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C.D. Gerstner, C.M. Hunter, K.A. Howes; S100B, Amphoterin, and RAGE in ARPE–19 Cell Differentiation . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5286.
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Purpose: Preliminary evidence suggests that RAGE and its CNS ligands S100B and amphoterin may play a role in RPE differentiation as seen in our immunoctyochemical studies in the mouse retina. S100B and amphoterin have been previously reported to mediate neurogenesis and myogenesis through RAGE. In myogenesis, S100B is an inhibitor of differentiation acting in a manner that is inversely related to amphoterin levels and directly related to RAGE expression levels. While for many cell types, RAGE is downregulated upon differentiation, reexpression of RAGE occurs in response to abnormal ligand accumulation. We propose that RPE RAGE upregulation in response to enhanced levels of the RAGE ligands, 1) S100B, in transgenic mice and 2) AGE products in aging and AMD retinas may alter RPE differentiation status. In this study, we examined effects of RAGE ligands on RAGE–mediated RPE differentiation status. Methods:ARPE–19 cells differentiate in culture, showing apical/basal polarity and expression of RPE specific proteins. ARPE–19 cells were collected at day 0, and at 4 day intervals for approximately 3 weeks to examine mRNA and protein levels of S100B, amphoterin, RAGE and the early differentiation specific marker, CRALBP by real time RT–PCR. Proteins were separated on SDS–PAGE and labeled with monospecific antibodies for amphoterin, S100B, RAGE, and CRALBP by western blot. In our next set of experiments, ARPE–19 cells will be treated with amphoterin, S100B, or various concentrations of both ligands, to examine differentiation responses. Results:Preliminary results from these studies show that amphoterin mRNA levels, undetectable at day 0, increase with ARPE–19 differentiation. In contrast, S100B and RAGE mRNA and protein levels progressively diminish with ARPE–19 differentiation. Differentiated ARPE–19 cells treated with S100B and AGE products significantly upregulate RAGE mRNA and proteins levels. Conclusions:These results suggest that S100B, amphoterin, and RAGE may play a normal role in RPE differentiation. We will pursue the hypothesis that reinstatement of RAGE expression in ARPE–19 cell culture with exposure to S100B and AGE products, may represent a mechanism for RPE dysfunction via alteration of differentiation status in S100B transgenics and AMD maculas.
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