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Molecular regulation of vascular endothelial growth factor expression in the retinal pigment epithelium.

Ford KM, D'Amore PA - Mol. Vis. (2012)

Bottom Line: Overexpression of a dominant negative that targets the MITF-Tfe family resulted in a 50% reduction in VEGF expression.The role of the MITF-Tfe family in VEGF regulation in the RPE was corroborated in studies with the VEGF-luciferase reporter constructs, where deletion of the distal VEGF promoter region containing putative binding sites for the MITF-Tfe family resulted in a 50% reduction in VEGF promoter activity. siRNA knockdown of the MITF-Tfe family individually, and in combination, revealed that downregulation of Tfe3 resulted in reduced VEGF expression.Our results indicate that Tfe3, in conjunction with other MITF-Tfe members, regulates VEGF expression in the RPE and are consistent with the hypothesis that VEGF expression in RPE cells is regulated as part of their differentiation.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Insititute/Massachusetts Eye and Ear, Boston, MA 02114, USA.

ABSTRACT

Purpose: Vascular endothelial growth factor (VEGF) plays an important role in homeostasis and diseases of the retinal pigment epithelium (RPE), choriocapillaris, and, most notably, age-related macular degeneration (AMD). Although much is known about VEGF regulation in pathologies, little is known about the control of VEGF expression under normal conditions. VEGF expression has been previously shown to be regulated in coordination with cell differentiation in the muscle and kidney. We therefore tested the hypothesis that VEGF in the adult RPE would similarly be regulated in conjunction with differentiation.

Methods: A human retinal pigment epithelium cell line (ARPE-19), a line of immortalized human RPE cells, was used for all experiments. RPE cells were polarized in culture for 4 weeks on laminin-coated Transwells. Levels of VEGF mRNA and protein were determined with real-time PCR and enzyme-linked immunosorbent assay, respectively. VEGF-luciferase reporter constructs were used to identify regions of the VEGF promoter that control VEGF expression in the RPE. Microphthalmia-associated transcription factor (MITF)-Tfe transcription factors were blocked using either a pan MITF-Tfe dominant negative or specific small interfering RNA (siRNA).

Results: VEGF mRNA and protein secretion increased over time in the RPE cells cultured on Transwells, with protein secretion occurring in a polarized fashion primarily toward the basolateral side. Overexpression of a dominant negative that targets the MITF-Tfe family resulted in a 50% reduction in VEGF expression. The role of the MITF-Tfe family in VEGF regulation in the RPE was corroborated in studies with the VEGF-luciferase reporter constructs, where deletion of the distal VEGF promoter region containing putative binding sites for the MITF-Tfe family resulted in a 50% reduction in VEGF promoter activity. siRNA knockdown of the MITF-Tfe family individually, and in combination, revealed that downregulation of Tfe3 resulted in reduced VEGF expression.

Conclusions: Our results indicate that Tfe3, in conjunction with other MITF-Tfe members, regulates VEGF expression in the RPE and are consistent with the hypothesis that VEGF expression in RPE cells is regulated as part of their differentiation.

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Overexpression of dnMITF leads to downregulation of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium (RPE). Human retinal pigment epithelium (ARPE-19) cells were transfected with a dominant negative against the MITF-Tfe transcription factor family (dnMITF) or an empty vector control (pBS). RNA was isolated 48 h post-transfection and analyzed with qRT–PCR. A 50% reduction in VEGF mRNA was observed in samples overexpressing the dnMITF; there was an 80% decrease in the expression of known targets for the MITF-Tfe family, tyrosinase (TYR), and tyrosinase-related protein-1 (TRP-1). The experiment was performed in triplicate. A representative experiment is shown with data expressed as the mean±standard deviation. *: p<0.05; ***: p<0.001.
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f7: Overexpression of dnMITF leads to downregulation of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium (RPE). Human retinal pigment epithelium (ARPE-19) cells were transfected with a dominant negative against the MITF-Tfe transcription factor family (dnMITF) or an empty vector control (pBS). RNA was isolated 48 h post-transfection and analyzed with qRT–PCR. A 50% reduction in VEGF mRNA was observed in samples overexpressing the dnMITF; there was an 80% decrease in the expression of known targets for the MITF-Tfe family, tyrosinase (TYR), and tyrosinase-related protein-1 (TRP-1). The experiment was performed in triplicate. A representative experiment is shown with data expressed as the mean±standard deviation. *: p<0.05; ***: p<0.001.

Mentions: To determine if the transcription factors important for RPE specification might also be involved in regulating VEGF expression, the entire MITF-Tfe family was inhibited with dominant negative, or knocked down individually with siRNA. Overexpression of a dominant negative construct targeting the MITF-Tfe family led to a significant (50%) reduction in VEGF expression (Figure 7). The MITF-Tfe family of transcription factors are basic helix–loop–helix-leucine zipper transcription factors, including MITF, Tfeb, Tfe3, and Tfec [23]. These transcription factors may bind as homodimers or heterodimers to regulate gene transcription, making the pan MITF-Tfe dominant negative highly useful in determining the role of this transcription factor family in modulating gene expression. Notably, the downregulation of VEGF expression following pan MITF-Tfe blockade was seen only when transfections were performed in low serum (0.1%), indicating a role for serum factor in regulating VEGF expression. The extent of VEGF downregulation achieved with dominant negative pan MITF-Tfe closely paralleled that observed with the truncated 5.2 kb VEGF-luciferase reporter construct that lacks the region containing the binding sites for the MITF-Tfe family. Taken together, these findings support the notion that this family is involved in regulating VEGF expression, either directly or indirectly.


Molecular regulation of vascular endothelial growth factor expression in the retinal pigment epithelium.

Ford KM, D'Amore PA - Mol. Vis. (2012)

Overexpression of dnMITF leads to downregulation of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium (RPE). Human retinal pigment epithelium (ARPE-19) cells were transfected with a dominant negative against the MITF-Tfe transcription factor family (dnMITF) or an empty vector control (pBS). RNA was isolated 48 h post-transfection and analyzed with qRT–PCR. A 50% reduction in VEGF mRNA was observed in samples overexpressing the dnMITF; there was an 80% decrease in the expression of known targets for the MITF-Tfe family, tyrosinase (TYR), and tyrosinase-related protein-1 (TRP-1). The experiment was performed in triplicate. A representative experiment is shown with data expressed as the mean±standard deviation. *: p<0.05; ***: p<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298425&req=5

f7: Overexpression of dnMITF leads to downregulation of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium (RPE). Human retinal pigment epithelium (ARPE-19) cells were transfected with a dominant negative against the MITF-Tfe transcription factor family (dnMITF) or an empty vector control (pBS). RNA was isolated 48 h post-transfection and analyzed with qRT–PCR. A 50% reduction in VEGF mRNA was observed in samples overexpressing the dnMITF; there was an 80% decrease in the expression of known targets for the MITF-Tfe family, tyrosinase (TYR), and tyrosinase-related protein-1 (TRP-1). The experiment was performed in triplicate. A representative experiment is shown with data expressed as the mean±standard deviation. *: p<0.05; ***: p<0.001.
Mentions: To determine if the transcription factors important for RPE specification might also be involved in regulating VEGF expression, the entire MITF-Tfe family was inhibited with dominant negative, or knocked down individually with siRNA. Overexpression of a dominant negative construct targeting the MITF-Tfe family led to a significant (50%) reduction in VEGF expression (Figure 7). The MITF-Tfe family of transcription factors are basic helix–loop–helix-leucine zipper transcription factors, including MITF, Tfeb, Tfe3, and Tfec [23]. These transcription factors may bind as homodimers or heterodimers to regulate gene transcription, making the pan MITF-Tfe dominant negative highly useful in determining the role of this transcription factor family in modulating gene expression. Notably, the downregulation of VEGF expression following pan MITF-Tfe blockade was seen only when transfections were performed in low serum (0.1%), indicating a role for serum factor in regulating VEGF expression. The extent of VEGF downregulation achieved with dominant negative pan MITF-Tfe closely paralleled that observed with the truncated 5.2 kb VEGF-luciferase reporter construct that lacks the region containing the binding sites for the MITF-Tfe family. Taken together, these findings support the notion that this family is involved in regulating VEGF expression, either directly or indirectly.

Bottom Line: Overexpression of a dominant negative that targets the MITF-Tfe family resulted in a 50% reduction in VEGF expression.The role of the MITF-Tfe family in VEGF regulation in the RPE was corroborated in studies with the VEGF-luciferase reporter constructs, where deletion of the distal VEGF promoter region containing putative binding sites for the MITF-Tfe family resulted in a 50% reduction in VEGF promoter activity. siRNA knockdown of the MITF-Tfe family individually, and in combination, revealed that downregulation of Tfe3 resulted in reduced VEGF expression.Our results indicate that Tfe3, in conjunction with other MITF-Tfe members, regulates VEGF expression in the RPE and are consistent with the hypothesis that VEGF expression in RPE cells is regulated as part of their differentiation.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Insititute/Massachusetts Eye and Ear, Boston, MA 02114, USA.

ABSTRACT

Purpose: Vascular endothelial growth factor (VEGF) plays an important role in homeostasis and diseases of the retinal pigment epithelium (RPE), choriocapillaris, and, most notably, age-related macular degeneration (AMD). Although much is known about VEGF regulation in pathologies, little is known about the control of VEGF expression under normal conditions. VEGF expression has been previously shown to be regulated in coordination with cell differentiation in the muscle and kidney. We therefore tested the hypothesis that VEGF in the adult RPE would similarly be regulated in conjunction with differentiation.

Methods: A human retinal pigment epithelium cell line (ARPE-19), a line of immortalized human RPE cells, was used for all experiments. RPE cells were polarized in culture for 4 weeks on laminin-coated Transwells. Levels of VEGF mRNA and protein were determined with real-time PCR and enzyme-linked immunosorbent assay, respectively. VEGF-luciferase reporter constructs were used to identify regions of the VEGF promoter that control VEGF expression in the RPE. Microphthalmia-associated transcription factor (MITF)-Tfe transcription factors were blocked using either a pan MITF-Tfe dominant negative or specific small interfering RNA (siRNA).

Results: VEGF mRNA and protein secretion increased over time in the RPE cells cultured on Transwells, with protein secretion occurring in a polarized fashion primarily toward the basolateral side. Overexpression of a dominant negative that targets the MITF-Tfe family resulted in a 50% reduction in VEGF expression. The role of the MITF-Tfe family in VEGF regulation in the RPE was corroborated in studies with the VEGF-luciferase reporter constructs, where deletion of the distal VEGF promoter region containing putative binding sites for the MITF-Tfe family resulted in a 50% reduction in VEGF promoter activity. siRNA knockdown of the MITF-Tfe family individually, and in combination, revealed that downregulation of Tfe3 resulted in reduced VEGF expression.

Conclusions: Our results indicate that Tfe3, in conjunction with other MITF-Tfe members, regulates VEGF expression in the RPE and are consistent with the hypothesis that VEGF expression in RPE cells is regulated as part of their differentiation.

Show MeSH
Related in: MedlinePlus