Shear stress regulateing the phosphorylation of endothelial nitric oxide synthase Ser633 and Ser1177 in human umbilical vein endothelial cells through Pim1/Akt pathway&nbsp;&nbsp;

DU Da-Peng; SUN Yu; ZHANG Min; WANG Han-Qin

doi:10.16098/j.issn.0529-1356.2023.05.007

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Acta Anatomica Sinica ›› 2023, Vol. 54 ›› Issue (5) : 546-552. DOI: 10.16098/j.issn.0529-1356.2023.05.007

Shear stress regulateing the phosphorylation of endothelial nitric oxide synthase Ser633 and Ser1177 in human umbilical vein endothelial cells through Pim1/Akt pathway

DU Da-peng^1,2SUN Yu²ZHANG Min² WANG Han-qin^1,2*

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Abstract

Objective To explore the effect of different modes of blood flow shear stress on the Pim1 expression in human umbilical vein endothelial cells (HUVECs) and the regulation of phosphorylation at Ser1177. and Ser633 of endothelial nitric oxide synthase (eNOS). Methods HUVECs were isolated from fresh human umbilical cord. The parallel plate flow chamber system was used to load 15 dyn/cm2 laminar shear stress (LSS) and (0.5 ± 4) dyn/cm2 oscillatory shear stress (OSS) on HUVECs. Western blotting was used to evaluate the protein levels of Pim1, phosphorylated Akt at Ser473, and phosphorylated eNOS at Ser633 and Ser1177. Small interfering RNA (siRNA) was used to knockdown of Pim1 and Akt. Results LSS obviously induced Pim1 protein expression in HUVECs (P<0.01) compared with OSS stimulation. LSS also significantly increased Akt phosphorylation at Ser473 and eNOS phosphorylation both at Ser633 and Ser1177 (P<0.05 or P<0.01). Pim1 silencing, or treatment with SMI-4a, an inhibitor of Pim1, abolished the above effects of LSS-stimulated HUVECs (P<0.05 or P<0.01). Knockdown of Akt also prevented the LSS-induced Akt phosphorylation at Ser473 and eNOS phosphorylation both at Ser1177 and Ser633 (P<0.05 or P<0.01). However, Akt knockdown did not alter the expression of Pim1 induced by LSS. Conclusion Shear stress can regulate the phosphorylation of eNOS Ser633 and Ser1177 in HUVECs through the Pim1/Akt signaling pathway.

Key words

Shear stress / Pim1 / Endothelial cell / Endothelial nitric oxide synthase / Western blotting / Human

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DU Da-peng SUN Yu ZHANG Min WANG Han-qin.

Shear stress regulateing the phosphorylation of endothelial nitric oxide synthase Ser633 and Ser1177 in human umbilical vein endothelial cells through Pim1/Akt pathway

[J]. Acta Anatomica Sinica. 2023, 54(5): 546-552 https://doi.org/10.16098/j.issn.0529-1356.2023.05.007

References

［1］Singh R, Tubbs RS. Histological verification of atherosclerosis due to bends and bifurcations in carotid arteries predicted by hemodynamic model ［J］. Vasc Bras, 2018, 17(4):280-289.

［2］Malek AM, Alper S L, Izumo S. Hemodynamic shear stress and its role in atherosclerosis ［J］. JAMA, 1999, 282(21):2035-2042.

［3］Fulton D, Ruan L, Sood SG, et al. Agonist-stimulated endothelial nitric oxide synthase activation and vascular relaxation. Role of eNOS phosphorylation at Tyr83 ［J］. Circ Res, 2008, 102(4):497-504.

［4］Michell BJ, Harris MB, Chen ZP, et al. Identification of regulatory sites of phosphorylation of the bovine endothelial nitric-oxide synthase at serine 617 and serine 635 ［J］. Biol Chem, 2002, 277(44):42344-42351.

［5］Ghimire K, Zaric J, Alday-Parejo B, et al. MAGI1 mediates enos activation and no production in endothelial cells in response to fluid shear stress ［J］. Cells, 2019, 8(5):388.

［6］Jin YJ, Chennupati R, Li R, et al. Protein kinase N2 mediates flow-induced endothelial NOS activation and vascular tone regulation ［J］. Clin Invest, 2021, 131(21):e145734.

［7］Fulton D,Gratton JP,McCabe T,et al. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt ［J］. Nature, 1999, 399(6736):597-601.

［8］Boo YC,Hwang J,Sykes M,et al. Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism ［J］. Am J Physiol Heart Circ Physiol, 2002, 283(5):H1819-H1828.

［9］Zippo A, De Robertis A, Bardelli M, et al. Identification of Flk-1 target genes in vasculogenesis: Pim-1 is required for endothelial and mural cell differentiation in vitro ［J］. Blood, 2004, 103(12):4536-4544.

［10］Li H, Liu F, Xu JJ. Upregulation of provirus integration site 1 moloney murine leukemia virus expression by ciliary neurotrophic factor and neuritin can promote the neurite regeneration of neuron-like cells［J］. Acta Anatomica Sinica, 2020, 51(2): 153-161. (in Chinese)

李贺，刘芳，许家军. 睫状神经营养因子及神经突起素上调莫洛尼小鼠白血病病毒前病毒插入位点1表达促进受损神经元样细胞突起再生［J］. 解剖学报, 2020, 51(2): 153-161.

［11］Chen M, Yi B, Zhu N, et al. Pim1 kinase promotes angiogenesis through phosphorylation of endothelial nitric oxide synthase at Ser-633 ［J］. Cardiovasc Res, 2016, 109(1):141-150.

［12］Lu Y, Xu ZD, Shen FY, et al. Propofol protects against TNF-α-induced blood-brain barrier disruption via the PIM-1/eNOS/NO pathway ［J］. Curr Neurovasc Res, 2020, 17(4):471-479.

［13］Zhang M, Sun Y, Tang PJ, et al. Shear stress regulates NO production in vascular endothelial cells through Pim1/eNOS signaling pathway ［J］. Chinese Journal of Pathophysiology, 2020, 36(1):17-21. (in Chinese)

张敏, 孙玉, 唐平静, 等. 切应力通过Pim1/eNOS途径调节人脐静脉内皮细胞NO分泌［J］. 中国病理生理杂志, 2020, 36(1):17-21.

［14］Warfel NA, Kraft AS. PIM kinase (and Akt) biology and signaling in tumors ［J］. Pharmacol Ther, 2015, 151:41-49.

［15］Muraski JA,Rota M,Misao Y,et al. Pim-1 regulates cardiomyocyte survival downstream of Akt ［J］. Nat Med, 2007, 13(12):1467-1475.

［16］Sussman MA. Mitochondrial integrity: preservation through Akt/Pim-1 kinase signaling in the cardiomyocyte ［J］. Expert Rev Cardiovasc Ther, 2009, 7(8):929-938.