Dataset Information

Enhanced Autophagy in GAB1-Deficient Vascular Endothelial Cells Is Responsible for Atherosclerosis Progression.

ABSTRACT: Autophagy is a host machinery that controls cellular health. Dysfunction of autophagy is responsible for the pathogenesis of many human diseases that include atherosclerosis obliterans (ASO). Physiologically, host autophagy removes aging organelles and delays the formation of atherosclerotic plaque. However, in ischemia event, dysregulated autophagy can be induced to trigger autosis, leading to an inevitable cellular death. Grb2-associated binder 1 (GAB1) is a docking/scaffolding adaptor protein that regulates many cell processes including autophagy. Our study first reported that the protein expression of GAB1 significantly decreased in ASO. Mechanically, our results showed that inhibition of Akt (protein kinase B), the upstream of mTOR (mechanistic target of rapamycin), significantly enhanced autophagy by demonstrating the downregulation of p62/Sequestosome 1 expression and the upregulation of the ratio of LC3II/LC3I. Conversely, we found that the inhibition of ERK1/2 (extracellular signal-regulated kinases1/2), p38, and JNK (c-Jun N-terminal kinase) signaling pathway, respectively, significantly inhibited autophagy by demonstrating the upregulation of p62 expression and the downregulation of the ratio of LC3II/LC3I. Further, we demonstrated that knockdown of GAB1 significantly increased autophagy in HUVECs (human umbilical vein endothelial cells) via activation of MAPK (mitogen-activated protein kinase) pathways that include ERK1/2, p38, and JNK. Moreover, we found that knockdown of GAB1 profoundly inhibited HUVEC proliferation, migration, and tube formation. Taken together, this study first suggests that GAB1 is a key regulator of autophagy in HUVECs. Targeting GAB1 may serve as a potential strategy for the atherosclerosis treatment.

SUBMITTER: Qian X

PROVIDER: S-EPMC7877249 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Enhanced Autophagy in GAB1-Deficient Vascular Endothelial Cells Is Responsible for Atherosclerosis Progression.

Qian Xin X Wang Han H Wang Yuli Y Chen Jiaquan J Guo Xiangjiang X Deng Haoyu H

Frontiers in physiology 20210202

Autophagy is a host machinery that controls cellular health. Dysfunction of autophagy is responsible for the pathogenesis of many human diseases that include atherosclerosis obliterans (ASO). Physiologically, host autophagy removes aging organelles and delays the formation of atherosclerotic plaque. However, in ischemia event, dysregulated autophagy can be induced to trigger autosis, leading to an inevitable cellular death. Grb2-associated binder 1 (GAB1) is a docking/scaffolding adaptor protein ...[more]

PMID: 33584322

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Project description:RationaleThe NLRP3 (NLR [NOD-like receptor] family, pyrin domain containing 3) inflammasome is an important driver of atherosclerosis. Our previous study shows that chaperone-mediated autophagy (CMA), one of the main lysosomal degradative process, has a regulatory role in lipid metabolism of macrophages. However, whether the NLRP3 inflammasome is regulated by CMA, and the role of CMA in atherosclerosis remains unclear.ObjectiveTo determine the role of CMA in the regulation of NLRP3 inflammasome and atherosclerosis.Methods and resultsThe expression of CMA marker, LAMP-2A (lysosome-associated membrane protein type 2A), was first analyzed in ApoE-/- mouse aortas and human coronary atherosclerotic plaques, and a significant downregulation of LAMP-2A in advanced atherosclerosis in both mice and humans was observed. To selectively block CMA, we generated macrophage-specific conditional LAMP-2A knockout mouse strains in C57BL/6 mice and ApoE-/- mice. Deletion of macrophage LAMP-2A accelerated atherosclerotic lesion formation in the aortic root and the whole aorta in ApoE-/- mice. Mechanistically, LAMP-2A deficiency promoted NLRP3 inflammasome activation and subsequent release of mature IL (interleukin)-1β in macrophages and atherosclerotic plaques. Furthermore, gain-of-function studies verified that restoration of LAMP-2A levels in LAMP-2A-deficient macrophages greatly attenuated NLRP3 inflammasome activation. Importantly, we identified the NLRP3 protein as a CMA substrate and demonstrated that LAMP-2A deficiency did not affect the NLRP3 mRNA levels but hindered degradation of the NLRP3 protein through CMA pathway.ConclusionsCMA function becomes impaired during the progression of atherosclerosis, which increases NLRP3 inflammasome activation and secretion of IL-1β, promoting vascular inflammation and atherosclerosis progression. Our study unveils a new mechanism by which NLRP3 inflammasome is regulated in macrophages and atherosclerosis, thus providing a new insight into the role of autophagy-lysosomal pathway in atherosclerosis. Pharmacological activation of CMA may provide a novel therapeutic strategy for atherosclerosis and other NLRP3 inflammasome/IL-1β-driven diseases.

Project description:Ammonium tetrathiomolybdate (TTM) is a copper chelator in clinical trials for treatment of Wilson's disease, tumors and other diseases. In the current study, we innovatively discovered that TTM is a novel NRF2 activator and illustrated that autophagy contributed to TTM-induced NRF2 activation. We showed that TTM treatment promoted NRF2 nuclear translocation and upregulated transcription level of NRF2 target genes including HMOX1, GCLM, and SLC7A11 in vascular endothelial cells (HUVECs). Moreover, NRF2 deficiency directly hindered TTM-mediated antioxidative effects. Followingly, we revealed that overexpression of KEAP1, a negative regulator of NRF2, significantly repressed NRF2 activation induced by TTM. Further mutation analysis revealed that KEAP1 Cys151 is a major sensor responsible for TTM-initiated NRF2 signaling, suggesting that KEAP1 is involved in TTM-mediated NRF2 activation. Notably, we found that TTM can trigger autophagy as evidenced by accumulation of autophagosomes, elevation of LC3BI-II/I, increase of LC3 puncta and activation of AMPK/mTOR/ULK1 pathway. Autophagic flux assay indicated that TTM significantly enhanced autophagic flux in HUVECs. Inhibition of autophagy with knockout of autophagy key gene ATG5 resulted in suppression of TTM-induced NRF2 activation. TTM also induced phosphorylation of autophagy receptor SQSTM1 at Ser349, while SQSTM1-deficiency inhibited KEAP1 degradation and blocked NRF2 signaling pathway, suggesting that TTM-induced NRF2 activation is autophagy dependent. As the novel NRF2 activator, TTM protected against sodium arsenite (NaAsO2)-induced oxidative stress and cell death, while NRF2 deficiency weakened TTM antioxidative effects. Finally, we showed that autophagy-dependent NRF2 activation contributed to the protective effects of TTM against NaAsO2-induced oxidative injury, because of ATG5 or SQSTM1 knockout aggravated NaAsO2-induced elevation of HMOX1, cleaved PARP and γH2AX. Taken together, our findings highlight copper chelator TTM is a novel autophagy-dependent NRF2 activator and shed a new light on the cure for oxidative damage-related diseases.

Dataset Information

Enhanced Autophagy in GAB1-Deficient Vascular Endothelial Cells Is Responsible for Atherosclerosis Progression.

Publications

Enhanced Autophagy in GAB1-Deficient Vascular Endothelial Cells Is Responsible for Atherosclerosis Progression.

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