PURPOSE: To determine the relative expression of metallothionein isoforms and their differential induction by oxidative stress in cultured RPE cells and to localize the isoforms in the human chorioretinal complex. METHODS: Total RNA was isolated from cultured human retinal pigment epithelial cells using TRI-Reagent. An "anchor-oligo-dT primer" was used for the synthesis of cDNA, reverse transcribed using avian reverse transcriptase and subsequently subjected to PCR analysis using oligonucleotides specific for metallothionein (MT) I, MT II, and MT III. The selected transcripts were then used to assess the expression of the above elements in fixed tissue sections by in situ hybridization. Cultured RPE cells were allowed to phagocytose bovine photoreceptor outer segments (ROS) or were treated with H(2)O(2) for 6 hours and then analyzed by RT-PCR or in situ hybridization to ascertain the effect of oxidative stress on metallothionein mRNA isoform expression. RESULTS: Relative density analysis of amplified products demonstrate the presence of MT I, MT II and MT III in RPE cells, with an apparent relative expression MT II > MT I > MT III [corrected]. Expression of MT I and MT II mRNA was increased by both phagocytosis and hydrogen peroxide, however MT III was not induced by either stress. In situ hybridization corroborated the findings of the RT-PCR analysis and showed that MTs were mainly localized in the RPE and the photoreceptor layer of the retina. CONCLUSIONS: The localization of MT and the response of MT to oxidative stress are consistent with a role for MT as an antioxidant in the RPE and retina. Studies are ongoing to determine the specific mechanisms of action of these antioxidants in RPE cells.
PURPOSE: To determine the relative expression of metallothionein isoforms and their differential induction by oxidative stress in cultured RPE cells and to localize the isoforms in the humanchorioretinal complex. METHODS: Total RNA was isolated from cultured human retinal pigment epithelial cells using TRI-Reagent. An "anchor-oligo-dT primer" was used for the synthesis of cDNA, reverse transcribed using avian reverse transcriptase and subsequently subjected to PCR analysis using oligonucleotides specific for metallothionein (MT) I, MT II, and MT III. The selected transcripts were then used to assess the expression of the above elements in fixed tissue sections by in situ hybridization. Cultured RPE cells were allowed to phagocytose bovine photoreceptor outer segments (ROS) or were treated with H(2)O(2) for 6 hours and then analyzed by RT-PCR or in situ hybridization to ascertain the effect of oxidative stress on metallothionein mRNA isoform expression. RESULTS: Relative density analysis of amplified products demonstrate the presence of MT I, MT II and MT III in RPE cells, with an apparent relative expression MT II > MT I > MT III [corrected]. Expression of MT I and MT II mRNA was increased by both phagocytosis and hydrogen peroxide, however MT III was not induced by either stress. In situ hybridization corroborated the findings of the RT-PCR analysis and showed that MTs were mainly localized in the RPE and the photoreceptor layer of the retina. CONCLUSIONS: The localization of MT and the response of MT to oxidative stress are consistent with a role for MT as an antioxidant in the RPE and retina. Studies are ongoing to determine the specific mechanisms of action of these antioxidants in RPE cells.
Authors: Michele Nachman-Clewner; Frank J Giblin; C Kathleen Dorey; Robert H I Blanks; Loan Dang; Christopher J Dougherty; Janet C Blanks Journal: Invest Ophthalmol Vis Sci Date: 2008-07 Impact factor: 4.799