Abstract
Increased cellular production of vascular endothelial growth factor (VEGF) is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4) protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4216, which represses VEGF promoter activity. The TEAD4216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE), which is the sequence critical to hypoxia inducible factor (HIF)-mediated effects. The TEAD4216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4216 isoform can competitively repress the stimulatory activity of the TEAD4434 and TEAD4148 enhancers. Synthesis of the native VEGF165 protein and cellular proliferation is suppressed by the TEAD4216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases.
Highlights
Alternate splicing to generate multiple mRNAs and subsequent protein products from one gene is exploited in many organisms, and in different tissues within the same organism, to augment the functional complexity of the translated genome. [1,2,3] Transcriptional enhancer factor 1 (TEF-1) is a member of the TEA DNAbinding family (TEAD), in which tissue and disease specific isoforms, generated by alternate splicing, have been observed. [4,5] The TEAD family of proteins is remarkably conserved from yeast to humans and, depending upon interaction with other proteins, can either activate or repress gene expression
[12] TEAD4 expression was up-regulated under hypoxic conditions, and TEAD4 mediated stimulation of vascular endothelial growth factor (VEGF) expression was independent of the classic hypoxia responsive element (HRE) and hypoxia-inducible factor (HIF-1) mechanism
We report the discovery of a novel TEAD4 isoform, isolated from vascular endothelial cells derived from human eyes, that represses gene expression from the VEGF promoter
Summary
Alternate splicing to generate multiple mRNAs and subsequent protein products from one gene is exploited in many organisms, and in different tissues within the same organism, to augment the functional complexity of the translated genome. [1,2,3] Transcriptional enhancer factor 1 (TEF-1) is a member of the TEA DNAbinding family (TEAD), in which tissue and disease specific isoforms, generated by alternate splicing, have been observed. [4,5] The TEAD family of proteins is remarkably conserved from yeast to humans and, depending upon interaction with other proteins, can either activate or repress gene expression. [6] Four TEAD genes exist in mammals (TEAD 1 to 4) and expression of these genes has been characterized in various mammalian tissues and cell types.[7,8,9] Transcriptional enhancer factor 1-related (RTEF-1 or TEAD4) protein was originally reported to regulate muscle-specific genes in cardiac and smooth muscle cells. [8] The TEAD4 transcription factor requires the presence of myocytespecific (M-CAT) sequences and muscle-specific cofactors to facilitate cell-specific gene regulation. [1,2,3] Transcriptional enhancer factor 1 (TEF-1) is a member of the TEA DNAbinding family (TEAD), in which tissue and disease specific isoforms, generated by alternate splicing, have been observed. The TEAD4148 isoform was identified in human ocular endothelial cells cultured under hypoxic conditions, suggesting that environment-specific alternate splicing may occur within human tissue to generate specific transcription factors with altered functions. We report an alternatively spliced TEAD4 transcript that generates a novel isoform (TEAD4216) able to significantly repress promoter activity. This novel protein reduces native VEGF production and cell proliferation. In addition we demonstrate the presence of TEAD4 protein in choroidal neovascular membrane in human age-related macular degeneration, suggesting a role in human disease
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