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Deborah A. Ferrington, Marcela Maldonado, Marcia R. Terluk, Rebecca J. Kapphahn, Neal D. Huess, Dale S. Gregerson, Ching Yuan; Aberrent Regulation of NFkB Activity in Immunoproteasome-deficient Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5587.
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The immunoproteasome is a protease that is abundant in cells of the immune system, found in non-immune tissues, and known to generate peptides for presentation on MHC class I molecules. Recent evidence supports a novel role in the cellular stress response. Activation of the NFkB pathway, which is controlled by proteasomal degradation of NFkB regulators, is the primary response to a myriad of stressors. The current study tests the hypothesis that immunoproteasome assists in regulating NFkB.
Retinal pigment epithelial (RPE) cells derived from WT and KO mice lacking either one (L2, lmp2-/-) or two (L7M1, lmp7-/-/mecl-/-) catalytic subunits of the immunoproteasome were used. Phagocytic activity was evaluated by flow cytometric-detection of internalized one micron fluorescent-labeled beads. Immunohistochemistry, qRT-PCR, and Western blotting were used to evaluate morphological characteristics and expression of prototypic epithelial cell markers, including pigment epithelium derived factor (PEDF) and RPE65. Cells were incubated with TNFa to activate NFkB. Activation of NFkB family members (p65, p50, p52, cRel, RelB) was evaluated from their nuclear localization using Western blotting and the ELISA-based TransAM Activation Assay. Expression of NFkB-regulated genes was monitored by ELISA for the secreted protein IL6 or quantitative real-time PCR for IL6, COX2, iNOS, and IkBa.
Cultured RPE cells from WT and KO mice maintain their phagocytic activity, epithelial morphology, and expression of PEDF and RPE 65. Incubation of RPE with TNFa showed significant differences in response between WT and immunoproteasome-deficient cells. Comparing levels of secreted IL6 and COX2 expression, L7M1-deficient RPE cells were hyper responsive to TNFa compared with WT and L2-deficient RPE. L2-deficient cells exhibited no upregulation of iNOS and IkBa compared to the robust response from WT cells. Activation of NFkB showed early nuclear translocation for p65 and p52 and a late response for RelB in all cells. However, responses unique to L2-deficient cells include activation of p50 and a dramatic decrease in cRel.
These results show aberrant regulation of NFkB activity in immunoproteasome- deficient RPE and suggest a key role for immunoproteasome in regulating NFkB signaling.
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