ABSTRACT
Atherogenesis is a chronic inflammatory process that involves complex interactions between endothelial dysfunction, lipid deposition and vascular smooth-muscle cell (VSMC) proliferation. However, the molecular mechanism is still unclear. We found that a pro-atherosclerotic factor (oxLDL) induced the expression of Krüppel-like factor 5 (KLF5), which in turn increased miR-29a expression levels. The increased miR-29a was retained within HASMCs and down-regulated Fbw7/CDC4 expression by targeting the 3´UTR of Fbw7/CDC4, subsequently increasing KLF5 stability by reducing the Fbw7/CDC4-dependent ubiquitination of KLF5, forming a positive feedback loop to enhance VSMC proliferation and promote atherogenesis. These results indicate a potentially important role for the oxLDL-activated feedback mechanism in VSMC proliferation and atherogenesis. Suppression of miR-29a may be an effective way to attenuate atherosclerosis. In conclusion, our data are the first to reveal that the regulatory crosstalk between KLF5, miR-29a, and Fbw7/CDC4 cooperatively promotes atherosclerotic development.
Subject(s)
Atherosclerosis/metabolism , F-Box-WD Repeat-Containing Protein 7/metabolism , Gene Expression Regulation , Kruppel-Like Transcription Factors/metabolism , MicroRNAs/metabolism , 3' Untranslated Regions , Animals , Aorta/cytology , Biomarkers/metabolism , Cell Differentiation , Cell Proliferation , Cells, Cultured , F-Box-WD Repeat-Containing Protein 7/genetics , Gene Expression Profiling , Humans , Inflammation , Kruppel-Like Transcription Factors/genetics , Lipoproteins, LDL/metabolism , Macrophages/metabolism , Mice , Mice, Knockout, ApoE , MicroRNAs/genetics , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , NIH 3T3 Cells , UbiquitinationABSTRACT
OBJECTIVES: The formation of vascular neointima is mainly related to impairment of the vascular endothelial barrier and abnormal proliferation and migration of smooth muscle cells. The objective of this study was to investigate whether miR-29a exerts any promoting effect on the vascular neointimal hyperplasia and if so, its mechanism. MATERIALS AND METHODS: RT-qPCR was performed to determine expression of miR-29a in vascular smooth muscle cells (VSMC) and vascular neointimal hyperplasia. To further understand its role, we restored its expression in VSMCs by transfection with miR-29a mimics or inhibitors. Effects of miR-29a on cell proliferation were also determined. RESULTS: In this study, we used two kinds of model to observe the role of miR-29a in neointimal hyperplasia induced by carotid ligation or balloon injury. The major findings were that: (i) miR-29a overexpression promoted neointimal hyperplasia induced by carotid ligation; (ii) miR-29a increased proliferation of VSMCs, one aspect of which was by targeting expression of Ying and yang 1 protein (YY1), a negative regulator of Cyclin D1. A further aspect, was by increasing expression of Krüppel-like factor 5, a positive regulator of Cyclin D1, thereby allowing formation a synergistic effect. (iii) Tongxinluo (TXL), a traditional Chinese medicine reduced neointimal formation in ligated vessels by inhibiting VSMC proliferation and migration. CONCLUSIONS: These findings provide a new molecular mechanism of TXL in decreasing neointima hyperplasia.
Subject(s)
Hyperplasia/genetics , MicroRNAs/genetics , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , YY1 Transcription Factor/genetics , Animals , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Disease Models, Animal , Drugs, Chinese Herbal/pharmacology , Humans , Hyperplasia/drug therapy , Hyperplasia/pathology , Kruppel-Like Transcription Factors/deficiency , Kruppel-Like Transcription Factors/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , MicroRNAs/antagonists & inhibitors , Muscle, Smooth, Vascular/drug effects , Rats , Rats, Sprague-Dawley , YY1 Transcription Factor/deficiency , YY1 Transcription Factor/metabolismABSTRACT
The regulation of vascular smooth muscle cell (VSMC) proliferation is an important issue due to its major implications for the prevention of pathological vascular conditions. The objective of this work was to assess the function of small ubiquitin-like modifier (SUMO)ylated KrÏppel-like transcription factor 4 (KLF4) in the regulation of VSMC proliferation in cultured cells and in animal models with balloon injury. We found that under basal conditions, binding of non-SUMOylated KLF4 to p300 activated p21 (p21(WAF1/CIP1))transcription, leading to VSMC growth arrest. PDGF-BB promoted the interaction between Ubc9 and KLF4 and the SUMOylation of KLF4, which in turn recruited transcriptional corepressors to the p21 promoter. The reduction in p21 enhanced VSMC proliferation. Additionally, the SUMOylated KLF4 did not affect the expression of KLF4, thereby forming a positive feedback loop enhancing cell proliferation. These results demonstrated that SUMOylated KLF4 plays an important role in cell proliferation by reversing the transactivation action of KLF4 on p21 induced with PDGF-BB.
Subject(s)
Cell Proliferation , Gene Expression Regulation , Kruppel-Like Transcription Factors/metabolism , Myocytes, Smooth Muscle/physiology , Sumoylation , Animals , Becaplermin , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Femoral Artery/injuries , Femoral Artery/pathology , Humans , Kruppel-Like Factor 4 , Male , Mice, Inbred C57BL , Muscle, Smooth, Vascular/pathology , Promoter Regions, Genetic , Proto-Oncogene Proteins c-sis/physiology , Rats, Sprague-Dawley , Transcription, Genetic , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Vascular Diseases/metabolismABSTRACT
The Krüppel-like factor 4 is a DNA-binding transcriptional regulator that regulates a diverse array of cellular processes, including development, differentiation, proliferation, and apoptosis. The previous studies about KLF4 functions mainly focused on its role as a transcription factor, its functions in the cytoplasm are still unknown. In this study, we found that PDGF-BB could prompt the translocation of KLF4 to the cytoplasm through CRM1-mediated nuclear export pathway in vascular smooth muscle cells (VSMCs) and increased the interaction of KLF4 with actin in the cytoplasm. Further study showed that both KLF4 phosphorylation and SUMOylation induced by PDGF-BB participates in regulation of cytoskeletal organization by stabilizing the actin cytoskeleton in VSMCs. In conclusion, these results identify that KLF4 participates in the cytoskeletal organization by stabilizing cytoskeleton in the cytoplasm of VSMCs.