Limits...
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.

Show MeSH

Related in: MedlinePlus

Schematic of vascular endothelial growth factor (VEGF)-luciferase reporter constructs. VEGF-luciferase constructs indicating the size of upstream VEGF promoter sequence, transcription start site, and 5′ UTR. VEGF genomic DNA was ligated upstream of a promoterless luciferase gene in the pGL2-basic plasmid. Deletions were made by taking advantage of the restriction sites located within the VEGF promoter and the pGL2 multiple cloning site [16].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3298425&req=5

f4: Schematic of vascular endothelial growth factor (VEGF)-luciferase reporter constructs. VEGF-luciferase constructs indicating the size of upstream VEGF promoter sequence, transcription start site, and 5′ UTR. VEGF genomic DNA was ligated upstream of a promoterless luciferase gene in the pGL2-basic plasmid. Deletions were made by taking advantage of the restriction sites located within the VEGF promoter and the pGL2 multiple cloning site [16].

Mentions: To begin to examine the molecular basis of VEGF expression, constructs containing various truncations of the VEGF promoter controlling luciferase were used to determine the regions of the VEGF promoter that are essential for VEGF expression in the RPE. These constructs consisted of a 9 kb full-length VEGF promoter, as well as deletion mutants containing 5.2 kb, 2.1 kb, and 1.6 kb of upstream VEGF promoter (Figure 4). Analysis of these constructs in ARPE-19 cells revealed a twofold induction of promoter activity with the full-length 9 kb VEGF reporter construct over the 5.2 kb construct. Interestingly, similar induction of promoter activity was not observed in a control cell type (HEK 293), in which maximal promoter activity was obtained with the 2.1 kb construct (Figure 5). This observation suggests that RPE may express transcription factors or enhancers that bind specifically in the distal region of the VEGF promoter. Sequence analysis of the VEGF promoter revealed numerous binding sites for several transcription factors required for RPE specification; most notably, the putative binding sites for the MITF-Tfe transcription factor family were concentrated primarily in the -5 kb to -9 kb region (Figure 6).


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

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

Schematic of vascular endothelial growth factor (VEGF)-luciferase reporter constructs. VEGF-luciferase constructs indicating the size of upstream VEGF promoter sequence, transcription start site, and 5′ UTR. VEGF genomic DNA was ligated upstream of a promoterless luciferase gene in the pGL2-basic plasmid. Deletions were made by taking advantage of the restriction sites located within the VEGF promoter and the pGL2 multiple cloning site [16].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Schematic of vascular endothelial growth factor (VEGF)-luciferase reporter constructs. VEGF-luciferase constructs indicating the size of upstream VEGF promoter sequence, transcription start site, and 5′ UTR. VEGF genomic DNA was ligated upstream of a promoterless luciferase gene in the pGL2-basic plasmid. Deletions were made by taking advantage of the restriction sites located within the VEGF promoter and the pGL2 multiple cloning site [16].
Mentions: To begin to examine the molecular basis of VEGF expression, constructs containing various truncations of the VEGF promoter controlling luciferase were used to determine the regions of the VEGF promoter that are essential for VEGF expression in the RPE. These constructs consisted of a 9 kb full-length VEGF promoter, as well as deletion mutants containing 5.2 kb, 2.1 kb, and 1.6 kb of upstream VEGF promoter (Figure 4). Analysis of these constructs in ARPE-19 cells revealed a twofold induction of promoter activity with the full-length 9 kb VEGF reporter construct over the 5.2 kb construct. Interestingly, similar induction of promoter activity was not observed in a control cell type (HEK 293), in which maximal promoter activity was obtained with the 2.1 kb construct (Figure 5). This observation suggests that RPE may express transcription factors or enhancers that bind specifically in the distal region of the VEGF promoter. Sequence analysis of the VEGF promoter revealed numerous binding sites for several transcription factors required for RPE specification; most notably, the putative binding sites for the MITF-Tfe transcription factor family were concentrated primarily in the -5 kb to -9 kb region (Figure 6).

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