[Show abstract][Hide abstract]ABSTRACT: Previous studies have demonstrated that both retinoids and apelin possess potent cardiovascular properties and that retinoids can mediate the expression of many genes in the cardiovascular system. However, it is not clear whether and how retinoids regulate apelin expression in rat vascular smooth muscle cells (VSMCs). In this study, we investigated the molecular mechanism of apelin expression regulation by synthetic retinoid Am80 in VSMCs. The results showed that Am80 markedly upregulated apelin mRNA and protein levels in VSMCs. Furthermore, KLF5 and Sp1 cooperatively mediated Am80-induced apelin expression through their direct binding to the TGF-β control element (TCE) on the apelin promoter. Interestingly, upon Am80 stimulation, the retinoic acid receptor alpha (RARα) was recruited to the apelin promoter by interacting with KLF5 and Sp1 prebound to the TCE site of the apelin promoter to form a transcriptional activation complex, subsequently leading to the upregulation of apelin expression in VSMCs. Thus, these results describe a novel mechanism of apelin regulation by Am80 and further expand the network of RARα in the retinoid pathway.
Preview · Article · Aug 2013 · Biochemical Journal
[Show abstract][Hide abstract]ABSTRACT: Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a key event in atherosclerosis and restenosis. In this paper, we report that Y-box binding protein 1 (YB1) functions as a phenotypic regulator in VSMC proliferation-differentiation switching through targeting GC box-dependent genes. Oligo pull-down assays demonstrated that YB1 binds directly to GC boxes via amino acids 125-220. YB1 C-terminal tail domain (CTD, amino acids 125-324) regulates GC box-dependent target gene transcription and suppresses VSMC proliferation. These findings provide a novel insight into the regulation of GC box-related genes by YB1, and provide a new understanding of VSMC proliferation regulation.
[Show abstract][Hide abstract]ABSTRACT: The transcription factor Krüppel-like factor 4 (KLF4) plays a critical role in vascular smooth muscle cell (VSMC) differentiation induced by all-trans-retinoic acid (ATRA). Although it has been demonstrated that ATRA stimulation augments both KLF4 protein and mRNA levels in VSMCs, the molecular mechanisms by which ATRA regulates Klf4 transcription are unknown. In this study, we examined the roles of ATRA-selective nuclear retinoic acid receptors (RARs) in the transcriptional regulation of Klf4. The introduction of small interfering RNA and an RAR antagonist demonstrated that RARα, but not RARβ or RARγ, mediated ATRA-induced Klf4 expression. A luciferase assay for the Klf4 promoter showed that three GC boxes in the proximal Klf4 promoter were indispensible for ATRA-induced Klf4 transcription and that RARα enhanced Klf4 promoter activity in a GC box-dependent manner. Furthermore, chromatin immunoprecipitation and oligonucleotide pulldown assays demonstrated that the transcription factors KLF4, Sp1, and YB1 directly bound to the GC boxes of the proximal Klf4 promoter. Upon RARα agonist stimulation, RARα was recruited to the Klf4 promoter through its interaction with KLF4, Sp1, and YB1 to form a transcriptional activation complex on the three GC boxes of the Klf4 promoter. These results suggest that RARα serves as an essential co-activator for ATRA signaling and that the recruitment of RARα to the KLF4-Sp1-YB1 complex, which leads to Klf4 expression in VSMCs, is independent of a retinoic acid response element.
No preview · Article · Feb 2012 · Journal of Biological Chemistry
[Show abstract][Hide abstract]ABSTRACT: Smooth muscle 22 alpha (SM22 alpha) is not required for the development and basal homeostatic function of smooth muscle cells (SMCs). However, a recent study demonstrated that SM22 alpha plays a role in inhibiting the phenotypic modulation of vascular SMCs (VSMCs) from contractile to synthetic/proliferative cells. The present study investigated the mechanism underlying the SM22 alpha-mediated maintenance of the contractile phenotype of VSMCs.
The redifferentiation of synthetic SMCs was induced by serum deprivation for 48-72 h. The expression plasmids containing full-length cDNA of rat SM22 alpha and a vector expressing SM22 alpha antisense transcripts were constructed, respectively. Coimmunoprecipitation, cosedimentation assay and immunofluorescence analyses were used to detect the interaction of SM22 alpha with F-actin.
The results revealed that SM22 alpha directly interacted and colocalized with F-actin and thus participated in the organization of the actin cytoskeleton in differentiated VSMCs. SM22 alpha facilitated the assembly of actin filaments into bundles. The blockade of SM22 alpha expression by SM22 alpha antisense RNA led to the thinning and dispersion of actin filaments. Consequently, the ratio of F-actin to globular (G)-actin was reduced, and the cell contractility was lost.
The SM22 alpha-induced F-actin bundling enhances the contractility and mobility of VSMCs, and the activity of SM22 alpha is necessary for maintaining the differentiated phenotype of VSMCs.
[Show abstract][Hide abstract]ABSTRACT: To investigate the molecular mechanisms of smooth muscle 22 alpha (SM22alpha) whereby cytoskeleton remodeling of vascular smooth muscle cells (VSMCs) is regulated.
Synthetic (dedifferentiated) VSMCs were converted to contractile (differentiated) VSMCs by serum deprivation. Cells were transfected with pEGFP-SM22alpha, and localization of SM22alpha and its relationship with F-actin were observed through fluorescence microscopy. Fractional extraction of proteins and Western blotting were used to detect polymerization of SM alpha-actin in antisense-pcD2-SM22alpha-transfected VSMCs.Furthermore, effect of SM22alpha on F-actin cross-linking was observed by F-actin polymerization experiment.
Fluorescence microscopy showed that SM22alpha co-localized with F-actin in contractile VSMCs. Western blotting of protein extracts from F-/G-actin fractions revealed that polymerization of SM alpha-actin was lower in antisense-pcD2-SM22alpha-transfected VSMCs, in which SM alpha-actin mostly existed as soluble G-actin. Moreover, F-actin polymerization in vitro also showed that GST-SM22alpha could promote cross-linking of F-actin to form thick and bundled stress fibres,while extracts from VSMCs transfected with antisense-pcD2-SM22alpha could not effectively induce the polymerization of F-actin.
SM22alpha acts as a modulator to participate in VSMC cytoskeleton remodeling. It can not only induce polymerization of G-actin to F-actin, but also promote cross-linking of F-actin to bundled stress fibres, indicating its vital role in cytoskeleton remodeling.
No preview · Article · Nov 2008 · Zhongguo ying yong sheng li xue za zhi = Zhongguo yingyong shenglixue zazhi = Chinese journal of applied physiology
[Show abstract][Hide abstract]ABSTRACT: To investigate the interaction between C-terminal domains of SM22alpha and cytoskeleton F-actin.
Prokaryotic expression vector containing SM22alpha cDNA and GST sequence was constructed. The induction conditions were optimized to increase the product of soluble GST-SM22alpha fusion protein in E coli. Expression products were purified and rabbit anti-GST-SM22alpha polyclonal antibody was produced by the purified fusion protein. In order to explore the effect of SM22alpha on cytoskeleton reorganization, VSMCs were treated with serum withdrawal and then serum stimulation to induce contractile/synthetic phenotypic modulation. SM22alpha protein distribution in F-actin/G-actin fractions was detected by Western blotting. The interaction between SM22alpha and actin was examined by GST pull down assay and coimmunoprecipitation. Colocalization of endogenous SM22alpha with F-actin was observed by immunofluorescence.
The results showed that the expression of soluble GST-SM22alpha protein was the highest under condition induced by 30 degrees C, 0.5 mmol/L IPTG for 6 h. Immunofluorescence and Western blotting of protein extracts from F-actin/G-actin fractions revealed that SM22alpha colocalized with F-actin during VSMC redifferentiation. GST pull down assay and coimmunoprecipitation showed that SM22alpha interacted with F-actin by C-terminal domains to participate in cytoskeleton reorganization.
The recombinant SM22alpha C-terminal domains have the ability to bind F-actin, by which SM22alpha interacts with actin and participates in cytoskeleton reorganization.
No preview · Article · Aug 2007 · Zhongguo ying yong sheng li xue za zhi = Zhongguo yingyong shenglixue zazhi = Chinese journal of applied physiology
[Show abstract][Hide abstract]ABSTRACT: Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a key role in vascular remodeling diseases, such as atherosclerosis, hypertension and restenosis. Recent researches have focused on the expression regulation of VSMC-specific marker genes and cytoskeleton organization in association with phenotypic modulation of VSMCs. Smooth muscle 22 alpha (SM22 alpha) is a novel differentiated VSMC marker, which is characterized by its smooth muscle tissue-specific and VSMC phenotype-specific expression pattern, and serves as an actin-association protein to participate in VSMC cytoskeleton organization and vascular remodeling. This article reviews recent advances in the characterization of SM22 alpha structure and its mechanism in VSMC cytoskeleton organization and vascular remodeling.
No preview · Article · Aug 2006 · Sheng li ke xue jin zhan [Progress in physiology]