M Jin1, S He, V Wörpel, S J Ryan, D R Hinton. 1. Departments of Pathology and. Ophthalmology, Keck School of Medicine at the University of Southern California, Los Angeles, USA.
Abstract
PURPOSE: Adhesion and migration of retinal pigment epithelial (RPE) cells to provisional extracellular matrices (ECM) is important in the development of epiretinal membranes found in proliferative vitreoretinopathy (PVR). Tumor necrosis factor alpha (TNF-alpha) is found in PVR membranes and regulates many functions of RPE cells. In this study, the effects of TNF-alpha on adhesion and migration of RPE cells to various components of ECM were examined and elucidation of the mechanism of the response was attempted. METHODS: Mitogen activated protein kinase (ERK1/2; MAPK) activation was measured by immunoblot. RPE cells pretreated with TNF-alpha (10 ng/ml) or TNF-alpha + PD98059 (a specific inhibitor of MAPK, 30 microM) for 24 hours were compared with control RPE. Attachment was measured by modified MTT assay on fibronectin and collagen types I and IV. Spreading was measured by staining with fluo3-AM and confocal laser scanning microscopy. Migration of RPE cells on substrates was determined by Boyden chamber assay using PDGF-BB (20 ng/ml) as a chemotactic factor. Integrin expression was determined by flow cytometry and RT-PCR. RESULTS: TNF-alpha rapidly activated MAPK and increased the extent of attachment, spreading and migration on fibronectin and collagen type I (P < 0.01) but not on collagen type IV. TNF-stimulated RPE cells showed increased mRNA and surface protein expression for alpha1 and alpha5 integrin (P < 0.01) but not alpha3 integrin subunit. Neutralizing the anti-alpha1 antibody inhibited migration on collagen type I, whereas alpha5 antibody inhibited fibronectin-induced migration. Treatment with both TNF and PD98095 reduced attachment and migration on provisional ECM and reduced the upregulated integrin expression to control levels. CONCLUSIONS: After treatment with TNF-alpha, there is increased expression of specific integrins associated with increased adhesion and migration on provisional ECM (fibronectin and collagen type I). This effect is mediated, at least in part, by activation of MAPK signaling pathway.
PURPOSE: Adhesion and migration of retinal pigment epithelial (RPE) cells to provisional extracellular matrices (ECM) is important in the development of epiretinal membranes found in proliferative vitreoretinopathy (PVR). Tumor necrosis factor alpha (TNF-alpha) is found in PVR membranes and regulates many functions of RPE cells. In this study, the effects of TNF-alpha on adhesion and migration of RPE cells to various components of ECM were examined and elucidation of the mechanism of the response was attempted. METHODS: Mitogen activated protein kinase (ERK1/2; MAPK) activation was measured by immunoblot. RPE cells pretreated with TNF-alpha (10 ng/ml) or TNF-alpha + PD98059 (a specific inhibitor of MAPK, 30 microM) for 24 hours were compared with control RPE. Attachment was measured by modified MTT assay on fibronectin and collagen types I and IV. Spreading was measured by staining with fluo3-AM and confocal laser scanning microscopy. Migration of RPE cells on substrates was determined by Boyden chamber assay using PDGF-BB (20 ng/ml) as a chemotactic factor. Integrin expression was determined by flow cytometry and RT-PCR. RESULTS:TNF-alpha rapidly activated MAPK and increased the extent of attachment, spreading and migration on fibronectin and collagen type I (P < 0.01) but not on collagen type IV. TNF-stimulated RPE cells showed increased mRNA and surface protein expression for alpha1 and alpha5 integrin (P < 0.01) but not alpha3 integrin subunit. Neutralizing the anti-alpha1 antibody inhibited migration on collagen type I, whereas alpha5 antibody inhibited fibronectin-induced migration. Treatment with both TNF and PD98095 reduced attachment and migration on provisional ECM and reduced the upregulated integrin expression to control levels. CONCLUSIONS: After treatment with TNF-alpha, there is increased expression of specific integrins associated with increased adhesion and migration on provisional ECM (fibronectin and collagen type I). This effect is mediated, at least in part, by activation of MAPK signaling pathway.
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