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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

ALK1 rescues LDL uptake and promotes LDL uptake independent of its kinase activity.(a) Analysis of DiI-LDL uptake in EA.hy926 treated with ACVRL1 siRNA (+) in the presence of increasing amounts of expressed ALK1 (using various MOI of AdALK1-GFP). DiI-LDL uptake was normalized by Hoechst dye stained nuclei. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student's t-test. (b) ALK1 dose dependently increases DiI-LDL uptake in Ldlr-KO MEFs. DiI-LDL data are normalized for ALK1-GFP expression in Ldlr-KO MEFs by using various MOI of AdALK1-GFP. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test. (c) HeLa cells were transfected with either GFP, WT, constitutively active (CA, Q201D) and inactive variant (IA, R374Q) ALK1 constructs and p-SMAD1/5 levels were examined in the absence or presence of BMP9 (10 ng ml−1) (Data are mean±s.e.m., experiment was performed three times). A non-cropped western blot for this experiment can be found in Supplementary Fig. 10a. (d) DiI-LDL uptake analysis of cells expressing GFP and the different variants of ALK1. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student's t-test. (e) Western bot analysis of p-SMAD1/5 after starvation using BMP9 (10 ng ml−1) or pharmacological inhibitors (ALK1ecto,400 ng ml−1: tenfold molar excess or LDN193189, 50 nM). A non-cropped western blot for this experiment can be found in Supplementary Fig. 10b. (f) BMP9 stimulation or inhibition does not affect DiI-LDL uptake into endothelial cells. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test. ns, not significant.
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f4: ALK1 rescues LDL uptake and promotes LDL uptake independent of its kinase activity.(a) Analysis of DiI-LDL uptake in EA.hy926 treated with ACVRL1 siRNA (+) in the presence of increasing amounts of expressed ALK1 (using various MOI of AdALK1-GFP). DiI-LDL uptake was normalized by Hoechst dye stained nuclei. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student's t-test. (b) ALK1 dose dependently increases DiI-LDL uptake in Ldlr-KO MEFs. DiI-LDL data are normalized for ALK1-GFP expression in Ldlr-KO MEFs by using various MOI of AdALK1-GFP. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test. (c) HeLa cells were transfected with either GFP, WT, constitutively active (CA, Q201D) and inactive variant (IA, R374Q) ALK1 constructs and p-SMAD1/5 levels were examined in the absence or presence of BMP9 (10 ng ml−1) (Data are mean±s.e.m., experiment was performed three times). A non-cropped western blot for this experiment can be found in Supplementary Fig. 10a. (d) DiI-LDL uptake analysis of cells expressing GFP and the different variants of ALK1. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student's t-test. (e) Western bot analysis of p-SMAD1/5 after starvation using BMP9 (10 ng ml−1) or pharmacological inhibitors (ALK1ecto,400 ng ml−1: tenfold molar excess or LDN193189, 50 nM). A non-cropped western blot for this experiment can be found in Supplementary Fig. 10b. (f) BMP9 stimulation or inhibition does not affect DiI-LDL uptake into endothelial cells. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test. ns, not significant.

Mentions: To examine the sufficiency of ALK1 for LDL uptake, rescue and gain-of-function experiments were performed. As shown in Fig. 4a, DiI-LDL uptake is significantly decreased in endothelial cells after knockdown of ALK1, an effect rescued, in a dose-dependent manner (0–100 MOI of adenovirus), by the expression of an adenovirus encoding ALK1-GFP (AdALK1). Moreover, infection of AdALK1-GFP into Ldlr-KO mouse lung fibroblasts (MEF) also dose-dependently increased DiI-LDL uptake (Fig. 4b), showing again that ALK1 mediates LDL uptake independently of the LDLR.


Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells
ALK1 rescues LDL uptake and promotes LDL uptake independent of its kinase activity.(a) Analysis of DiI-LDL uptake in EA.hy926 treated with ACVRL1 siRNA (+) in the presence of increasing amounts of expressed ALK1 (using various MOI of AdALK1-GFP). DiI-LDL uptake was normalized by Hoechst dye stained nuclei. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student's t-test. (b) ALK1 dose dependently increases DiI-LDL uptake in Ldlr-KO MEFs. DiI-LDL data are normalized for ALK1-GFP expression in Ldlr-KO MEFs by using various MOI of AdALK1-GFP. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test. (c) HeLa cells were transfected with either GFP, WT, constitutively active (CA, Q201D) and inactive variant (IA, R374Q) ALK1 constructs and p-SMAD1/5 levels were examined in the absence or presence of BMP9 (10 ng ml−1) (Data are mean±s.e.m., experiment was performed three times). A non-cropped western blot for this experiment can be found in Supplementary Fig. 10a. (d) DiI-LDL uptake analysis of cells expressing GFP and the different variants of ALK1. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student's t-test. (e) Western bot analysis of p-SMAD1/5 after starvation using BMP9 (10 ng ml−1) or pharmacological inhibitors (ALK1ecto,400 ng ml−1: tenfold molar excess or LDN193189, 50 nM). A non-cropped western blot for this experiment can be found in Supplementary Fig. 10b. (f) BMP9 stimulation or inhibition does not affect DiI-LDL uptake into endothelial cells. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test. ns, not significant.
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f4: ALK1 rescues LDL uptake and promotes LDL uptake independent of its kinase activity.(a) Analysis of DiI-LDL uptake in EA.hy926 treated with ACVRL1 siRNA (+) in the presence of increasing amounts of expressed ALK1 (using various MOI of AdALK1-GFP). DiI-LDL uptake was normalized by Hoechst dye stained nuclei. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student's t-test. (b) ALK1 dose dependently increases DiI-LDL uptake in Ldlr-KO MEFs. DiI-LDL data are normalized for ALK1-GFP expression in Ldlr-KO MEFs by using various MOI of AdALK1-GFP. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test. (c) HeLa cells were transfected with either GFP, WT, constitutively active (CA, Q201D) and inactive variant (IA, R374Q) ALK1 constructs and p-SMAD1/5 levels were examined in the absence or presence of BMP9 (10 ng ml−1) (Data are mean±s.e.m., experiment was performed three times). A non-cropped western blot for this experiment can be found in Supplementary Fig. 10a. (d) DiI-LDL uptake analysis of cells expressing GFP and the different variants of ALK1. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student's t-test. (e) Western bot analysis of p-SMAD1/5 after starvation using BMP9 (10 ng ml−1) or pharmacological inhibitors (ALK1ecto,400 ng ml−1: tenfold molar excess or LDN193189, 50 nM). A non-cropped western blot for this experiment can be found in Supplementary Fig. 10b. (f) BMP9 stimulation or inhibition does not affect DiI-LDL uptake into endothelial cells. Data represent the mean±s.e.m. and are representative of three experiments. *P<0.05, Student's t-test. ns, not significant.
Mentions: To examine the sufficiency of ALK1 for LDL uptake, rescue and gain-of-function experiments were performed. As shown in Fig. 4a, DiI-LDL uptake is significantly decreased in endothelial cells after knockdown of ALK1, an effect rescued, in a dose-dependent manner (0–100 MOI of adenovirus), by the expression of an adenovirus encoding ALK1-GFP (AdALK1). Moreover, infection of AdALK1-GFP into Ldlr-KO mouse lung fibroblasts (MEF) also dose-dependently increased DiI-LDL uptake (Fig. 4b), showing again that ALK1 mediates LDL uptake independently of the LDLR.

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