ABCC4 impairs the clearance of plasma LDL cholesterol through suppressing LDLR abundance on hepatic surface
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ABSTRACT:
PROVIDER: PRJNA1265130 | ENA |
REPOSITORIES: ENA
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Project description:Low expression level of low-density lipoprotein receptor (LDLR) on the surface of hepatocytes contributes to hypercholesterolemia and ultimately is the primary determinant of atherosclerotic cardiovascular disease (ASCVD)，which is the leading cause of death in humans globally. Here, we report that inhibition of hepatocyte ABCC4, identified as a top hit from large-scale CRISPR/Cas9 screens, significantly increases hepatic LDLR abundance and LDL cholesterol clearance. As a hepatic transporter for cAMP efflux, ABCC4 silencing alters its intracellular distribution and activates downstream Epac2/Rap1a signaling pathway, which ultimately downregulates PCSK9 protein expression, thereby blocking lysosomal degradation of LDLR. Furthermore, in both animal and cell models, we demonstrate that liver-specific disruption of ABCC4 and pharmacological inhibition of ABCC4 enhance hepatic plasma membrane LDLR expression and lower plasma LDL cholesterol level through ABCC4-cAMP-PCKS9 pathway. Collectively, genome-wide CRISPR screening offers a valuable resource for identifying LDLR modifiers, providing potential insights for therapeutic strategies in hypercholesterolemia and atherosclerosis.
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Project description:In familial hypercholesterolemia (FH), mutations in the low-density lipoprotein (LDL) receptor (LDLr) gene result in increased plasma LDL cholesterol. Clinical and preclinical studies revealed an association between FH and hippocampus-related memory and mood impairment. We here asked whether hippocampal pathology in FH might be a consequence of compromised adult hippocampal neurogenesis. We evaluated hippocampus-dependent behaviour and neurogenesis in adult C57BL/6JRj and LDLr-/- mice. Behavioural tests revealed that adult LDLr-/- mice showed reduced performance in a dentate gyrus (DG)-dependent metric change task. This phenotype was accompanied by a reduction in cell proliferation and adult neurogenesis in the DG of LDLr-/- mice, suggesting a potential direct impact of LDLr mutation on NPC. We thus investigated the effects of elevated cholesterol and the function of LDLr in neural precursor cells (NPC) isolated from adult C57BL/6JRj mice in vitro. Exposure of NPC to LDL as well as LDLr gene knockdown reduced proliferation and disrupted transcriptional activity of genes involved in endogenous cholesterol synthesis and metabolism. The LDL treatment also induced an increase in intracellular lipid storage. Functional analysis of differentially expressed genes revealed parallel modulation of distinct regulatory networks upon LDL treatment and LDLr knockdown. Together, these results suggest that high LDL levels and a loss of LDLr function, which are characteristic to individuals with FH, might contribute to a disease-related impairment in adult hippocampal neurogenesis and, consequently, cognitive functions.
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2023-09-06 | GSE227578 | GEO
Project description:In familial hypercholesterolemia (FH), mutations in the low-density lipoprotein (LDL) receptor (LDLr) gene result in increased plasma LDL cholesterol. Clinical and preclinical studies revealed an association between FH and hippocampus-related memory and mood impairment. We here asked whether hippocampal pathology in FH might be a consequence of compromised adult hippocampal neurogenesis. We evaluated hippocampus-dependent behaviour and neurogenesis in adult C57BL/6JRj and LDLr-/- mice. Behavioural tests revealed that adult LDLr-/- mice showed reduced performance in a dentate gyrus (DG)-dependent metric change task. This phenotype was accompanied by a reduction in cell proliferation and adult neurogenesis in the DG of LDLr-/- mice, suggesting a potential direct impact of LDLr mutation on NPC. We thus investigated the effects of elevated cholesterol and the function of LDLr in neural precursor cells (NPC) isolated from adult C57BL/6JRj mice in vitro. Exposure of NPC to LDL as well as LDLr gene knockdown reduced proliferation and disrupted transcriptional activity of genes involved in endogenous cholesterol synthesis and metabolism. The LDL treatment also induced an increase in intracellular lipid storage. Functional analysis of differentially expressed genes revealed parallel modulation of distinct regulatory networks upon LDL treatment and LDLr knockdown. Together, these results suggest that high LDL levels and a loss of LDLr function, which are characteristic to individuals with FH, might contribute to a disease-related impairment in adult hippocampal neurogenesis and, consequently, cognitive functions.
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Project description:The ATP-binding cassette transporter ABCC4 regulates cAMP membrane microdomain formation after adenylate cyclase activation but how ABCC4 localizes to these membrane microdomains are still unclear. Herein we demonstrate that the assembly and spatiotemporal organization of ABCC4’s protein neighborhood by PKA requires its PDZ motif and PKA activation. Density gradient ultracentrifugation revealed the formation of ABCC4 PDZ motif–specific, large protein complex upon PKA activation. To elucidate how PKA ushers ABCC4 to form this membrane supercomplex we identified proteins that closely interact with ABCC4 and proteins in ABCC4’s “neighborhood” network using Avi-tagged or APEX-tagged ABCC4 constructs, and their deletion mutants without the PDZ motif. We found that ABCC4 interacts with numerous PDZ domain containing proteins (PDZ proteins), and the PKA-induced enrichment of actin filament-binding proteins in the vicinity of ABCC4 requires the PDZ motif. The assembly of the supercomplex was crucially dependent on the actin-binding protein Moesin (MSN), which also interacts with the PDZ motif. This supercomplex can be disrupted by a specific potent inhibitor of ABCC4. These findings indicate that PKA activation promotes ABCC4 membrane localization by enhancing the assembly of a protein scaffold through PDZ proteins, MSN and actin cytoskeletal proteins.
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Project description:The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.
2024-01-05 | GSE234560 | GEO
Project description:The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.
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