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S. A. Medearis, I. Han, P. Yang, N. Zhang, J. J. Peairs, G. J. Jaffe; The Critical Role of Bcl-xL in Mouse Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):471.
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We have previously demonstrated a crucial role for Bcl-xL as a human RPE (hRPE) cell survival protein. Mouse cells and tissues are frequently used as a model for human disease. Herein, we determined the role of Bcl-xL as a survival protein in cultured mouse RPE (mRPE) cells.
Cultured mRPE cells were treated with media alone, or with human IL-1-β, human TNF-α or mouse TNF-α for 24 hours. Real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to determine survival factor gene expression. Cultured human and mouse cells were transfected with modified, 2’-O-methoxyethoxy Bcl-xL-mismatched control antisense oligonucleotides (ASOs) and Bcl-xL-specific ASOs, and protein and RNA were isolated. Relative gene expression of anti- and pro-apoptotic survival genes was determined by qRT-PCR, and western blot-analyzed proteins. Cell count quantified cell viability post-transfection.
In mRPE cells, Bcl-xL was the most highly expressed of all survival factors. Expression of the anti-apoptotic genes Traf-1, c-IAP1, and c-IAP2 was upregulated in mRPE cells treated with TNF-α, while Bcl-xL expression was not significantly affected, similar to what we have found in hRPE cells. Upon treatment with Bcl-xL-specific ASOs, Bcl-xL gene expression and protein levels were markedly decreased after 3 days post-transfection. Following this same treatment, Traf-1 expression was increased, while expression of all other anti-apoptotic genes was either unchanged, or modestly decreased, relative to controls. The number of human and mouse cells treated with Bcl-xL-specific ASO was significantly decreased when compared to the number of cells treated with control ASOs (p < 0.05).
Cultured mRPE cells are highly similar to cultured hRPE cells with respect to the role and function of the survival factor Bcl-xL. Bcl-xL is a critical protein present in mRPE cells that promotes the cell’s survival. Based on the similarity between survival factors within hRPE in situ and in vitro to that of mRPE in vitro, cultured mRPE may serve as a useful model for hRPE cell studies and human pathology, particularly for macular diseases in which RPE cell survival is critical.
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