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PK Mukherjee, SG Barreiro, M Soriano, Z Campbell, MK Topham, SM Prescott, NG Bazan; Diacylglycerol Kinase Epsilon Gene Specifically Modulates Morphology and Survival in ARPE-19 Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4586.
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Purpose:Several seven-transmembrane-spanning G-protein-linked receptors regulate cell function by generating arachidonoyl-DAG, which in turn modulates downstream protein kinase C phosphorylation events. DAGK-ϵ, one of the eight genes encoding an enzyme that catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid, selectively phosphorylates arachidonoyl-DAG. To define the significance of the gene DAGK-ϵ, we transfected human retinal pigment epithelium (ARPE-19) cells with DAGK-ϵ or DAGK-z (encoding a DAGK not selective for arachidonic acid) expression vectors. Methods:ARPE-19 cells were transfected with expression vectors of DAG kinases using DOTAP. Promoterless green fluorescent protein (GFP) expression vector was co-transfected to assess transfection efficiency. Four hours later, cells were fed normal medium and incubated 12 h. Then cells were serum-starved for 8 h before the addition of inducers. Genetically engineered EcR 293 cells containing DAGK-ϵ and DAGK-z expression vectors, used for comparison, were grown in 6-well plates to near confluency, serum-starved for 8 h, and challenged with phorbol ester (100 nM) for 8 h. Morphological changes were detected by phase-contrast microscopy and apoptotic cell death was monitored by Hoechst staining with confocal microscopy. Results:Overexpression of DAGK-ϵ but not DAGK-z by 100 nM TPA caused a profound alteration of cell morphology in ARPE-19 cells. Hoechst staining of the TPA-induced DAGK-ϵ transfected cells revealed an accelerated apoptotic cell death as compared to DAGK-z-transfected cells. Genetically engineered EcR 293, which contain DAGK-ϵ expression vector, follow the same pattern under the internal overexpression of the DAGK-ϵ induced by TPA. Conclusion:DAGK-ϵ but not DAGK-z specifically exerts an effect on cell morphology and apoptotic cell death. The overexpression of DAGK-ϵ may lead to abnormal expression of other genes that participate in apoptosis. These events may modulate critical signaling in RPE function and in pathologic conditions (e.g., pathoangiogenesis, aging). Supported by EY05121, Marilyn Rosenson Fund, and Eye, Ear, Nose and Throat Foundation.
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