Endothelial senescence is induced by phosphorylation and nuclear export of telomeric repeat binding factor 2–interacting protein
Sivareddy Kotla, … , Nhat-Tu Le, Jun-ichi Abe
Sivareddy Kotla, … , Nhat-Tu Le, Jun-ichi Abe
Published May 2, 2019
Citation Information: JCI Insight. 2019;4(9):e124867. https://doi.org/10.1172/jci.insight.124867.
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Research Article Cardiology Vascular biology

Endothelial senescence is induced by phosphorylation and nuclear export of telomeric repeat binding factor 2–interacting protein

Abstract

The interplay among signaling events for endothelial cell (EC) senescence, apoptosis, and activation and how these pathological conditions promote atherosclerosis in the area exposed to disturbed flow (d-flow) in concert remain unclear. The aim of this study was to determine whether telomeric repeat-binding factor 2–interacting protein (TERF2IP), a member of the shelterin complex at the telomere, can regulate EC senescence, apoptosis, and activation simultaneously, and if so, by what molecular mechanisms. We found that d-flow induced p90RSK and TERF2IP interaction in a p90RSK kinase activity–dependent manner. An in vitro kinase assay revealed that p90RSK directly phosphorylated TERF2IP at the serine 205 (S205) residue, and d-flow increased TERF2IP S205 phosphorylation as well as EC senescence, apoptosis, and activation by activating p90RSK. TERF2IP phosphorylation was crucial for nuclear export of the TERF2IP-TRF2 complex, which led to EC activation by cytosolic TERF2IP-mediated NF-κB activation and also to senescence and apoptosis of ECs by depleting TRF2 from the nucleus. Lastly, using EC-specific TERF2IP-knockout (TERF2IP-KO) mice, we found that the depletion of TERF2IP inhibited d-flow–induced EC senescence, apoptosis, and activation, as well as atherosclerotic plaque formation. These findings demonstrate that TERF2IP is an important molecular switch that simultaneously accelerates EC senescence, apoptosis, and activation by S205 phosphorylation.

Authors

Sivareddy Kotla, Hang Thi Vu, Kyung Ae Ko, Yin Wang, Masaki Imanishi, Kyung-Sun Heo, Yuka Fujii, Tamlyn N. Thomas, Young Jin Gi, Hira Mazhar, Jesus Paez-Mayorga, Ji-Hyun Shin, Yunting Tao, Carolyn J. Giancursio, Jan L.M. Medina, Jack Taunton, Aldos J. Lusis, John P. Cooke, Keigi Fujiwara, Nhat-Tu Le, Jun-ichi Abe

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Figure 4

TERF2IP S205 phosphorylation plays a crucial role in EC senescence.

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TERF2IP S205 phosphorylation plays a crucial role in EC senescence. (A–C...
(A–C) Human aortic endothelial cells (HAECs) were incubated with TERF2IP or control siRNA for 48 hours and transduced by adenovirus containing p90RSK (Ad-WT-p90RSK) or GFP (Ad-GFP) for 24 hours. (A) EC senescence was evaluated by SA–β-gal staining. Scale bars: 100 μm. (B) Reduced expression of TERF2IP after siRNA transfection and expression levels of exogenous p90RSK were detected by Western blotting. (C) The percentages of cells positive for SA–β-gal staining are shown. More than 200 cells/sample were counted. The data represent mean ± SD, n = 3. (D–F) HUVECs were transduced by Ad-TERF2IP WT or Ad-TERF2IP S205A mutant for 24 hours and then exposed to d-flow for 16 hours. (D) Cell senescence was evaluated by SA–β-gal staining and quantified as in C. The data represent mean ± SD, n = 3. (E) Expression of TERF2IP WT and S205A and β-actin as determined by Western blotting. (F) Graph shows fold increase of annexin V–positive cells analyzed by flow cytometry among Ad-TERF2IP WT–transduced HUVECs exposed to d-flow (24 hours) compared with similarly treated cells expressing the Ad-TERF2IP S205A mutant. The data represent mean ± SD, n = 3–5. (G) Annexin V–positive cells among Ad-TERF2IP WT– and -S205D–transfected HUVECs were detected. The data represent mean ± SD, n = 3–5. (H and I) HUVECs were transduced with Ad-TERF2IP WT or Ad-TERF2IP S205A for 18 hours and then stimulated by d-flow or no flow for 36 hours, followed by TUNEL staining. DAPI staining was used to identify nuclei. Quantification of apoptosis is shown as the percentage of TUNEL-positive cells (I). More than 200 cells were counted for each category. Data represent mean ± SD, n = 5, **P < 0.01, *P < 0.05. All statistical analyses in this figure were done by 1-way ANOVA followed by Bonferroni post hoc test.