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 2

p90RSK associates with TERF2IP, and the p90RSK-TERF2IP Myb domain interaction has a critical role in regulating NF-κB activation.

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p90RSK associates with TERF2IP, and the p90RSK-TERF2IP Myb domain intera...
(A) HUVECs were exposed to d-flow for various lengths of time, as indicated, and cell lysates were immunoprecipitated with anti-TERF2IP or control IgG and immunoblotted with anti-p90RSK. Total cell lysates were immunoblotted with the indicated antibodies. Upper panel shows representative data from 3 independent experiments. Lower panel shows densitometric quantification of coimmunoprecipitated p90RSK with TERF2IP, which was normalized by total TERF2IP protein levels. Data represent mean ± SD, n = 3, **P < 0.01. (B) HUVECs were pretreated with FMK-MEA and exposed to d-flow for 10 minutes. TERF2IP and p90RSK binding was detected as described in A. Data represent mean ± SD, n = 3, **P < 0.01. Quantification analysis was also performed (lower panel). (C–E) HUVECs were transfected with plasmids containing Gal4-p90RSK WT, VP16-TERF2IP-FL (full length) (C), VP16-TERF2IP Myb fragment (D), Flag-tagged TEERF2IP Myb fragment (Flag-Myb) (E), or empty vector and Gal4-responsive luciferase reporter pG5-luc for 24 hours. Cells were stimulated with or without TNF for 2 hours (C), and luciferase activities were quantified. Data represent mean ± SD, n = 6–12. (F) HUVECs were transfected with plasmids containing WT p90RSK, VP16-TERF2IP Myb fragment, or VP16-empty vector (Cont) with the NF-κB activity reporter gene. After 24 hours of transfection, NF-κB activity was detected by a luciferase assay. Data represent mean ± SD, n = 5. All statistical analyses in this figure were done by 1-way ANOVA followed by Bonferroni post hoc test.