PURPOSE: We have previously demonstrated the importance of P2Y(2) receptors in the corneal re-epithelialization effect triggered by diadenosine tetraphosphate (Ap(4)A). In addition, we have also shown the ERK1/2 and ROCK-I activation in Ap(4)A-wound repair response. Phospholipase C/Protein Kinase C (PLC/PKC) pathway activation has been suggested as a molecular mechanism of growth factors-modulated corneal cell migration and P2Y(2) agonists. Hence, the aim of this study is to investigate the role of PLC/PKC cascade in the modification of re-epithelialization rate triggered by Ap(4)A in an established corneal epithelial cell line (Statens Seruminstitut rabbit cornea [SIRC] cells). METHODS: In wounded confluent SIRC cell monolayers and in the presence or absence of Ap(4)A 100 μM, a group of PLC/PKC inhibitors (U73122 3 μM, Staurosporine 1 nM and Bisindolylmaleimide-I 10 μM) and activator (PDBU 1 μM) were assayed and the migration rate was evaluated. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with or without Ap(4)A, U73122, Staurosporine, Bisindolylmaleimide-I and PDBU. RESULTS: Pre-treatment of wounded SIRC cells with PLC/PKC inhibitors significantly diminished the Ap(4)A-stimulated cell migration rate. Furthermore, PLC/PKC inhibitors also reduced ERK1/2 phosphorylation and ROCK-I activation triggered by Ap(4)A. CONCLUSIONS: The present study shows the involvement of PLC/PKC pathway in the activation of ERK1/2 and ROCK-I downstream signal transduction pathways stimulated by Ap(4)A/P2Y(2) receptor during corneal epithelial wound repair.
PURPOSE: We have previously demonstrated the importance of P2Y(2) receptors in the corneal re-epithelialization effect triggered by diadenosine tetraphosphate (Ap(4)A). In addition, we have also shown the ERK1/2 and ROCK-I activation in Ap(4)A-wound repair response. Phospholipase C/Protein Kinase C (PLC/PKC) pathway activation has been suggested as a molecular mechanism of growth factors-modulated corneal cell migration and P2Y(2) agonists. Hence, the aim of this study is to investigate the role of PLC/PKC cascade in the modification of re-epithelialization rate triggered by Ap(4)A in an established corneal epithelial cell line (Statens Seruminstitut rabbit cornea [SIRC] cells). METHODS: In wounded confluent SIRC cell monolayers and in the presence or absence of Ap(4)A 100 μM, a group of PLC/PKC inhibitors (U73122 3 μM, Staurosporine 1 nM and Bisindolylmaleimide-I 10 μM) and activator (PDBU 1 μM) were assayed and the migration rate was evaluated. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with or without Ap(4)A, U73122, Staurosporine, Bisindolylmaleimide-I and PDBU. RESULTS: Pre-treatment of wounded SIRC cells with PLC/PKC inhibitors significantly diminished the Ap(4)A-stimulated cell migration rate. Furthermore, PLC/PKC inhibitors also reduced ERK1/2 phosphorylation and ROCK-I activation triggered by Ap(4)A. CONCLUSIONS: The present study shows the involvement of PLC/PKC pathway in the activation of ERK1/2 and ROCK-I downstream signal transduction pathways stimulated by Ap(4)A/P2Y(2) receptor during corneal epithelial wound repair.
Authors: Carlos Carpena-Torres; Jesus Pintor; Fernando Huete-Toral; Alba Martin-Gil; Candela Rodríguez-Pomar; Alejandro Martínez-Águila; Gonzalo Carracedo Journal: Int J Mol Sci Date: 2021-11-05 Impact factor: 5.923