Endothelial senescence is induced by phosphorylation and nuclear export of telomeric repeat binding factor 2–interacting protein
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
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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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 8

Depletion of TERF2IP inhibits EC senescence and activation in vitro.

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Depletion of TERF2IP inhibits EC senescence and activation in vitro. (A–...
(A–C) Mouse lung endothelial cells (MLECs) were isolated from WT or Terf2iphomo–EC-specific KO (EKO) mice. After treatment of cells with d-flow or static conditions for 12 hours, protein expression was analyzed by Western blotting using specific antibodies as indicated A, and the level of each protein expression under different conditions was quantified B. Data represent mean ± SD, n = 3. (C) Cell apoptosis, TL length, and NF-κB activity were quantified. Data represent mean ± SD, n = 3–6, **P < 0.01. (D) MLECs were isolated from WT or Terf2iphomo–EKO (TERF2IP-EKO) mice. After transduction of Ad-p90RSK WT or Ad-GFP as a control for 12 hours, Western blotting was performed using specific antibodies as indicated. (E) Graphs represent densitometry data of immunoblots from 3 independent experiments. Data represent mean ± SD. (F) Left and middle panels show that, after transduction with Ad-p90RSK-WT or Ad-GFP as a control for 12 hours, MLECs were assayed and quantified for apoptosis by FITC annexin V staining (left) and for telomere lengths (middle). Data represent mean ± SD, n = 6. Right panel shows that MLECs were transfected with the NF-κB reporter gene for 24 hours, followed by transduction with Ad-p90RSK WT or Ad-GFP as a control for 12 hours. NF-κB activity was assayed using the dual-luciferase reporter assay. Data represent mean ± SD, n = 6, **P < 0.01. All statistical analyses in this figure were done by 1-way ANOVA followed by Bonferroni post hoc test.