Korean J Physiol Pharmacol.  2022 Sep;26(5):347-355. 10.4196/kjpp.2022.26.5.347.

Activating transcription factor 4 aggravates angiotensin IIinduced cell dysfunction in human vascular aortic smooth muscle cells via transcriptionally activating fibroblast growth factor 21

Affiliations
  • 1Department of General Surgery, Changshu Hospital Affiliated to Soochow University, Changshu 215500, China
  • 2Department of Vascular Surgery, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou 215008, China

Abstract

Abdominal aortic aneurysm (AAA) is a life-threatening disorder worldwide. Fibroblast growth factor 21 (FGF21) was shown to display a high level in the plasma of patients with AAA; however, its detailed functions underlying AAA pathogenesis are unclear. An in vitro AAA model was established in human aortic vascular smooth muscle cells (HASMCs) by angiotensin II (Ang-II) stimulation. Cell counting kit-8, wound healing, and Transwell assays were utilized for measuring cell proliferation and migration. RT-qPCR was used for detecting mRNA expression of FGF21 and activating transcription factor 4 (ATF4). Western blotting was utilized for assessing protein levels of FGF21, ATF4, and markers for the contractile phenotype of HASMCs. ChIP and luciferase reporter assays were implemented for identifying the binding relation between AFT4 and FGF21 promoters. FGF21 and ATF4 were both upregulated in Ang-II-treated HASMCs. Knocking down FGF21 attenuated Ang-IIinduced proliferation, migration, and phenotype switch of HASMCs. ATF4 activated FGF21 transcription by binding to its promoter. FGF21 overexpression reversed AFT4 silencing-mediated inhibition of cell proliferation, migration, and phenotype switch. ATF4 transcriptionally upregulates FGF21 to promote the proliferation, migration, and phenotype switch of Ang-II-treated HASMCs.

Keyword

Abdominal aortic aneurysm; Activating transcription factor 4; FGF21; Phenotype switch; VSMCs

Figure

  • Fig. 1 Ang-II induced cell proliferation increase and FGF21 upregulation. (A) CCK-8 assay for assessing the proliferation of HASMCs under treatment of different concentrations of Ang-II (0-100 nM). (B–D) RT-qPCR analysis and western blotting of FGF21 mRNA and protein expression levels in HASMCs treated with different concentrations of Ang-II (0–100 nM), respectively. Ang-II, angiotensin II; FGF21, fibroblast growth factor 21; CCK-8, cell counting kit-8; HASMCs, human aortic vascular smooth muscle cells; RT-qPCR, real time quantitative polymerase chain reaction. *p < 0.05, **p < 0.01.

  • Fig. 2 FGF21 silencing inhibits HASMC proliferation, migration and phenotype switch. HASMCs were transfected with si-FGF21 or si-NC for 48 h before treatment of 100 nM Ang-II for 24 h. (A–C) RT-qPCR and western blotting for evaluating the efficiency of FGF21 knockdown in HASMCs. (D) CCK-8 assay for assessing cell proliferation in each group. (E–G) Wound healing and Transwell assays for examining cell migratory ability. Magnification for wound healing: ×40; magnification for Transwell: ×200. (H) Western blotting of SM22α and α-SMA protein expression in HASMCs. FGF21, fibroblast growth factor 21; HASMCs, human aortic vascular smooth muscle cells; NC, negative control; Ang-II, angiotensin II; RT-qPCR, real time quantitative polymerase chain reaction; CCK-8, cell counting kit-8. **p < 0.01.

  • Fig. 3 ATF4 transcriptionally activates FGF21 in HASMCs. (A–C) RT-qPCR and Western blotting of ATF4 expression in HASMCs treated with different concentrations (0–100 nM) of Ang-II. (D, E) JASPAR website for prediction of ATF4 binding site in FGF21 promoter region. (F) ChIP assay for identifying the binding relation between ATF4 and FGF21 promoter. (G, H) Western blotting of ATF4 and FGF21 protein expression in ATF4-depleted HASMCs. (I) Luciferase reporter assay for identifying the binding relation between ATF4 and FGF21 promoter. ATF4, activating transcription factor 4; FGF21, fibroblast growth factor 21; HASMCs, human aortic vascular smooth muscle cells; RT-qPCR, real time quantitative polymerase chain reaction; Ang-II, angiotensin II; TFBS, transcription factor binding site; TSS, transcription start site; ChIP, chromatin immunoprecipitation. *p < 0.05, **p < 0.01, ***p < 0.001.

  • Fig. 4 ATF4 promotes proliferation, migration and phenotype switch of HASMCs by activating FGF21 transcription. HASMCs were transfected with si-NC, si-ATF4 or si-ATF4 + pcDNA3.1/FGF21 for 48 h before treatment of 100 nM Ang-II for 24 h. (A–C) RT-qPCR analysis and western blotting for assessing the efficiency of FGF21 overexpression in HASMCs. (D) CCK-8 assay for evaluating cell proliferative ability in each group. (E–G) Wound healing and Transwell assays for assessing cell migration in each group. Magnification for wound healing: ×40; magnification for Transwell: × 200. (H) Western blotting of SM22α and α-SMA protein expression in HASMCs of each group. ATF4, activating transcription factor 4; HASMCs, human aortic vascular smooth muscle cells; FGF21, fibroblast growth factor 21; NC, negative control; Ang-II, angiotensin II; RT-qPCR, real time quantitative polymerase chain reaction; CCK-8, cell counting kit-8. *p < 0.05, **p < 0.01, , ***p < 0.001.

  • Fig. 5 A schematic view of the model investigated in this study. ATF4 activates FGF21 transcription by binding to its promoter in the nuclei of HASMCs. Increased FGF21 mRNA levels leads to the upregulation of FGF21 protein expression, which promotes proliferation, migration and phenotype switch of HASMCs. ATF4, activating transcription factor 4; HASMCs, human aortic vascular smooth muscle cells; FGF21, fibroblast growth factor 21.


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