Abstract
Purpose: :
Hemochromatosis is a genetic disorder of iron overload resulting in oxidative stress. Mutations in HFE, which result in excessive iron accumulation in tissues, are found in ~ 85% of hemochromatosis patients. HFE is also expressed in retina, specifically in RPE, and plays an obligatory role in maintenance of retinal iron homeostasis. Hfe-/- mice accumulate excessive iron in retina and exhibit several features found in age-related macular degeneration, including hyperproliferation of retinal pigment epithelium (RPE). Here the mechanism underlying this particular phenotype in RPE was investigated.
Methods: :
The following biochemical characteristics were compared between primary cultures of mouse RPE prepared from wild type mice and Hfe-/- mice. Cellular senescence was monitored by senescence-specific β-galactosidase activity. Expression and location of survivin were determined by RT-PCR, Western blot, and immunofluorescence. Migration and invasion were monitored using appropriate kits. Expression and function of glucose transporters were determined by RT-PCR and uptake of 3-O-methyl-D-glucose. Expression and function of histone deacetylases (HDACs) were monitored by RT-PCR, catalytic activity, and acetylation status of histones H3 and H4. Expression of DNA methyltransferases (DNMTs) was studied by RT-PCR.
Results: :
Compared to wild type RPE cells, Hfe-/- RPE cells exhibited slower senescence rate, accompanied with increased expression of the anti-apoptotic protein survivin. Hfe-/- cells migrated faster than wild type cells; however, the invasion rate was comparable. Hfe-/- cells possessed greater glucose uptake activity, accompanied with increased expression of glucose transporters SGLT1, GLUT1, GLUT3, and GLUT4. Levels of HDAC1 and HDAC3 were higher in Hfe-/- cells, demonstrable both at mRNA level and activity level. Similarly, expression of DNMT1 and DNMT3a was higher in Hfe-/- cells.
Conclusions: :
Hfe-/- RPE cells exhibit several features of tumor cells: decreased senescence, enhanced migration, increased glucose uptake, and elevated levels of epigenetic enzymes HDACs and DNMTs. These biochemical parameters underlie the hyperproliferative phenotype of Hfe-/- RPE cells.
Keywords: retinal pigment epithelium • age-related macular degeneration • retina