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F. Taheri, H.E. P. Bazan; Nuclear expression of a platelet–activating factor receptor in corneal epithelial cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3816.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Platelet–activating factor (PAF), a potent lipid mediator that accumulates in cornea after injury, induces the expression of several genes in rabbit corneal epithelial cells, including a selective up–regulation of gelatinase B (matrix metalloproteinase–9; MMP–9) and tissue inhibitor of metalloproteinases–1 and –2. These gene expressions are inhibited by PAF antagonists that compete for the PAF receptor (PAF–R) at the cell membrane. We investigated the possibility that PAF–R is expressed on the nuclear membrane of corneal epithelial cells to activate the signaling cascade that initiate the transcription of some genes within the cell. Methods: Immunocytochemistry was performed using anti–PAF–R antibody on untreated or NP–40–permeabilized cells from primary rabbit corneal epithelial cells (RCEC) or an immortalized human corneal epithelial cell line (HCEC) as well as isolated nuclei from HCEC. Expression of PAF–R was analyzed by Western blotting in whole–cell lysate, cytosolic and membrane fractions, and in purified nuclear extract of the cells. Results: Immunostaining of untreated RCEC and HCEC with antibody specific to PAF–R revealed a generally distributed expression of the receptor on the plasma membrane with some patchy foci throughout the cytoplasm that were more prominent in the perinuclear area. After permeabilization of the cells, however, the expression was confined to the nuclear membrane in both cell types. Immunostaining with anti–actin and Hoechst labeling of the DNA was utilized for demarcation of the nuclear boundaries. Isolated nuclei from RCEC also showed the expression of PAF–R on the nuclear membrane. Nuclear extracts tested by Western blotting demonstrated the presence of PAF–R. Conclusions: Our findings indicate that PAF–R is present on the nuclear membranes of both rabbit and human corneal epithelial cells. This suggests that synthesis of PAF after activation of phospholipase A2 leads to the release of PAF into the cytoplasm of the cells which activates its nuclear receptor to trigger a series of signaling pathways that, in turn, induce the over–expression of specific genes. Supported by NIH grant EY04928.
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