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 7

TERF2IP S205 phosphorylation is required for nuclear export of the TERF2IP-TRF2 complex.

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TERF2IP S205 phosphorylation is required for nuclear export of the TERF2...
(A) HUVECs were exposed to d-flow for 0–90 min.; binding between TERF2IP and TRF2 was analyzed by co-IP with anti-TRF2, followed by Western blotting with anti-TERF2IP. Total TERF2IP in each sample is shown (bottom). Representative images from 3 independent experiments are shown. (B) Quantification of TERF2IP-TRF2 binding is shown after normalization to levels at 0 min. n = 3–4. (C) Reduced expression of TERF2IP and TRF2 following transfection with TERF2IP and TRF2 siRNA was detected by Western blotting. Representative images from 3 independent experiments are shown. (D) After 24 hours of transfection with the NF-κB reporter gene together with control or TRF2 siRNA, HUVECs were exposed to d-flow for 24 hours, and NF-κB activity was measured by the luciferase assay (left). TRF2 and tubulin expression were detected by Western blotting (right). n = 4. (E and F) FITC annexin V–positive cells (E) and TL length (F) in HUVECs transfected with TRF2 or control siRNA were determined after 24 hours of d-flow or static culture. (G and H) After 48 hours of TERF2IP or control siRNA transfection, HUVECs were exposed to d-flow for 4 hours and stained with anti-TRF2 and DAPI. (G) In ECs exposed to d-flow, anti-TRF2 staining became detectable in control cells but not siTERF2IP-treated cells. Scale bars: 50 μm. (H) Extranuclear localization of TRF2 (%) was calculated by counting more than 200 cells/sample for each group. n = 3. (I) After transduction with Ad-TERF2IP WT and -S205A, HUVECs were exposed to d-flow for 4 hours and stained with anti-TRF2 and DAPI. Note d-flow–induced translocation of TRF2 into the cytoplasm. Scale bars: 50 μm. (J) Extranuclear localization of TRF2 (%) was calculated by counting more than 200 cells/sample for each group. n = 3. Data represent mean of percentages ± SD. **P < 0.01, *P < 0.05. All statistical analyses were done by 1-way ANOVA followed by Bonferroni post hoc test. (K) Proposed scheme of the TERF2IP-TRF2 complex nuclear export. Nuclear TRF2 is critical for protecting TLs from shortening. TERF2IP can deliver TRF2 to the cytoplasm. TERF2IP S205 phosphorylation causes the TERF2IP-TRF2 complex to move out of the nucleus and induces TL shortening and subsequent apoptosis (middle 2 panels). Both depletion of TERF2IP and inhibition of TERF2IP S205 phosphorylation prevent TRF2 nuclear export and protect TLs (bottom 2 panels).