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 3

p90RSK phosphorylates TERF2IP S205 and increases NF-κB activation.

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p90RSK phosphorylates TERF2IP S205 and increases NF-κB activation. (A an...
(A and B) After 24 hours of transfection of NF-κB activity reporter gene and either TERF2IP WT or an S205A mutant treated with and without p90RSK or LacZ as a control (A), or S205D (B) mutant with and without d-flow, NF-κB activity was detected by luciferase assay. Expression of TERF2IP WT, TERF2IP-S205D, and tubulin was determined by Western blotting and with and without d-flow (B, left panel). Data represent mean ± SD, n = 5, **P < 0.01. (C and D) Protein expression of p-TERF2IP-S205, TERF2IP, and tubulin (loading control) (C) and Flag-KD-p90RSK (D) in lysates of HUVECs transduced with Ad-Flag-TERF2IP WT or -S205A mutant (C) or KD-p90RSK (D) and exposed to H2O2 (200 μM) (C) or d-flow (D) was assessed by Western blotting. Graph (E) shows densitometric quantification of phosphorylated TERF2IP-S205, which was normalized by total TERF2IP protein levels. Shown are representative data from 3 independent experiments. Data represent mean ± SD, n = 3–5, **P < 0.01. (F) An in vitro kinase assay was performed using immunoprecipitated TERF2IP in the absence or presence of recombinant p90RSK followed by immunoblotting with anti–p-TERF2IP (S205) (upper panel). The same amount of recombinant TERF2IP was immunoprecipitated by anti-TERF2IP, as indicated by anti-TERF2IP immunoblotting (lower panel). (G) Graph shows densitometric quantification of phosphorylated TERF2IP-S205, which was normalized by total TERF2IP protein levels. Data represent mean ± SD, n = 3, **P < 0.01. Statistical analyses in A–F were done by 1-way ANOVA followed by Bonferroni post hoc test, and statistical analysis in G was performed by unpaired 2-tailed t test.