May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Alteration of Ferritin Subunit Composition Changes Iron Metabolism in Lens Epithelial Cells With Downstream Effects on Glutathione Levels and Vascular Endothelial Growth Factor Secretion
Author Affiliations & Notes
  • M. McGahan
    Dept of Molec Biomed Sci, North Carolina State University, Raleigh, North Carolina
  • J. Ferrell
    Dept of Molec Biomed Sci, North Carolina State University, Raleigh, North Carolina
  • M. Mukunnemkeril
    Dept of Molec Biomed Sci, North Carolina State University, Raleigh, North Carolina
  • L. Fleisher
    Dept of Molec Biomed Sci, North Carolina State University, Raleigh, North Carolina
  • S. Nagar
    Dept of Molec Biomed Sci, North Carolina State University, Raleigh, North Carolina
  • M. Goralska
    Dept of Molec Biomed Sci, North Carolina State University, Raleigh, North Carolina
  • J. Harned
    Dept of Molec Biomed Sci, North Carolina State University, Raleigh, North Carolina
  • Footnotes
    Commercial Relationships M. McGahan, None; J. Ferrell, None; M. Mukunnemkeril, None; L. Fleisher, None; S. Nagar, None; M. Goralska, None; J. Harned, None.
  • Footnotes
    Support NIH Grant EY-04900 and Funds from the State of North Carolina
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 2436. doi:
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    • Get Citation

      M. McGahan, J. Ferrell, M. Mukunnemkeril, L. Fleisher, S. Nagar, M. Goralska, J. Harned; Alteration of Ferritin Subunit Composition Changes Iron Metabolism in Lens Epithelial Cells With Downstream Effects on Glutathione Levels and Vascular Endothelial Growth Factor Secretion. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2436.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: Ferritin is a multimeric iron storage protein consisting of 24 subunits of two types, heavy (H) and light (L). The ratio of these subunits is tissue specific and is thought to control iron storage ability. We have recently demonstrated that in addition to catalyzing free radical damage, iron increases glutathione (GSH) levels and decreases VEGF secretion from cultured lens epithelial cells (LEC). The purpose of this study was to alter the H:L ratio using siRNA for each chain and to determine the effects of these changes on iron storage and downstream effects on intracellular GSH levels in and VEGF secretion by cultured canine LEC.

Methods:: Custom siRNAs for canine ferritin H- and L-chains were obtained from Dharmacon. A non-targeting siRNA served as a control. De novo synthesis of H- and L-chain ferritins was determined by labeling with 35S-methionine. The amount of GSH in the LEC was determined fluorometrically and the amount of VEGF in the bathing media was determined using ELISA.

Results:: Transfection with siRNA for H-chain ferritin resulted in a 54% decrease in H-chain synthesis. Iron incorporation into ferritin was decreased by 53%. Four/five days after transfection GSH levels were increased while VEGF secretion was reduced by 54%. In contrast, transfection with siRNA for L-chain caused a 37% decrease in L-chain synthesis. There was a concomitant increase in iron incorporation into ferritin (53%). This treatment did not alter GSH levels in these cells and had only a minor effect on VEGF secretion.

Conclusions:: Reducing synthesis of H-chain ferritin decreased iron incorporation into ferritin. This should increase iron availability and could account for the observed increase in GSH levels and reduction in VEGF secretion. Conversely, decreasing L-chain synthesis increased iron incorporation into ferritin, which should decrease iron availablility. However, the siRNA-induced decrease in L-chain synthesis did not have significant effects on either GSH levels within, or VEGF secretion by LEC. These results may reflect differential effects of these two treatments on discrete intracellular iron pools resulting in differential downstream effects, and could provide important clues about how intracellular iron availability affects normal physiological functions.

Keywords: metabolism • oxidation/oxidative or free radical damage • protein structure/function 
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