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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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Related in: MedlinePlus

Knockdown of MITF and/or Tfeb does not affect vascular endothelial growth factor (VEGF) expression. Human retinal pigment epithelium (ARPE-19) cells were transfected with an siControl or a SMARTpool targeting MITF and/or Tfeb. Depletion of (A) MITF and/or (B) Tfeb did not reduce VEGF expression. 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; ns: p>0.05.
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f8: Knockdown of MITF and/or Tfeb does not affect vascular endothelial growth factor (VEGF) expression. Human retinal pigment epithelium (ARPE-19) cells were transfected with an siControl or a SMARTpool targeting MITF and/or Tfeb. Depletion of (A) MITF and/or (B) Tfeb did not reduce VEGF expression. 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; ns: p>0.05.

Mentions: To determine which member(s) of the MITF-Tfe family may mediate VEGF regulation, pooled siRNA against MITF and Tfeb was used. MITF and Tfeb were successfully knocked down, individually and in combination, yet no effect of their knockdown on VEGF expression was observed (Figure 8). Tfe3 knockdown with siRNA led to a 20% reduction in VEGF mRNA; however, there was also a three- to fourfold increase in the known target genes of Tfe3 (tyrosinase-related protein 1 and tyrosinase; Figure 9). Although the knockdown of MITF, Tfeb, and Tfe3 was significant at 60%–80%, there is a possibility that even a low level of expression of these factors may be sufficient to influence VEGF expression in the RPE. Alternatively, there could be a redundancy among the family members so that inhibiting all members of the family would be necessary to affect VEGF expression. Nevertheless, these data provide compelling support for the concept that members of this family of transcription factors, particularly Tfe3, are involved in regulating VEGF expression in the RPE.


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

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

Knockdown of MITF and/or Tfeb does not affect vascular endothelial growth factor (VEGF) expression. Human retinal pigment epithelium (ARPE-19) cells were transfected with an siControl or a SMARTpool targeting MITF and/or Tfeb. Depletion of (A) MITF and/or (B) Tfeb did not reduce VEGF expression. 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; ns: p>0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Knockdown of MITF and/or Tfeb does not affect vascular endothelial growth factor (VEGF) expression. Human retinal pigment epithelium (ARPE-19) cells were transfected with an siControl or a SMARTpool targeting MITF and/or Tfeb. Depletion of (A) MITF and/or (B) Tfeb did not reduce VEGF expression. 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; ns: p>0.05.
Mentions: To determine which member(s) of the MITF-Tfe family may mediate VEGF regulation, pooled siRNA against MITF and Tfeb was used. MITF and Tfeb were successfully knocked down, individually and in combination, yet no effect of their knockdown on VEGF expression was observed (Figure 8). Tfe3 knockdown with siRNA led to a 20% reduction in VEGF mRNA; however, there was also a three- to fourfold increase in the known target genes of Tfe3 (tyrosinase-related protein 1 and tyrosinase; Figure 9). Although the knockdown of MITF, Tfeb, and Tfe3 was significant at 60%–80%, there is a possibility that even a low level of expression of these factors may be sufficient to influence VEGF expression in the RPE. Alternatively, there could be a redundancy among the family members so that inhibiting all members of the family would be necessary to affect VEGF expression. Nevertheless, these data provide compelling support for the concept that members of this family of transcription factors, particularly Tfe3, are involved in regulating VEGF expression in the RPE.

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