Abstract
Krüppel-like factor 5 (KLF5), originally isolated as a regulator of phenotypic modulation of vascular smooth muscle cells, induces pathological cell growth and is expressed in the neointima. Although induction of KLF5 up-regulates growth factors like platelet-derived growth factor-A chain, how KLF5 actually contributes to vascular remodeling, notably its direct effects on cell proliferation, had been poorly clarified. To investigate the effects of KLF5 on neointimal formation, we at first performed adenoviral overexpression of KLF5 to rats subjected to carotid balloon injury. Neointimal formation and proliferating cell nuclear antigen-positive rate were significantly increased at 14 days after injury in the KLF5-treated animals. At the cellular level, overexpression of KLF5 also resulted in markedly increased cell proliferation and cell cycle progression. As a molecular mechanism, we showed that KLF5 directly bound to the promoter and up-regulated gene expression of cyclin D1, as well as showing specific transactivation of cyclins and cyclin-dependent kinase inhibitors in cardiovascular cells. Conversely, knockdown of KLF5 by RNA interference specifically down-regulated cyclin D1 and impaired vascular smooth muscle cell proliferation. Furthermore, KLF5 attenuated cleavage of caspase-3 under conditions of apoptotic stimulation. Moreover, KLF5-administered animals exhibited a significant decrease in terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling-positive cells in the medial layer, suggesting inhibition of apoptosis in the early phase after denudation. These findings collectively suggest that KLF5 plays a central role in cardiovascular pathologies through direct and specific stimulation of cell growth as well as inhibition of apoptosis.
Highlights
Kruppel-like factor 5 (KLF5)) is a zinc finger transcription factor that belongs to a family known as the Sp/KLF factors, which are comprised of ϳ20 members in mammals [1, 2]
Fax: 81-3-3815-2087; E-mail: nagai-tky@umin.ac.jp. 4 The abbreviations used are: KLF5, Kruppel-like factor 5; SMC, smooth muscle cell; VSMC, vascular SMC; PDGF, platelet-derived growth factor; PCNA, proliferating cell nuclear antigen; CKI, cyclin-dependent kinase inhibitors; KLF5) is a zinc finger transcription factor that belongs to a family known as the Sp/KLF factors, which are comprised of ϳ20 members in mammals [1, 2]
Direct Promotion of VSMC Proliferation by KLF5 via Transactivation of Cyclin D1 Transcription—In the present study, KLF5 overexpression resulted in promotion of cell proliferation, and gene silencing of KLF5 with RNA interference showed marked suppression of cyclin D1 expression and decreased cell growth
Summary
Preparation of Plasmid Constructs and Recombinant Adenoviruses—pCAG-KLF5 and adenoviral KLF5 and Sp1 constructs were constructed as described previously [16]. Vascular Smooth Muscle Cell Proliferation Assay—2 ϫ 105 rat VSMCs were at first infected with each adenoviral construct (100 multiplicities of infection) for 12 h, reseeded in duplicate in 6-well plates, and starved from serum for 24 h. We overexpressed KLF5 in rats subjected to carotid balloon injury by adenoviral transfer to address the direct effects of KLF5 on vascular remodeling. Under conditions in which overexpression of KLF5 in rat carotid arteries was confirmed by immunohistochemical staining and Western blotting (Fig. 1, A and B), histological examination showed that neointimal formation was markedly increased by adenoviral transfer of KLF5 at 2 weeks after balloon injury (Fig. 1C). Apoptotic Assays—5 ϫ 104 rat VSMCs were transfected with induction resulted in increased cell proliferation after serum adenoviral constructs and treated with 2 mol/liter ani- stimulation as compared with those of Sp1 or empty groups somysin (Sigma). The threshold of significance was taken as p Ͻ 0.05
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