FLRT2 prevents endothelial cell senescence and vascular aging by regulating the ITGB4/mTORC2/p53 signaling pathway
Hyun Jung Hwang, Donghee Kang, Jae-Ryong Kim, Joon Hyuk Choi, Ji-Kan Ryu, Allison B. Herman, Young-Gyu Ko, Heon Joo Park, Myriam Gorospe, Jae-Seon Lee
Hyun Jung Hwang, Donghee Kang, Jae-Ryong Kim, Joon Hyuk Choi, Ji-Kan Ryu, Allison B. Herman, Young-Gyu Ko, Heon Joo Park, Myriam Gorospe, Jae-Seon Lee
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Research Article Cell biology Vascular biology

FLRT2 prevents endothelial cell senescence and vascular aging by regulating the ITGB4/mTORC2/p53 signaling pathway

Abstract

The roles of fibronectin leucine-rich transmembrane protein 2 (FLRT2) in physiological and pathological processes are not well known. Here, we identify a potentially novel function of FLRT2 in preventing endothelial cell senescence and vascular aging. We found that FLRT2 expression was lower in cultured senescent endothelial cells as well as in aged rat and human vascular tissues. FLRT2 mediated endothelial cell senescence via the mTOR complex 2, AKT, and p53 signaling pathway in human endothelial cells. We uncovered that FLRT2 directly associated with integrin subunit beta 4 (ITGB4) and thereby promoted ITGB4 phosphorylation, while inhibition of ITGB4 substantially mitigated the induction of senescence triggered by FLRT2 depletion. Importantly, FLRT2 silencing in mice promoted vascular aging, and overexpression of FLRT2 rescued a premature vascular aging phenotype. Therefore, we propose that FLRT2 could be targeted therapeutically to prevent senescence-associated vascular aging.

Authors

Hyun Jung Hwang, Donghee Kang, Jae-Ryong Kim, Joon Hyuk Choi, Ji-Kan Ryu, Allison B. Herman, Young-Gyu Ko, Heon Joo Park, Myriam Gorospe, Jae-Seon Lee

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Figure 4

Signaling through ITGB4, mTORC2, and AKT is required for a senescence program triggered by FLRT2 depletion.

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Signaling through ITGB4, mTORC2, and AKT is required for a senescence pr...
(A and B) HUVECs were transfected with Con Si, ITGB1 Si, ITGB3 Si, ITGB4 Si, or ITGB5 Si (first transfection) 6 hours before transfection with Con Si or FLRT2 Si (second transfection). At day 2 after transfection, cells were harvested and subjected to immunoblotting (A). At day 3 after transfection, SA-β-Gal activity was examined (B). (C) HUVECs were transfected with Con Si or FLRT2 Si, and reverse transcription (RT) followed by real-time quantitative polymerase chain reaction (qPCR) (RT-qPCR) analysis was carried out 2 days after transfection. (D and E) HUVECs were transfected with Con Si or FLRT2 Si, followed by treatment with an inhibitor of ITGB4 (ASC-8). Immunoblot assay (D) and SA-β-Gal assay (E) were performed at days 2 and 3 after transfection, respectively. Scale bar: 10 μm. (F) HUVECs were transfected with Con Si or ITGB4 Si 1 day before transfection with empty vector (EV), a vector encoding wild-type ITGB4 (WT), or a vector encoding a truncated mutant of ITGB4 lacking the residues downstream of amino acid 1355 (ΔCYT), followed by transfection with Con Si or FLRT2 Si. At day 2 after transfection, the cells were subjected to immunoblotting analysis. (G) HUVECs were transfected with Con Si or ITGB4 Si. At 6 hours after transfection, one-half of each group was transfected with Con Si, while the other half was transfected with FLRT2 Si. At day 2 after transfection, cell lysates were subjected to immunoprecipitation with anti-Rictor antibody. Mean ± SD (n = 3; #P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001). One-way ANOVA.