title: Phospholipase C/Protein Kinase C pathway is essential for corneal re-epithelialization induced by Ap(4)A.
abstract: OBJECTIVE
  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: Departamento de Bioquímica, EU Óptica, Universidad Complutense de Madrid, Madrid, Spain.
authors: Aránzazu
authors: A
authors: Mediero
authors: Almudena
authors: A
authors: Crooke
authors: Ana
authors: A
authors: Guzmán-Aránguez
authors: Jesús
authors: J
authors: Pintor
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002465:Cell Movement; D003315:Cornea / Q000201:enzymology / Q000473:pathology; D065306:Corneal Injuries; D015226:Dinucleoside Phosphates / Q000494:pharmacology*; D004195:Disease Models, Animal; D019573:Epithelium, Corneal / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D005131:Eye Injuries / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D015290:Second Messenger Systems; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*; D014945:Wound Healing / Q000187:drug effects*
chemical_list: D015226:Dinucleoside Phosphates; D010975:Platelet Aggregation Inhibitors; C020733:diadenosine tetraphosphate; D011493:Protein Kinase C; D010738:Type C Phospholipases