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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 deficiency does not affect sterol sensing in the endothelium.(a) Quantitative PCR analysis of SREBP2-dependent genes after knockdown of DNM2 or ACVRL1. The loss of DNM2 increases SREBP2-dependent gene expression, whereas the loss of ALK1 does not. Data represent the mean±s.e.m. and are representative of three experiments in duplicates. *P<0.05, Student's t-test. (b) Western blot analysis of the BMP9 (10 ng ml−1) induced phosphorylation of SMAD 1/5. HUVEC were incubated in LPDS and exposed to BMP9 for 60 min. In lane 2, cells were pretreated with LDL (25 μg ml−1) to downregulate LDLR. In lanes 3 and 4, ALK1 or LDLR was silenced with siRNA, respectively. A non-cropped western blot for this experiment can be found in Supplementary Fig. 9b. (c) The loss of ALK1 does not influence LDLR on the cell surface. Flow cytometric analysis of cell surface LDLR levels in endothelial cells treated with control, ACVRL1 or LDLR siRNAs. The Ab C7 was used for LDLR and IgG is an isotype control and data quantified in right panel. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student's t-test. (d) 125I-LDL internalization and degradation in cells treated with control, ACVRL1 or LDLR siRNAs. EA.hy926 cells were pre-incubated with LDL (25 μg ml−1) overnight and the internalization and degradation of 125I-LDL was after 4 h of incubation. ACVRL1 siRNA reduced internalization and had no effect on LDL degradation, whereas the LDLR siRNA (as a positive control) reduced LDL internalization and led to less degradation of LDL. Data represent the mean±s.e.m. and are representative of three experiments in duplicates. *P<0.05, Student's t-test. (e) Loss of ALK1 does not increase cellular free cholesterol. Filipin-III staining was examined in endothelial cells treated with siRNAs for ACVRL1, LDLR, DNM2 and NPC2 siRNA or treated with U18666 to enhance free cholesterol. Scale bar, 10 μm. Data are representative of at least four experiments. ns, not significant.
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f3: ALK1 deficiency does not affect sterol sensing in the endothelium.(a) Quantitative PCR analysis of SREBP2-dependent genes after knockdown of DNM2 or ACVRL1. The loss of DNM2 increases SREBP2-dependent gene expression, whereas the loss of ALK1 does not. Data represent the mean±s.e.m. and are representative of three experiments in duplicates. *P<0.05, Student's t-test. (b) Western blot analysis of the BMP9 (10 ng ml−1) induced phosphorylation of SMAD 1/5. HUVEC were incubated in LPDS and exposed to BMP9 for 60 min. In lane 2, cells were pretreated with LDL (25 μg ml−1) to downregulate LDLR. In lanes 3 and 4, ALK1 or LDLR was silenced with siRNA, respectively. A non-cropped western blot for this experiment can be found in Supplementary Fig. 9b. (c) The loss of ALK1 does not influence LDLR on the cell surface. Flow cytometric analysis of cell surface LDLR levels in endothelial cells treated with control, ACVRL1 or LDLR siRNAs. The Ab C7 was used for LDLR and IgG is an isotype control and data quantified in right panel. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student's t-test. (d) 125I-LDL internalization and degradation in cells treated with control, ACVRL1 or LDLR siRNAs. EA.hy926 cells were pre-incubated with LDL (25 μg ml−1) overnight and the internalization and degradation of 125I-LDL was after 4 h of incubation. ACVRL1 siRNA reduced internalization and had no effect on LDL degradation, whereas the LDLR siRNA (as a positive control) reduced LDL internalization and led to less degradation of LDL. Data represent the mean±s.e.m. and are representative of three experiments in duplicates. *P<0.05, Student's t-test. (e) Loss of ALK1 does not increase cellular free cholesterol. Filipin-III staining was examined in endothelial cells treated with siRNAs for ACVRL1, LDLR, DNM2 and NPC2 siRNA or treated with U18666 to enhance free cholesterol. Scale bar, 10 μm. Data are representative of at least four experiments. ns, not significant.

Mentions: As cholesterol homeostasis is crucial for cell growth and maintenance, cholesterol uptake, synthesis and metabolism, is largely regulated through sterol regulatory element-binding protein 2 (SREBP2) of sterol responsive genes. As seen in Fig. 3a, knockdown of dynamin 2 (DNM2) in EA.hy926 cells grown in complete media and serum, did not influence ACVRL1 gene expression but upregulated the transcript levels of several SREBP2-dependent genes (LDLR, HMGCR, INSIG1 and PCSK9), whereas the loss of ACVRL1 did not affect SREBP2-dependent gene expression. A further analysis was performed in EA.hy926 cell cultured in LPDS or LPDS containing LDL (25 μg ml−1, Supplementary Fig. 6). As expected, the addition of LDL to LPDS resulted in downregulation of LDLR, HMGCR and INSIG1—all SREBP2-dependent genes. Importantly, the loss of DNM2, but not ACVRL1, blunted the ability of LDL to reduce gene expression showing that ALK1 does not influence sterol sensing. Moreover, silencing of ACVRL1 had no effect on the total levels of LDLR in cell lysates by western blotting (Fig. 3b) or on cell surface LDLR quantified by fluorescence-activated cell sorting (FACS) (Fig. 3c). Because the uptake of LDL by the LDLR results in its lysosomal degradation15, we assessed the uptake and degradation of 125I-LDL in cells deficient in LDLR or ACVRL1. EA.hy926 were transfected with control, LDLR or ACVRL1 siRNAs, and then exposed to excess LDL (25 μg ml−1) overnight. The next morning, cells were incubated with 125I-LDL and the uptake and degradation (after 4 h) was assessed6. The net uptake of 125I-LDL in LDL pretreated EA.hy926 cells (Fig. 3d) was lower than that in MLEC cultured in LPDS, Fig. 2d). Knockdown of ALK1 in LDL pretreated EA.hy926 cells reduced 125I-LDL internalization, whereas knockdown of LDLR did not. However, the degradation of 125I-LDL, assessed by free 125I-tyrosine in the medium, was reduced by the loss of LDLR, but not the loss of ALK1. The data suggests that the suppression of LDLR by pre-treatment with LDL was incomplete, and so the subsequent addition of siRNA against LDLR further suppressed LDL internalization and degradation via the LDLR. As ALK1-mediated LDL uptake did not affect sterol sensing or LDL degradation, the pool of non-esterified, free cholesterol was analysed using Filipin-III staining (Fig. 3e). The knockdown of ACVRL1, LDLR or DNM2 did not increase the pool of free cholesterol, whereas both positive controls (U18666 (ref. 16) and NPC2 siRNA) did. These data demonstrate that ALK1 facilitates LDL uptake but does not target LDL for degradation.


Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells
ALK1 deficiency does not affect sterol sensing in the endothelium.(a) Quantitative PCR analysis of SREBP2-dependent genes after knockdown of DNM2 or ACVRL1. The loss of DNM2 increases SREBP2-dependent gene expression, whereas the loss of ALK1 does not. Data represent the mean±s.e.m. and are representative of three experiments in duplicates. *P<0.05, Student's t-test. (b) Western blot analysis of the BMP9 (10 ng ml−1) induced phosphorylation of SMAD 1/5. HUVEC were incubated in LPDS and exposed to BMP9 for 60 min. In lane 2, cells were pretreated with LDL (25 μg ml−1) to downregulate LDLR. In lanes 3 and 4, ALK1 or LDLR was silenced with siRNA, respectively. A non-cropped western blot for this experiment can be found in Supplementary Fig. 9b. (c) The loss of ALK1 does not influence LDLR on the cell surface. Flow cytometric analysis of cell surface LDLR levels in endothelial cells treated with control, ACVRL1 or LDLR siRNAs. The Ab C7 was used for LDLR and IgG is an isotype control and data quantified in right panel. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student's t-test. (d) 125I-LDL internalization and degradation in cells treated with control, ACVRL1 or LDLR siRNAs. EA.hy926 cells were pre-incubated with LDL (25 μg ml−1) overnight and the internalization and degradation of 125I-LDL was after 4 h of incubation. ACVRL1 siRNA reduced internalization and had no effect on LDL degradation, whereas the LDLR siRNA (as a positive control) reduced LDL internalization and led to less degradation of LDL. Data represent the mean±s.e.m. and are representative of three experiments in duplicates. *P<0.05, Student's t-test. (e) Loss of ALK1 does not increase cellular free cholesterol. Filipin-III staining was examined in endothelial cells treated with siRNAs for ACVRL1, LDLR, DNM2 and NPC2 siRNA or treated with U18666 to enhance free cholesterol. Scale bar, 10 μm. Data are representative of at least four experiments. ns, not significant.
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f3: ALK1 deficiency does not affect sterol sensing in the endothelium.(a) Quantitative PCR analysis of SREBP2-dependent genes after knockdown of DNM2 or ACVRL1. The loss of DNM2 increases SREBP2-dependent gene expression, whereas the loss of ALK1 does not. Data represent the mean±s.e.m. and are representative of three experiments in duplicates. *P<0.05, Student's t-test. (b) Western blot analysis of the BMP9 (10 ng ml−1) induced phosphorylation of SMAD 1/5. HUVEC were incubated in LPDS and exposed to BMP9 for 60 min. In lane 2, cells were pretreated with LDL (25 μg ml−1) to downregulate LDLR. In lanes 3 and 4, ALK1 or LDLR was silenced with siRNA, respectively. A non-cropped western blot for this experiment can be found in Supplementary Fig. 9b. (c) The loss of ALK1 does not influence LDLR on the cell surface. Flow cytometric analysis of cell surface LDLR levels in endothelial cells treated with control, ACVRL1 or LDLR siRNAs. The Ab C7 was used for LDLR and IgG is an isotype control and data quantified in right panel. Data represent the mean±s.e.m. and are representative of three experiments in triplicates. *P<0.05, Student's t-test. (d) 125I-LDL internalization and degradation in cells treated with control, ACVRL1 or LDLR siRNAs. EA.hy926 cells were pre-incubated with LDL (25 μg ml−1) overnight and the internalization and degradation of 125I-LDL was after 4 h of incubation. ACVRL1 siRNA reduced internalization and had no effect on LDL degradation, whereas the LDLR siRNA (as a positive control) reduced LDL internalization and led to less degradation of LDL. Data represent the mean±s.e.m. and are representative of three experiments in duplicates. *P<0.05, Student's t-test. (e) Loss of ALK1 does not increase cellular free cholesterol. Filipin-III staining was examined in endothelial cells treated with siRNAs for ACVRL1, LDLR, DNM2 and NPC2 siRNA or treated with U18666 to enhance free cholesterol. Scale bar, 10 μm. Data are representative of at least four experiments. ns, not significant.
Mentions: As cholesterol homeostasis is crucial for cell growth and maintenance, cholesterol uptake, synthesis and metabolism, is largely regulated through sterol regulatory element-binding protein 2 (SREBP2) of sterol responsive genes. As seen in Fig. 3a, knockdown of dynamin 2 (DNM2) in EA.hy926 cells grown in complete media and serum, did not influence ACVRL1 gene expression but upregulated the transcript levels of several SREBP2-dependent genes (LDLR, HMGCR, INSIG1 and PCSK9), whereas the loss of ACVRL1 did not affect SREBP2-dependent gene expression. A further analysis was performed in EA.hy926 cell cultured in LPDS or LPDS containing LDL (25 μg ml−1, Supplementary Fig. 6). As expected, the addition of LDL to LPDS resulted in downregulation of LDLR, HMGCR and INSIG1—all SREBP2-dependent genes. Importantly, the loss of DNM2, but not ACVRL1, blunted the ability of LDL to reduce gene expression showing that ALK1 does not influence sterol sensing. Moreover, silencing of ACVRL1 had no effect on the total levels of LDLR in cell lysates by western blotting (Fig. 3b) or on cell surface LDLR quantified by fluorescence-activated cell sorting (FACS) (Fig. 3c). Because the uptake of LDL by the LDLR results in its lysosomal degradation15, we assessed the uptake and degradation of 125I-LDL in cells deficient in LDLR or ACVRL1. EA.hy926 were transfected with control, LDLR or ACVRL1 siRNAs, and then exposed to excess LDL (25 μg ml−1) overnight. The next morning, cells were incubated with 125I-LDL and the uptake and degradation (after 4 h) was assessed6. The net uptake of 125I-LDL in LDL pretreated EA.hy926 cells (Fig. 3d) was lower than that in MLEC cultured in LPDS, Fig. 2d). Knockdown of ALK1 in LDL pretreated EA.hy926 cells reduced 125I-LDL internalization, whereas knockdown of LDLR did not. However, the degradation of 125I-LDL, assessed by free 125I-tyrosine in the medium, was reduced by the loss of LDLR, but not the loss of ALK1. The data suggests that the suppression of LDLR by pre-treatment with LDL was incomplete, and so the subsequent addition of siRNA against LDLR further suppressed LDL internalization and degradation via the LDLR. As ALK1-mediated LDL uptake did not affect sterol sensing or LDL degradation, the pool of non-esterified, free cholesterol was analysed using Filipin-III staining (Fig. 3e). The knockdown of ACVRL1, LDLR or DNM2 did not increase the pool of free cholesterol, whereas both positive controls (U18666 (ref. 16) and NPC2 siRNA) did. These data demonstrate that ALK1 facilitates LDL uptake but does not target LDL for degradation.

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