Search

Objective: Related transcriptional enhancer factor 1 (RTEF-1) is a key transcriptional regulator in endothelial function. In this study, we investigated a possible role for RTEF-1 in the regulation of microvascular relaxation and the underlying mechanism involved. Activation of fibroblast growth factor receptor 1 (FGFR1) by FGFs increases vasodilation, although transcriptional control of the molecular mechanisms underlying FGFR1 is still unclear., Materials and Methods: We demonstrated that RTEF-1 stimulated FGFR1 expression at the transcriptional level, specifically an area including Sp1 elements, as evidenced by promoter assays. Additionally, RTEF-1 increased FGFR1 mRNA and protein expression in vitro and in VE-cadherin-promoted RTEF-1 (VE-Cad/RTEF-1) transgenic mice, whereas RTEF-1 siRNA blocked the upregulation of FGFR1 expression. Furthermore, increased endothelial-dependent microvessel relaxation was observed in the coronary arteries of VE-Cad/RTEF-1 mice, and increased proliferation was observed in RTEF-1-overexpressing cells, both of which correlated to increased FGF/FGFR1 signaling and endothelial nitric oxide synthase (eNOS) upregulation. Our results indicate that RTEF-1 acts as a transcriptional stimulator of FGFR1 and is involved in FGF pathways by increasing microvessel dilatation via eNOS., Conclusions: These findings suggest that RTEF-1 plays an important role in FGFR1- stimulated vasodilatation. Understanding the effect of RTEF-1 in microvessel relaxation may provide beneficial knowledge in improving treatments in regards to ischemic vascular disorders., (Copyright © 2012 S. Karger AG, Basel.)

Fibroblast growth factor receptor-1 (FGFR-1) has been implicated in the process of cardiogenesis, although the underlying molecular mechanisms are poorly understood. In this study, we report the regulation of FGFR-1 expression by related transcriptional enhancer factor-1 (RTEF-1) in vitro (endothelial cells) and in vivo (RTEF-1 transgenic mice). FGFR-1 promoter activity, FGFR-1 mRNA and protein level were measured in bovine aortic endothelial cells (BAEC) in response to RTEF-1 and in endothelial cells isolated from livers in RTEF-1 transgenic mice. RTEF-1 stimulated FGFR-1 promoter activity in a dose-dependent manner. RTEF-1 enhanced FGFR-1 mRNA (4-fold) and protein expression (3.5-fold) whereas RTEF-1 siRNA decreased FGFR-1 protein expression (4-fold). FGFR-1 mRNA and protein expression were also increased in endothelial cells isolated from livers of RTEF-1 transgenic mice. Furthermore, RTEF-1 enhanced tubule formation whereas this was decreased by RTEF-1 knockdown. Moreover, increased relaxation of microvessels was found in RTEF-1 transgenic mice compared to wild-type mice. Our results indicate that RTEF-1 acts as a transcriptional stimulator of FGFR-1 in endothelial cells through its activation of the FGFR-1 promoter. RTEF-1 thus plays an important role in the regulation of FGFR-1 expression. These findings help further understand FGFR activity in angiogenesis and may lead to new therapeutic targets in ischemic vascular disorders.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor known to be up-regulated in ischemic heart and hypoxic endothelial cells. However, the transcriptional regulation of VEGF in hypoxia-induced angiogenesis is not fully understood. Transcriptional enhancer factor-1 (TEF-1) is a transcriptional factor family that can regulate many genes expressed in cardiac and skeletal muscle cells by binding to myocyte-specific chloramphenicol acetyltransferase heptamer elements in the promoters of these genes. In this study, we demonstrated that related TEF-1 (RTEF-1), a member of the TEF-1 family, is up-regulated in hypoxic endothelial cells. Overexpression of RTEF-1 increases VEGF promoter activity and VEGF expression. Sequential deletion and site-directed mutation analyses of the VEGF promoter demonstrated that a GC-rich region containing four Sp1 response elements, located between -114 and -50, was essential for RTEF-1 function. This region is beyond the hypoxia-inducible factor-1alpha binding site and does not consist of M-CAT-related elements. By electrophoretic mobility shift assay, RTEF-1 was found to interact with the first Sp1 residue (-97 to -87) of the four consecutive Sp1 elements. Binding activity of RTEF-1 to VEGF promoter is also confirmed by chromatin immunoprecipitation. In addition, induction of VEGF promoter activity by RTEF-1 results in an increase of angiogenic processes including endothelial cells proliferation and vascular structure formation. These results indicate that RTEF-1 acts as a transcriptional stimulator of VEGF by regulating VEGF promoter activity through binding to Sp1 site. In addition, RTEF-1-induced VEGF promoter activity was enhanced in a hypoxic condition, indicating that RTEF-1 may play an important role in the regulation of VEGF under hypoxia.

Gut-enriched Krüppel-like factor (GKLF, KLF4) is an epithelial-specific transcription factor that expresses in the gastrointestinal tract and mediates growth arrest of colonic epithelium. The molecular mechanisms governing its growth inhibitory effect have not been fully elucidated. In the present study, we showed that induction of GKLF mRNA and protein expression by interferon-gamma treatment was associated with reduction of ornithine decarboxylase (ODC) gene expression and enzyme activity in colon cancer HT-29 cells. Overexpression of GKLF in HT-29 cells significantly reduced ODC mRNA and protein levels as well as enzyme activity and resulted in growth arrest, indicating that ODC might be a downstream target of GKLF. This conclusion was further supported by data showing that GKLF mRNA and protein concentrations were the highest at the G(1)/S boundary of the cell cycle, where ODC mRNA and protein levels were the lowest and that overexpression of GKLF resulted in cell arrested at the G(1) phase. Reporter gene transfection studies and electrophoretic mobility gel shift assays demonstrated that GKLF repressed ODC promoter activity and that these effects appeared to be mediated through interaction with a GC box in the proximal portion of the promoter. Transfection studies using reporter constructs and chromatin immunoprecipitation assays also demonstrated that GKLF inhibited transactivation of the ODC gene by interfering with the binding of Sp1 to the ODC promoter. These results indicate that GKLF may function as a G(1)/S checkpoint regulator and exert its growth arrest effect through down-regulation of ODC gene expression. Furthermore, GKLF is a transcriptional repressor of the ODC gene, and these effects are mediated by interaction with the GC-rich region on the promoter.

Gut-enriched Krüppel-like factor (GKLF/KLF4), a member of the Krüppel-like factor family of transcription factors, has recently been shown to be associated with growth arrest in the colonic mucosa. Our laboratory has previously demonstrated that GKLF is one of the down-stream targets of interferon-gamma (IFN-gamma) in colon cancer cells, but the signaling pathway regulating GKLF expression is not known. In this report, we showed that IFN-gamma increased GKLF and interferon regulatory factor-1 mRNA levels in a parallel fashion and these effects were abolished in STAT1 knockout mouse fibroblast cell lines, indicating that STAT1 mediated IFN-gamma-stimulated GKLF gene expression. IFN-gamma treatment induced phosphorylation of STAT1 and IFN-gamma-induced GKLF mRNA expression was attenuated by tyrosine protein kinase inhibitors, suggesting that phosphorylation of STAT1 played an essential role in this process. To further evaluate the effect of STAT1 on GKLF gene expression, a 2622-bp mouse GKLF promoter was isolated from a liver genomic library. In a transient transfection system, IFN-gamma treatment increased GKLF promoter activity by 3.5-fold. Sequential deletion and mutation analysis of the GKLF promoter has identified the sequence between -1675 and -1580, a region containing a GAS element, to be essential for IFN-gamma function. By electrophoretic mobility gel shift assay, nuclear extracts from IFN-gamma-stimulated HT-29 cells were found to bind to the GAS motif on the GKLF promoter and this protein-DNA complex was supershifted by the STAT1 antiserum. These results indicate that IFN-gamma-induced GKLF expression required phosphorylated STAT1 and that these effects were mediated, in part, through interaction of STAT1 with the GAS element on the GKLF promoter.