Abstract
Autophagy plays critical roles in the maintenance of endothelial cells in response to cellular stress caused by blood flow. There is growing evidence that both cell adhesion and cell detachment can modulate autophagy, but the mechanisms responsible for this regulation remain unclear. Immunoglobulin and proline-rich receptor-1 (IGPR-1) is a cell adhesion molecule that regulates angiogenesis and endothelial barrier function. In this study, using various biochemical and cellular assays, we demonstrate that IGPR-1 is activated by autophagy-inducing stimuli, such as amino acid starvation, nutrient deprivation, rapamycin, and lipopolysaccharide. Manipulating the IκB kinase β activity coupled with in vivo and in vitro kinase assays demonstrated that IκB kinase β is a key serine/threonine kinase activated by autophagy stimuli and that it catalyzes phosphorylation of IGPR-1 at Ser220. The subsequent activation of IGPR-1, in turn, stimulates phosphorylation of AMP-activated protein kinase, which leads to phosphorylation of the major pro-autophagy proteins ULK1 and Beclin-1 (BECN1), increased LC3-II levels, and accumulation of LC3 punctum. Thus, our data demonstrate that IGPR-1 is activated by autophagy-inducing stimuli and in response regulates autophagy, connecting cell adhesion to autophagy. These findings may have important significance for autophagy-driven pathologies such cardiovascular diseases and cancer and suggest that IGPR-1 may serve as a promising therapeutic target.
Highlights
Autophagy, the lysosomal degradation of cytoplasmic organelles or cytosolic components, is an evolutionarily conserved cytoprotective mechanism that is induced in response to cellular stress, such as nutrient withdrawal, loss of cell adhesion, and flow shear stress, or by therapeutic genotoxic agents and others [1,2,3,4]
Immunoglobulin and proline-rich receptor-1 (IGPR-1) is activated by and regulates autophagy as doxorubicin [17] and flow shear stress [18] simulate phosphorylation of IGPR-1at Ser220. Because both shear stress and genotoxic agents are well-known for their roles in autophagy, we asked whether IGPR-1 is activated in response to autophagy
We examined the role of IGPR-1 in the autophagosome formation by measuring expression of LC3-phosphatidylethanolamine conjugate (LC3-II) and p62 endogenously expressed in human embryonic kidney epithelial-293 (HEK-293) cells, which are required for autophagosome development during autophagy [34], by Western blotting analysis
Summary
Autophagy ( called macroautophagy), the lysosomal degradation of cytoplasmic organelles or cytosolic components, is an evolutionarily conserved cytoprotective mechanism that is induced in response to cellular stress, such as nutrient withdrawal, loss of cell adhesion, and flow shear stress, or by therapeutic genotoxic agents and others [1,2,3,4]. Known autophagy-inducing conditions or agents such as nutrient withdrawal including amino acid and serum starvation, immunosuppressant rapamycin/Sirolimus, and LPS all activate several key kinases such as the IkB kinase (IKK) complex [1]. IGPR-1 responds to various cellular stresses, because its phosphorylation (i.e. Ser220) is significantly increased by flow shear stress [18] and exposure to doxorubicin [17, 18] Both shear stress [19] and doxorubicin [20, 21] are well-known potent inducers of autophagy, raising a possibility for the involvement of IGPR-1 in autophagy. We demonstrate that upon the induction of autophagy, IGPR-1 is phosphorylated at Ser220 via a mechanism that involves activation of IKKb. IKKbdependent phosphorylation of IGPR-1 stimulates phosphorylation of AMPK, leading to activation of BECN1 and ULK1, connecting cell adhesion and energy sensing to autophagy
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