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Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells

View Article: PubMed Central - PubMed

ABSTRACT

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.

No MeSH data available.


Related in: MedlinePlus

Time-dependent internalization and co-localization of DiI-LDL and ALK1.(a) Ldlr-KO MEFs were infected with ALK1-GFP, incubated in LPDS overnight and the time-dependent internalization of DiI-LDL examined at 37 °C. Cells were imaged for ALK1-GFP (green), DiI-LDL (red) and white reflecting co-localization of ALK1-GFP/DiI-LDL (Menders correlation). The internalization of DiI-LDL and its co-localization with DiI-LDL shows at 10–20 min and accumulates in a perinuclear compartment after 60 min. Scale bar, 10 μm. The data is representative for three independent experiments. (b) Analysis of ALK1-GFP localization in control (untreated), LDL (25 μg ml−1) or BMP9 (10 ng ml−1) treated EA.hy926 cells. Cells were infected for 48 h and transferred to LPDS for the remaining 24 h. Cells were treated as described for 1 h at 37 °C, fixed and imaged. Upper panels show original confocal laser scanning microscopy images (green, ALK1-GFP; red, EEA1; blue, nuclei). Lower panels show Menders correlation (green, ALK1-GFP alone; red, EEA1 alone; white, ALK1-GFP/EEA1 co-localized). Scale bar, 20 μm. The data is representative for three independent experiments. (c) Bar graph shows Pearson correlation of these three conditions. The result indicates a co-localization of ALK1 with the early endosome marker EEA1 upon stimulation with either LDL or BMP9 within 1 h. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test.
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f6: Time-dependent internalization and co-localization of DiI-LDL and ALK1.(a) Ldlr-KO MEFs were infected with ALK1-GFP, incubated in LPDS overnight and the time-dependent internalization of DiI-LDL examined at 37 °C. Cells were imaged for ALK1-GFP (green), DiI-LDL (red) and white reflecting co-localization of ALK1-GFP/DiI-LDL (Menders correlation). The internalization of DiI-LDL and its co-localization with DiI-LDL shows at 10–20 min and accumulates in a perinuclear compartment after 60 min. Scale bar, 10 μm. The data is representative for three independent experiments. (b) Analysis of ALK1-GFP localization in control (untreated), LDL (25 μg ml−1) or BMP9 (10 ng ml−1) treated EA.hy926 cells. Cells were infected for 48 h and transferred to LPDS for the remaining 24 h. Cells were treated as described for 1 h at 37 °C, fixed and imaged. Upper panels show original confocal laser scanning microscopy images (green, ALK1-GFP; red, EEA1; blue, nuclei). Lower panels show Menders correlation (green, ALK1-GFP alone; red, EEA1 alone; white, ALK1-GFP/EEA1 co-localized). Scale bar, 20 μm. The data is representative for three independent experiments. (c) Bar graph shows Pearson correlation of these three conditions. The result indicates a co-localization of ALK1 with the early endosome marker EEA1 upon stimulation with either LDL or BMP9 within 1 h. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test.

Mentions: To examine if the binding of LDL to ALK1 influences its internalization, imaging experiments were performed in Ldlr-KO MEFs infected with AdALK1-GFP and incubated with DiI-LDL from 0–60 min. As seen in Fig. 6a, ALK1-GFP co-localizes in a time-dependent manner with DiI-LDL in the cell with a perinuclear accumulation at 60 min. At 60 min, ALK1-GFP localized in an early endosomal compartment marked by EEA1 (Fig. 6b). Analysis of the images using Pearson correlation (Fig. 6c) showed a significant increase in co-localization of ALK1-GFP and EEA1 after stimulation with either LDL or BMP9.


Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells
Time-dependent internalization and co-localization of DiI-LDL and ALK1.(a) Ldlr-KO MEFs were infected with ALK1-GFP, incubated in LPDS overnight and the time-dependent internalization of DiI-LDL examined at 37 °C. Cells were imaged for ALK1-GFP (green), DiI-LDL (red) and white reflecting co-localization of ALK1-GFP/DiI-LDL (Menders correlation). The internalization of DiI-LDL and its co-localization with DiI-LDL shows at 10–20 min and accumulates in a perinuclear compartment after 60 min. Scale bar, 10 μm. The data is representative for three independent experiments. (b) Analysis of ALK1-GFP localization in control (untreated), LDL (25 μg ml−1) or BMP9 (10 ng ml−1) treated EA.hy926 cells. Cells were infected for 48 h and transferred to LPDS for the remaining 24 h. Cells were treated as described for 1 h at 37 °C, fixed and imaged. Upper panels show original confocal laser scanning microscopy images (green, ALK1-GFP; red, EEA1; blue, nuclei). Lower panels show Menders correlation (green, ALK1-GFP alone; red, EEA1 alone; white, ALK1-GFP/EEA1 co-localized). Scale bar, 20 μm. The data is representative for three independent experiments. (c) Bar graph shows Pearson correlation of these three conditions. The result indicates a co-localization of ALK1 with the early endosome marker EEA1 upon stimulation with either LDL or BMP9 within 1 h. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test.
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f6: Time-dependent internalization and co-localization of DiI-LDL and ALK1.(a) Ldlr-KO MEFs were infected with ALK1-GFP, incubated in LPDS overnight and the time-dependent internalization of DiI-LDL examined at 37 °C. Cells were imaged for ALK1-GFP (green), DiI-LDL (red) and white reflecting co-localization of ALK1-GFP/DiI-LDL (Menders correlation). The internalization of DiI-LDL and its co-localization with DiI-LDL shows at 10–20 min and accumulates in a perinuclear compartment after 60 min. Scale bar, 10 μm. The data is representative for three independent experiments. (b) Analysis of ALK1-GFP localization in control (untreated), LDL (25 μg ml−1) or BMP9 (10 ng ml−1) treated EA.hy926 cells. Cells were infected for 48 h and transferred to LPDS for the remaining 24 h. Cells were treated as described for 1 h at 37 °C, fixed and imaged. Upper panels show original confocal laser scanning microscopy images (green, ALK1-GFP; red, EEA1; blue, nuclei). Lower panels show Menders correlation (green, ALK1-GFP alone; red, EEA1 alone; white, ALK1-GFP/EEA1 co-localized). Scale bar, 20 μm. The data is representative for three independent experiments. (c) Bar graph shows Pearson correlation of these three conditions. The result indicates a co-localization of ALK1 with the early endosome marker EEA1 upon stimulation with either LDL or BMP9 within 1 h. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test.
Mentions: To examine if the binding of LDL to ALK1 influences its internalization, imaging experiments were performed in Ldlr-KO MEFs infected with AdALK1-GFP and incubated with DiI-LDL from 0–60 min. As seen in Fig. 6a, ALK1-GFP co-localizes in a time-dependent manner with DiI-LDL in the cell with a perinuclear accumulation at 60 min. At 60 min, ALK1-GFP localized in an early endosomal compartment marked by EEA1 (Fig. 6b). Analysis of the images using Pearson correlation (Fig. 6c) showed a significant increase in co-localization of ALK1-GFP and EEA1 after stimulation with either LDL or BMP9.

View Article: PubMed Central - PubMed

ABSTRACT

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.

No MeSH data available.


Related in: MedlinePlus