Independent and Combined Effects of Menhaden Oil and High Fructose on Hepatic Lipid Metabolism

Download PD

The Lipogenic Effect of High-Fructose Consumption on NAFLD During Weight Loss

Download PD

The Related Transcriptional Enhancer Factor-1 Isoform, TEAD4216, Can Repress Vascular Endothelial Growth Factor Expression in Mammalian Cells

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

TEAD4 is upregulated and alternatively spliced within the eye in an animal model of ocular ischemic disease.

<p>RT-PCR for TEAD4 from choroidal, retinal and iris tissue isolated from a non-human primate eye (<i>Rhesus macaque</i>) 24 hrs after occlusion of the central retinal artery (CRAO), indicates that the full length TEAD4₄₃₄ transcript is increased and the TEAD4₁₄₈ enhancer isoform is produced in the lasered eye. <b>L</b> =  Lasered CRAO eye; <b>C</b>  =  control eye.</p

The TEAD4₂₁₆ isoform does not require the HRE sequence to function.

<p>The TEAD4₂₁₆ isoform can repress expression from the human VEGF promoter (F2–R3) that lacks the HRE (p<0.01, n = 6). The TEAD4₁₄₈ and TEAD4₃₁₁ enchancer isoforms do not require the HRE to promote reporter gene expression (p<0.001). However the full length TEAD4₄₃₄ did not significantly enhance expression (p>0.02).</p

The TEAD4₁₄₈ and TEAD4₄₃₄ isoforms that enhance VEGF expression are localized to the nucleus whereas the TEAD4₂₁₆ repressor isoform is maintained in the cytoplasm.

<p>TEAD4 isoforms were expressed as fusion proteins within 293T cells with various fluorescent proteins (FP) (TEAD4₄₃₄ -Green-FP, and TEAD4₁₄₈-Yellow-FP (pseudo-colored green) and TEAD4₂₁₆-Green-FP) to visualize cellular localization.</p

Human TEAD4 protein is present on new vessels in neovascular AMD lesion.

<p>Photomicrograph showing a subretinal neovascular membrane in a human ocular tissue section (Fast Red, TEAD4; hematoxylin counterstain; original magnification x 100). Insert (location indicated by rectangle; original magnification x 1000) shows positive staining for TEAD4 by vascular endothelium of a choroidal new vessel that has bridged the elastic lamina of Bruch’s membrane.</p

The TEAD4₂₁₆ isoform can reduce endogenous VEGF₁₆₅ protein.

<p>Transient plasmid transfection or stable lentiviral mediated introduction of TEAD4₂₁₆ into human cells results in reduction of native VEGF protein. Solid bars represent VEGF₁₆₅ levels, quantified by ELISA, within conditioned media collected 48 hours after transfection of pcDNA plasmid vector containing the TEAD4₂₁₆ isoform into (<b>A</b>) 293T cells, (<b>B</b>) ARPE-19 retinal pigment epithelial (RPE) cells in culture (n = 4), (p<0.05). (<b>C</b>) Lentiviral (LV) expression of the TEAD4₂₁₆ isoform in human D407 RPE cells can inhibit endogenous VEGF production. Solid bars represent VEGF₁₆₅ levels, quantified by ELISA, within conditioned media collected 48 hours after transduced RPE cells were FAC sorted and plated (n = 3),(p<0.02).</p