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 1

Expression of DNA damage response elements 8-oxo-2’-deoxyguanosine (8-oxo-dG) and γ-H2AX and apoptosis are upregulated by d-flow and inhibited by Ad-KD-p90RSK transduction and deletion of TERF2IP.

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Expression of DNA damage response elements 8-oxo-2’-deoxyguanosine (8-ox...
(A and B) Flow cytometric analysis of 8-oxo-dG staining in bovine aortic endothelial cells (BAECs) or Human umblical vein endothelial cells (HUVEC) exposed to d-flow (8 hours). Graphs in A (BAEC) and B (HUVEC) show percentages of 8-oxo-dG–positive BAECs transduced with Ad-LacZ or Ad-KD-p90RSK and exposed to d-flow (8 hours). Error bars represent mean ± SD, n = 3–5, **P < 0.01. (C) Flow cytometric analysis of TL shortening in HUVECs exposed to d-flow (24 hours). HUVECs were transduced by Ad-KD-p90RSK or Ad-LacZ, and TL length was assayed using the TL PNA kit/FITC for flow cytometry. The mean fluorescence intensity was used to estimate the relative TL length. Data were analyzed using FlowJo software. Error bars represent mean ± SD, n = 3. (D) HUVECs were transfected with TERF2IP or control siRNA and exposed to d-flow. Apoptosis was detected by Western blotting with anti–cleaved caspase 3. Images shown are representative of 3 independent experiments. (E) Quantification of the cleaved caspase 3 level is shown after normalization by total protein levels. Data represent mean ± SD, n = 3–4. (F) Percentage of annexin V–positive HUVECs exposed to d-flow (24 hours) after transfection with control siRNA (siCont) and siTERF2IP was determined by flow cytometry. Data represent mean ± SD, n = 3–4, *P < 0.05, **P < 0.01. (G–J) HUVECs were transfected with TERF2IP or control siRNA and then stimulated by d-flow or no flow for 24 hours (G, H, and I) or 8 hours (J), followed by TUNEL (G), γ-H2AX (H), or 8-oxo-dG (I) staining and TL length (J) measurements, as described above. Data represent the mean ± SD, n = 3–5, *P < 0.05, **P < 0.01. All statistical analyses in this figure were done by 1-way ANOVA followed by Bonferroni post hoc test.