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. 2019 Apr;43(4):1597-1610.
doi: 10.3892/ijmm.2019.4090. Epub 2019 Feb 4.

Nucleolin Promotes Ang II‑induced Phenotypic Transformation of Vascular Smooth Muscle Cells via Interaction With Tropoelastin mRNA

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Nucleolin Promotes Ang II‑induced Phenotypic Transformation of Vascular Smooth Muscle Cells via Interaction With Tropoelastin mRNA

Li Fang et al. Int J Mol Med. .
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Abstract

The current study aimed to clarify the role of nucleolin in the phenotypic transformation of vascular smooth muscle cells (VSMCs) and to preliminarily explore its underlying mechanism. The spatial and temporal expression patterns of nucleolin, and the effects of angiotensin II (Ang II) on the expression of VSMC phenotypic transformation markers, α‑smooth muscle‑actin, calponin, smooth muscle protein 22α and osteopontin were investigated. The effects of nucleolin on VSMC phenotypic transformation and the expression of phenotypic transformation‑associated genes, tropoelastin, epiregulin and fibroblast growth factor 2 (b‑FGF), were determined. Protein‑RNA co‑immunoprecipitation was used to investigate the potential target genes regulated by the nucleolin in phenotypic transformation of VSMCs. Finally, the stability of tropoelastin mRNA and the effects of nucleolin on the expression of tropoelastin were assayed. The results revealed that Ang II significantly promoted the phenotypic transformation of VSMCs. The expression of nucleolin was gradually upregulated in VSMCs treated with Ang II at different concentrations for various durations. Ang II induced nucleolin translocation from the nucleus to cytoplasm. Additionally, Ang II significantly promoted the phenotypic transformation of VSMCs. Overexpression and silencing of nucleolin regulated the expressions of tropoelastin, epiregulin and b‑FGF. There was an interaction between tropoelastin mRNA and nucleolin protein, promoting the stability of tropoelastin mRNA and enhancing the expression of tropoelastin at the protein level. Upregulation of nucleolin had an important role in Ang II‑induced VSMC phenotypic transformation, and its underlying mechanism may be through interacting with tropoelastin mRNA, leading to its increased stability and protein expression. The findings provide a new perspective into the regulatory mechanism of VSMC phenotypic transformation.

Figures

Figure 1
Figure 1
Effect of Ang II on the expressions of VSMC phenotypic transformation markers α-SM-actin and OPN. VSMCs were stimulated with (A) different concentrations of Ang II for 48 h and (B) Ang II (10−5 mmol/l) for different durations; reverse transcription-quantitative polymerase chain reaction was used to detect the expressions of contractile phenotype of VSMCs (α-SM-actin, calponin, SM22a) and synthetic phenotype of VSMCs OPN at the mRNA level. VSMCs were stimulated with (C) different concentrations of Ang II for 48 h and (D) Ang II (10−5 mmol/l) for different durations; the total protein was extracted, and the expressions of contractile phenotype of VSMCs (α-SM-actin, calponin, SM22a) and synthetic phenotype of VSMCs OPN at the protein level were analyzed by western blotting. (E) Dose-response study of VSMCs treated with Ang II of various concentrations and time-response study of VSMCs treated with Ang II for 0-72 h showed that there was a dose and time-dependent increase in the proliferation of VSMCs. Data are expressed as the mean ± standard error. *P<0.05 vs. control. VSMCs, vascular smooth muscle cells; Ang II, angiotensin II; α-SM-actin, α-smooth muscle-actin; SM22a, smooth muscle protein 22 α; OPN, osteopontin; OD, optical density.
Figure 1
Figure 1
Effect of Ang II on the expressions of VSMC phenotypic transformation markers α-SM-actin and OPN. VSMCs were stimulated with (A) different concentrations of Ang II for 48 h and (B) Ang II (10−5 mmol/l) for different durations; reverse transcription-quantitative polymerase chain reaction was used to detect the expressions of contractile phenotype of VSMCs (α-SM-actin, calponin, SM22a) and synthetic phenotype of VSMCs OPN at the mRNA level. VSMCs were stimulated with (C) different concentrations of Ang II for 48 h and (D) Ang II (10−5 mmol/l) for different durations; the total protein was extracted, and the expressions of contractile phenotype of VSMCs (α-SM-actin, calponin, SM22a) and synthetic phenotype of VSMCs OPN at the protein level were analyzed by western blotting. (E) Dose-response study of VSMCs treated with Ang II of various concentrations and time-response study of VSMCs treated with Ang II for 0-72 h showed that there was a dose and time-dependent increase in the proliferation of VSMCs. Data are expressed as the mean ± standard error. *P<0.05 vs. control. VSMCs, vascular smooth muscle cells; Ang II, angiotensin II; α-SM-actin, α-smooth muscle-actin; SM22a, smooth muscle protein 22 α; OPN, osteopontin; OD, optical density.
Figure 2
Figure 2
Effect of Ang II on the expression and subcellular localization of nucleolin in VSMCs. (A) VSMCs were stimulated with different concentrations of Ang II for different durations, the total RNA of cells was extracted, and cDNA was obtained after reverse transcription. Quantitative polymerase chain reaction was used to detect the expression of nucleolin. *P<0.05 vs. control group (0, 10−8 and 10−7 mmol/l). (B) VSMCs were stimulated with different concentrations of Ang II for different durations, the total protein was extracted, and the expression of nucleolin was analyzed by western blotting. *P<0.05 vs. control group (0, 10−8 and 10−7 mmol/l). (C) VSMCs were stimulated with Ang II (10−6 mmol/l) for 12, 24, 48 and 72 h, nuclear protein and cytoplasmic protein were extracted, western blotting was used to detect the expression of nucleolin, and β-tubulin and PCNA were used as the internal controls of cytoplasmic protein and nuclear protein, respectively. Data are expressed as the mean ± standard error, n=5. (D) VSMCs were stimulated with Ang II (10−6 mmol/l) for 48 h, and indirect immunofluorescence was used to observe the subcellular localization of nucleolin. Nucleolin, analysis of nucleolin with fluorescein isothiocyanate-labeled antibody (green); Hochest33258, nuclei were counterstained with Hoechst 33258 (violet); Merge, overlap of cytoplasmic and nuclear fractions. Magnification, ×400. VSMCs, vascular smooth muscle cells; Ang II, angiotensin II; PCNA, proliferating cell nuclear antigen.
Figure 3
Figure 3
Effect of nucleolin overexpression and silencing on Ang II-induced phenotypic transformation of VSMCs. (A) Top panel, effect of nucleolin overexpression on the expression of nucleolin in VSMCs; VSMCs were transfected with the control plasmid (pcDNA3.1) and the recombinant plasmid pcDNA3.1-Nuc; total protein was extracted from the transfected cells and western blotting was performed (data are expressed as the mean ± standard error, n=5; *P<0.05 vs. control and pcDNA3.1 group). Bottom panel, effect of nucleolin overexpression on Ang II-induced expression of nucleolin in VSMCs; VSMCs were transfected with pcDNA3.1 and pcDNA3.1-Nuc, and then cells were treated with 10−6 mmol/l Ang II for 48 h. (B) Effect of nucleolin overexpression on Ang II-induced expressions of VSMC phenotypic transformation markers α-SM-actin, calponin, SM22a and OPN. VSMCs were transfected with pcDNA3.1 and pcDNA3.1-Nuc, and then cells were treated with 10−6 mmol/l Ang II for 48 h. (C) Left panel, effect of silencing of nucleolin on expression of nucleolin in VSMCs. VSMCs were transfected with the control plasmid (PsiRNA) and nucleolin siRNA plasmid (PsiRNA-Nuc); total protein was extracted from the transfected cells, and then western blotting was performed. Right panel, effect of silencing of nucleolin on Ang II-induced expression of nucleolin in VSMCs. VSMCs were transfected with PsiRNA and PsiRNA-Nuc, and then cells were treated with 10−6 mmol/l Ang II for 48 h. (D) Effect of silencing of nucleolin on Ang II-induced expressions of VSMC phenotypic transformation markers α-SM-actin, calponin, SM22a and OPN. VSMCs were transfected with PsiRNA and PsiRNA-Nuc, and then cells were treated with 10−6 mmol/l Ang II for 48 h (data are expressed as the mean ± standard error, n=5; *P<0.05 vs. normal control group; #P<0.05 vs. untransfected cell group and pcDNA3.1 or PsiRNA group). VSMCs, vascular smooth muscle cells; control, untransfected cell group; Ang II, angiotensin II treatment; pcDNA3.1, control plasmid group; pcDNA3.1-Nuc, nucleolin overexpression plasmid; OPN, osteopontin; α-SM-actin, α-smooth muscle-actin; SM22a, smooth muscle protein 22α; PsiRNA, control plasmid group; PsiRNA-Nuc, nucleolin RNA interference plasmid.
Figure 3
Figure 3
Effect of nucleolin overexpression and silencing on Ang II-induced phenotypic transformation of VSMCs. (A) Top panel, effect of nucleolin overexpression on the expression of nucleolin in VSMCs; VSMCs were transfected with the control plasmid (pcDNA3.1) and the recombinant plasmid pcDNA3.1-Nuc; total protein was extracted from the transfected cells and western blotting was performed (data are expressed as the mean ± standard error, n=5; *P<0.05 vs. control and pcDNA3.1 group). Bottom panel, effect of nucleolin overexpression on Ang II-induced expression of nucleolin in VSMCs; VSMCs were transfected with pcDNA3.1 and pcDNA3.1-Nuc, and then cells were treated with 10−6 mmol/l Ang II for 48 h. (B) Effect of nucleolin overexpression on Ang II-induced expressions of VSMC phenotypic transformation markers α-SM-actin, calponin, SM22a and OPN. VSMCs were transfected with pcDNA3.1 and pcDNA3.1-Nuc, and then cells were treated with 10−6 mmol/l Ang II for 48 h. (C) Left panel, effect of silencing of nucleolin on expression of nucleolin in VSMCs. VSMCs were transfected with the control plasmid (PsiRNA) and nucleolin siRNA plasmid (PsiRNA-Nuc); total protein was extracted from the transfected cells, and then western blotting was performed. Right panel, effect of silencing of nucleolin on Ang II-induced expression of nucleolin in VSMCs. VSMCs were transfected with PsiRNA and PsiRNA-Nuc, and then cells were treated with 10−6 mmol/l Ang II for 48 h. (D) Effect of silencing of nucleolin on Ang II-induced expressions of VSMC phenotypic transformation markers α-SM-actin, calponin, SM22a and OPN. VSMCs were transfected with PsiRNA and PsiRNA-Nuc, and then cells were treated with 10−6 mmol/l Ang II for 48 h (data are expressed as the mean ± standard error, n=5; *P<0.05 vs. normal control group; #P<0.05 vs. untransfected cell group and pcDNA3.1 or PsiRNA group). VSMCs, vascular smooth muscle cells; control, untransfected cell group; Ang II, angiotensin II treatment; pcDNA3.1, control plasmid group; pcDNA3.1-Nuc, nucleolin overexpression plasmid; OPN, osteopontin; α-SM-actin, α-smooth muscle-actin; SM22a, smooth muscle protein 22α; PsiRNA, control plasmid group; PsiRNA-Nuc, nucleolin RNA interference plasmid.
Figure 4
Figure 4
Effects of nucleolin on Ang II-induced expressions of phenotypic transformation-associated genes and the binding of nucleolin protein with tropoelastin, epiregulin and b-FGF mRNA in VSMCs. (A) Left, effects of silencing of nucleolin on Ang II-induced expressions of phenotypic transformation-related genes tropoelastin, epiregulin and b-FGF in VSMCs. VSMCs were transfected with PsiRNA and PsiRNA-Nuc, the total RNA was extracted from the transfected cells after 48 h, and then RT-qPCR was performed. Right, effect of nucleolin overexpression on Ang II-induced expressions of phenotypic transformation-associated genes tropoelastin, epiregulin and b-FGF in VSMCs. VSMCs were transfected with pcDNA3.1 and pcDNA3.1-Nuc, the total RNA was extracted from the transfected cells after 48 h, and then the RT-qPCR was performed. Data are expressed as the mean ± standard error, n=5; *P<0.05 vs. normal control group; #P<0.05 vs. untransfected cell group and pcDNA3.1 or PsiRNA group). (B) Normal VSMCs and Ang II-treated VSMCs were collected to prepare the cell extracts, cell extracts were divided into three equal groups: Input group, negative control IgG group and nucleolin antibody group. Immunoprecipitation was performed out using rabbit anti-nucleolin monoclonal antibody, and total mRNA was extracted from the sediment. Representative of three separate experiments. VSMCs, vascular smooth muscle cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Ang II, angiotensin II treatment; pcDNA3.1, control plasmid group; pcDNA3.1-Nuc, nucleolin overexpression plasmid; PsiRNA, control plasmid group; PsiRNA-Nuc, nucleolin RNA interference plasmid; Input, positive control; IgG, immunoglobulin G negative control; Nuc-Ab, nucleolin antibody; Ctrl, control cells; IP, immunoprecipitation; b-FGF, fibroblast growth factor 2.
Figure 5
Figure 5
Effect of nucleolin on tropoelastin mRNA stability and tropoelastin protein expression in VSMCs. (A) Effect of nucleolin overexpression on tropoelastin mRNA stability in VSMCs. VSMCs were transfected with pcDNA3.1 and pcDNA3.1-Nuc for 48 h, and then incubated with actinomycin D (5 µg/ml) for various periods of time (0, 0.5, 1, 2 and 3 h). The mRNA levels of tropoelastin were determined by RT-qPCR. *P<0.05 vs. Vect group, #P<0.01 vs. Vect group, n=5. (B) Effect of Ang II on tropoelastin mRNA stability in VSMCs. VSMCs were treated with 10−6 mM Ang II for 48 h. The cells were then incubated with actinomycin D (5 µg/ml) for various periods of time (0, 0.5, 1, 2 and 3 h). The mRNA levels of tropoelastin were determined by RT-qPCR. *P<0.05 vs. Ctrl group, n=5. (C) Effect of low expression of nucleolin on tropoelastin mRNA stability in VSMCs. VSMCs were transfected with nucleolin siRNA plasmid for 24 h, cells were treated with 10−6 mmol/l Ang II for 48 h, and then incubated with actinomycin D (5 µg/ml) for various periods of time (0, 0.5, 1, 2 and 3 h). The mRNA levels of tropoelastin were determined by RT-qPCR. *P<0.05 vs. PsiRNA group, n=5. Effect of nucleolin (D) overexpression and (E) low expression on Ang II-induced expressions of tropoelastin. VSMCs were transfected with pcDNA3.1, pcDNA3.1-Nuc, PsiRNA and PsiRNA-Nuc, and then cells were treated with 10−6 mmol/l Ang II for 48 h. Data are expressed as the mean ± standard error, n=5; *P<0.05 vs. normal control group, #P<0.05 vs. untransfected cell group and pcDNA3.1 group. VSMCs, vascular smooth muscle cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Ctrl, control; Vect, transfected with pcDNA3.1 plasmid; Nuc, transfected with pcDNA3.1-Nuc plasmid; Ang II, angiotensin II; PsiRNA, control plasmid group; psiRNA-Nuc, nucleolin RNA interference plasmid.

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