identity of putative alternative pathways, however, is poorly characterized.We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195 Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106.

ABSTRACT
Niemann-Pick disease type C (NPC) is caused by mutations in NPC1 or NPC2, which coordinate egress of low-density-lipoprotein (LDL)-cholesterol from late endosomes. We previously reported that the adenovirus-encoded protein RIDα rescues the cholesterol storage phenotype in NPC1-mutant fibroblasts. We show here that RIDα reconstitutes deficient endosome-to-endoplasmic reticulum (ER) transport, allowing excess LDL-cholesterol to be esterified by acyl-CoA:cholesterol acyltransferase and stored in lipid droplets (LDs) in NPC1-deficient cells. Furthermore, the RIDα pathway is regulated by the oxysterol-binding protein ORP1L. Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7. Our data, however, suggest that ORP1L may play a role in transport of LDL-cholesterol to a specific ER pool designated for LD formation. In contrast to NPC1, which is dispensable, the RIDα/ORP1L-dependent route requires functional NPC2. Although NPC1/NPC2 constitutes the major pathway, therapies that amplify minor egress routes for LDL-cholesterol could significantly improve clinical management of patients with loss-of-function NPC1 mutations. The molecular identity of putative alternative pathways, however, is poorly characterized. We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

Show MeSH
Related in: MedlinePlus