Crosstalk between the Akt/mTORC1 and NF-κB signaling pathways promotes hypoxia-induced pulmonary hypertension by increasing DPP4 expression in PASMCs

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Abstract

Abnormal wound healing by pulmonary artery smooth muscle cells (PASMCs) promotes vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Increasing evidence shows that both the mammalian target of rapamycin complex 1 (mTORC1) and nuclear factor-kappa B (NF-κB) are involved in the development of HPH. In this study, we explored the crosstalk between mTORC1 and NF-κB in PASMCs cultured under hypoxic condition and in a rat model of hypoxia-induced pulmonary hypertension (HPH). We showed that hypoxia promoted wound healing of PASMCs, which was dose-dependently blocked by the mTORC1 inhibitor rapamycin (5−20 nM). In PASMCs, hypoxia activated mTORC1, which in turn promoted the phosphorylation of NF-κB. Molecular docking revealed that mTOR interacted with IκB kinases (IKKs) and that was validated by immunoprecipitation. In vitro kinase assays and mass spectrometry demonstrated that mTOR phosphorylated IKKα and IKKβ separately. Inhibition of mTORC1 decreased the level of phosphorylated IKKα/β, thus reducing the phosphorylation and transcriptional activity of NF-κB. Bioinformatics study revealed that dipeptidyl peptidase-4 (DPP4) was a target gene of NF-κB; DPP4 inhibitor, sitagliptin (10−500 μM) effectively inhibited the abnormal wound healing of PASMCs under hypoxic condition. In the rat model of HPH, we showed that NF-κB activation (at 3 weeks) was preceded by mTOR signaling activation (after 1 or 2 weeks) in lungs, and administration of sitagliptin (1−5 mg/kg every day, ig) produced preventive effects against the development of HPH. In conclusion, hypoxia activates the crosstalk between mTORC1 and NF-κB, and increased DPP4 expression in PASMCs that leads to vascular remodeling. Sitagliptin, a DPP4 inhibitor, exerts preventive effect against HPH.

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References

  1. 1.

    Pugliese SC, Poth JM, Fini MA, Olschewski A, El Kasmi KC, Stenmark KR. The role of inflammation in hypoxic pulmonary hypertension: from cellular mechanisms to clinical phenotypes. Am J Physiol Lung Cell Mol Physiol. 2015;308:L229–52.

  2. 2.

    Voelkel NF, Tamosiuniene R, Nicolls MR. Challenges and opportunities in treating inflammation associated with pulmonary hypertension. Expert Rev Cardiovasc Ther. 2016;14:939–51.

  3. 3.

    Paulin R, Meloche J, Courboulin A, Lambert C, Haromy A, Courchesne A, et al. Targeting cell motility in pulmonary arterial hypertension. Eur Respir J. 2014;43:531–44.

  4. 4.

    Wang AP, Li XH, Yang YM, Li WQ, Zhang W, Hu CP, et al. A critical role of the mTOR/eIF2alpha pathway in hypoxia-induced pulmonary hypertension. PLoS ONE. 2015;10:e0130806.

  5. 5.

    Ma X, Yao J, Yue Y, Du S, Qin H, Hou J, et al. Rapamycin reduced pulmonary vascular remodelling by inhibiting cell proliferation via Akt/mTOR signalling pathway down-regulation in the carotid artery-jugular vein shunt pulmonary hypertension rat model. Interact Cardiovasc Thorac Surg. 2017;25:206–11.

  6. 6.

    Houssaini A, Abid S, Mouraret N, Wan F, Rideau D, Saker M, et al. Rapamycin reverses pulmonary artery smooth muscle cell proliferation in pulmonary hypertension. Am J Respir Cell Mol Biol. 2013;48:568–77.

  7. 7.

    Kudryashova TV, Goncharov DA, Pena A, Ihida-Stansbury K, DeLisser H, Kawut SM, et al. Profiling the role of mammalian target of rapamycin in the vascular smooth muscle metabolome in pulmonary arterial hypertension. Pulm Circ. 2015;5:667–80.

  8. 8.

    Ruygrok PN, Muller DW, Serruys PW. Rapamycin in cardiovascular medicine. Intern Med J. 2003;33:103–9.

  9. 9.

    Bee J, Fuller S, Miller S, Johnson SR. Lung function response and side effects to rapamycin for lymphangioleiomyomatosis: a prospective national cohort study. Thorax. 2018;73:369–75.

  10. 10.

    Fan J, Fan X, Li Y, Ding L, Zheng Q, Guo J, et al. Chronic normobaric hypoxia induces pulmonary hypertension in rats: role of NF-kappaB. High Alt Med Biol. 2016;17:43–9.

  11. 11.

    Farkas D, Alhussaini AA, Kraskauskas D, Kraskauskiene V, Cool CD, Nicolls MR, et al. Nuclear factor kappaB inhibition reduces lung vascular lumen obliteration in severe pulmonary hypertension in rats. Am J Respir Cell Mol Biol. 2014;51:413–25.

  12. 12.

    Zhu R, Bi L, Kong H, Xie W, Hong Y, Wang H. Ruscogenin exerts beneficial effects on monocrotaline-induced pulmonary hypertension by inhibiting NF-kappaB expression. Int J Clin Exp Pathol. 2015;8:12169–76.

  13. 13.

    Price LC, Caramori G, Perros F, Meng C, Gambaryan N, Dorfmuller P, et al. Nuclear factor kappa-B is activated in the pulmonary vessels of patients with end-stage idiopathic pulmonary arterial hypertension. PLoS ONE. 2013;8:e75415.

  14. 14.

    Luo Y, Xu DQ, Dong HY, Zhang B, Liu Y, Niu W, et al. Tanshinone IIA inhibits hypoxia-induced pulmonary artery smooth muscle cell proliferation via Akt/Skp2/p27-associated pathway. PLoS ONE. 2013;8:e56774.

  15. 15.

    Deng L, Blanco FJ, Stevens H, Lu R, Caudrillier A, McBride M, et al. MicroRNA-143 activation regulates smooth muscle and endothelial cell crosstalk in pulmonary arterial hypertension. Circ Res. 2015;117:870–83.

  16. 16.

    Yang H, Rudge DG, Koos JD, Vaidialingam B, Yang HJ, Pavletich NP. mTOR kinase structure, mechanism and regulation. Nature. 2013;497:217–23.

  17. 17.

    Liu S, Misquitta YR, Olland A, Johnson MA, Kelleher KS, Kriz R, et al. Crystal structure of a human IkappaB kinase beta asymmetric dimer. J Biol Chem. 2013;288:22758–67.

  18. 18.

    Moss BL, Gross S, Gammon ST, Vinjamoori A, Piwnica-Worms D. Identification of a ligand-induced transient refractory period in nuclear factor-kappaB signaling. J Biol Chem. 2008;283:8687–98.

  19. 19.

    Li Y, Yang Z, Li W, Xu S, Wang T, Wang T, et al. TOPK promotes lung cancer resistance to EGFR tyrosine kinase inhibitors by phosphorylating and activating c-Jun. Oncotarget. 2016;7:6748–64.

  20. 20.

    Xu S, Wang T, Yang Z, Li Y, Li W, Wang T, et al. miR-26a desensitizes non-small cell lung cancer cells to tyrosine kinase inhibitors by targeting PTPN13. Oncotarget. 2016. https://doi.org/10.18632/oncotarget.9920.

  21. 21.

    Webb B, Sali A. Comparative protein structure modeling using MODELLER. Curr Protoc Bioinforma / Ed board. 2014;47:5.6.1–5.6.32.

  22. 22.

    Pronk S, Pall S, Schulz R, Larsson P, Bjelkmar P, Apostolov R, et al. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics. 2013;29:845–54.

  23. 23.

    Brooks BR, Brooks CL 3rd, Mackerell AD Jr, Nilsson L, Petrella RJ, Roux B, et al. CHARMM: the biomolecular simulation program. J Comput Chem. 2009;30:1545–614.

  24. 24.

    Chen R, Li L, Weng Z. ZDOCK: an initial-stage protein-docking algorithm. Proteins. 2003;52:80–7.

  25. 25.

    Li L, Chen R, Weng Z. RDOCK: refinement of rigid-body protein docking predictions. Proteins. 2003;53:693–707.

  26. 26.

    Yang Z, Yang G, Zhou L. Mutation effects of neuraminidases and their docking with ligands: a molecular dynamics and free energy calculation study. J Comput Aided Mol Des. 2013;27:935–50.

  27. 27.

    Yang Z, Wu F, Yuan X, Zhang L, Zhang S. Novel binding patterns between ganoderic acids and neuraminidase: Insights from docking, molecular dynamics and MM/PBSA studies. J Mol Graph Model. 2016;65:27–34.

  28. 28.

    Accelrys. Discovery Studio 3.1. 2011. http://accelrys.com. Accessed 25 Jul 2013.

  29. 29.

    Park J-W, Kim CU, Isard W. Permit allocation in emissions trading using the Boltzmann distribution. Phys A. 2012;391:4883–90.

  30. 30.

    Krymskaya VP, Snow J, Cesarone G, Khavin I, Goncharov DA, Lim PN, et al. mTOR is required for pulmonary arterial vascular smooth muscle cell proliferation under chronic hypoxia. FASEB J. 2011;25:1922–33.

  31. 31.

    Samokhin AO, Stephens T, Wertheim BM, Wang RS, Vargas SO, Yung LM, et al. NEDD9 targets COL3A1 to promote endothelial fibrosis and pulmonary arterial hypertension. Sci Transl Med. 2018;10:eaap7294.

  32. 32.

    Jones MR, Liu C, Wilson AK. Molecular dynamics studies of the protein-protein interactions in inhibitor of kappaB kinase-beta. J Chem Inf Model. 2014;54:562–72.

  33. 33.

    Park H, Shin Y, Choe H, Hong S. Computational design and discovery of nanomolar inhibitors of IkappaB kinase beta. J Am Chem Soc. 2015;137:337–48.

  34. 34.

    Sapienza PJ, Mauldin RV, Lee AL. Multi-timescale dynamics study of FKBP12 along the rapamycin-mTOR binding coordinate. J Mol Biol. 2011;405:378–94.

  35. 35.

    Ghate M, Jain SV. Structure based lead optimization approach in discovery of selective DPP4 inhibitors. Mini Rev Med Chem. 2013;13:888–914.

  36. 36.

    Rohrborn D, Wronkowitz N, Eckel J. DPP4 in diabetes. Front Immunol. 2015;6:386.

  37. 37.

    Suresh PS, Srinivas NR, Mullangi R. A concise review of the bioanalytical methods for the quantitation of sitagliptin, an important dipeptidyl peptidase-4 (DPP4) inhibitor, utilized for the characterization of the drug. Biomed Chromatogr. 2016;30:749–71.

  38. 38.

    Aghamohammadzadeh R, Zhang YY, Stephens TE, Arons E, Zaman P, Polach KJ, et al. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth muscle cell survival patterns to promote pulmonary arterial hypertension. FASEB J. 2016;30:2511–27.

  39. 39.

    Liu ZQ, Liu B, Yu L, Wang XQ, Wang J, Liu HM. Simvastatin has beneficial effect on pulmonary artery hypertension by inhibiting NF-kappaB expression. Mol Cell Biochem. 2011;354:77–82.

  40. 40.

    Liu M, Yu P, Jiang H, Yang X, Zhao J, Zou Y, et al. The essential role of Pin1 via NF-kappaB signaling in vascular inflammation and atherosclerosis in ApoE-/- Mice. Int J Mol Sci. 2017;18:E644.

  41. 41.

    Huang M, Zeng S, Zou Y, Shi M, Qiu Q, Xiao Y, et al. The suppression of bromodomain and extra-terminal domain inhibits vascular inflammation by blocking NF-kappaB and MAPK activation. Br J Pharmacol. 2017;174:101–15.

  42. 42.

    Wang YL, Sun GY, Zhang Y, He JJ, Zheng S, Lin JN. Tormentic acid inhibits H2O2-induced oxidative stress and inflammation in rat vascular smooth muscle cells via inhibition of the NF-kappaB signaling pathway. Mol Med Rep. 2016;14:3559–64.

  43. 43.

    Wang Z, Castresana MR, Newman WH. NF-kappaB is required for TNF-alpha-directed smooth muscle cell migration. FEBS Lett. 2001;508:360–4.

  44. 44.

    Chandrasekar B, Mummidi S, Mahimainathan L, Patel DN, Bailey SR, Imam SZ, et al. Interleukin-18-induced human coronary artery smooth muscle cell migration is dependent on NF-kappaB- and AP-1-mediated matrix metalloproteinase-9 expression and is inhibited by atorvastatin. J Biol Chem. 2006;281:15099–109.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Nos. 81670045, 81272586, and 81470249) and the Chinese Postdoctoral Science Foundation (No. 2014M560759).

Author information

SQL designed the experiments and prepared the initial manuscript. YL and LY conducted the experiments. LD, JZ and NZ cultivated the cells. ZWY and SLZ were responsible for the experimental calculations. JWX, YG, MJN, XJZ, YYZ, XMW, YZZ and PZ were involved in the experimental analysis. YL, LY, LD, ZWY and JZ contributed equally to this manuscript. SQL is the corresponding author.

Correspondence to Sheng-qing Li.

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Keywords

  • pulmonary hypertension
  • mTORC1
  • NF-κB
  • IκB kinase
  • DPP4
  • sitagliptin
  • pulmonary artery smooth muscle cells