Nucleolin promotes Ang�II‑induced phenotypic transformation of vascular smooth muscle cells via interaction with tropoelastin mRNA

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.

Nucleolin promotes Ang II‐induced phenotypic transformation of vascular smooth muscle cells by regulating EGF and PDGF‐BB

“…The mRNA sequences of phenotypic transformation‐associated genes in VSMCs were screened by bioinformatics analysis, and multiple phenotypic transformation‐associated genes containing nucleolin‐binding elements ‘(T/G) CCCG (A/G)’ were identified. Bioinformatics analysis found that the mRNA sequences of 12 genes contained nucleolin‐binding elements, including EGF and PDGF‐BB . The analysis results suggested that nucleolin bound to the mRNA of these genes, regulated the expressions of these phenotype‐related genes and then modulated the phenotypic transformation of VSMCs.…”

“…Previous studies have confirmed that nucleolin overexpression can promote the up-regulation of nucleolin. After nucleolin RNA interference, the up-regulation of nucleolin induced by AngII is significantly inhibited in VSMCs 10. Results demonstrated that nucleolin was overexpressed in pcDNA3.1-Nuc-transfected cells compared with the control plasmid pcDNA3.1 and untransfected cells.Furthermore, the expressions of EGF and PDGF-BB at the protein level were significantly increased in pcDNA3.1-Nuc-transfected cells compared with the control plasmid pcDNA3.1 and untransfected cells.…”

“…Statistical analysis was performed by oneway ANOVA (LSD test) for multiple testing. P < .05 was considered as statistically significant.Bioinformatics analysis found that the mRNA sequences of 12 genes contained nucleolin-binding elements, including EGF and PDGF-BB 10. The analysis results suggested that nucleolin bound to the mRNA of these genes, regulated the expressions of these phenotype-related genes and then modulated the phenotypic transformation of VSMCs.…”

Anti-nucleolin aptamer, iSN04, inhibits the inflammatory responses in myoblasts by modulating the β-catenin/NF-κB signaling pathway

“…We investigated the effects of iSN04 on vascular smooth muscle cells (VSMCs) and pre-adipocytes, in which inflammation is a therapeutic target for atherosclerosis, diabetes, or obesity. Nucleolin has been reported to regulate the differentiation of VSMCs [28,29]. As shown in Fig.…”

“…Murine myoblast cell line C2C12 (DS Pharma Biomedical, Osaka, Japan) [20,23], rat embryonic thoracic aorta smooth muscle cell line A10 (CRL-1476; ATCC, Manassas, VA, USA) [28,29], and murine 3T3-L1 fibroblast cell line (IFO50416; JCRB Cell Bank, Osaka, Japan) as a model of pre-adipocytes [30] were cultured in DMEM (Nacalai, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare, Salt Lake City, UT, USA) and a mixture of 100 units/ml penicillin and 100 μg/ml streptomycin (Nacalai) at 37°C under 5% CO2 throughout the experiments.…”