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Corneal Re-epithelialization Stimulated by Diadenosine Polyphosphates Recruits RhoA/ROCK and ERK1/2 Pathways

From Departamentos de ¹Bioquímica y Biología Molecular IV y de ²Óptica II, E.U. Óptica, Universidad Complutense de Madrid, Madrid, Spain.

PURPOSE. To investigate the role of ERK1/2 and RhoA/ROCK intracellular pathways in the modification of corneal re-epithelialization when stimulated by the diadenosine polyphosphates Ap₄A and Ap₃A.

METHODS. In wounded confluent SIRC (Statens Seruminstitut rabbit cornea) cell monolayers and in the presence or absence of Ap₄A or Ap₃A 100 µM, a battery of P2 receptor antagonists and inhibitors of tyrosin kinases, MAPK, and cytoskeleton pathways (AG1478 100 µM, U0126 100 µM, Y27632 100 nM, and (–)-blebbistatin 10 µM; n = 8 each) were assayed. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with Ap₄A and Ap₃A (100 µM), with or without suramin, RB-2, U0126, and Y27632. The intracellular distribution of pERK and ROCK-I was examined in the presence of Ap₄A or Ap₃A (100 µM) with U0126 and Y27632 (100 nM).

RESULTS. In the presence of Ap₄A, U0126, Y27632, AG1478, and (–)-blebbistatin, reduced the migration rate compared to the effect of Ap₄A alone (P < 0.0001, P < 0.001, P < 0.01, and P < 0.1 versus Ap₄A, respectively). In the presence of Ap₃A 100 µM, U0126 and Y27632 accelerated the migration rate when compared with the effect of Ap₃A alone, whereas AG1478 and (–)-blebbistatin (P < 0.0001 versus Ap₃A) slowed the migration rate. Western blot assays demonstrated that both dinucleotides activated the ERK1/2 pathway but only Ap₄A activated the ROCK-I pathway. The intracellular distribution of pERK1/2 and ROCK-I reflected cross-talk between these two pathways.

CONCLUSIONS. The activation of the Ap₄A/P2Y₂ receptor, accelerates corneal epithelial cell migration during wound healing with the activation of MAPK and cytoskeleton pathways, whereas activation of the Ap₃A/P2Y₆ receptor signals only the MAPK pathway.