Abstract
Purpose: :
To study the modulation of the proliferation step in corneal epithelial wound healing after treatment with Ap4A and Ap3A in the presence and absence of a siRNA for P2Y2 receptor.
Methods: :
In vitro proliferation assays were performed in serum starved SIRC cells seed in 96 well plates coated with gelatine and treated with different treatments (n = 8 each) (Control; Ap4A or Ap3A 100 uM alone or in the presence of antagonists of the P2 receptors, inhibitors of MAPK, actin cytoskeleton and PLC/PKC) and to quantify the rate of proliferation at 24 and 36 hours from the beginning of the incubations, the CyQUANT NF Cell Proliferation Assay was used. In vivo proliferation assay was performed in wounded corneas treated with NaCl 0.9 % (control wounds), Ap4A or Ap3A 100 uM both alone or in the presence of P2Y2 siRNA, and relative quantification of CCNE1 and CDK2 gene expression was performed by qRT-PCR 24 and 36 hours after the wound.
Results: :
In vitro, both Ap4A and Ap3A produced an increased in proliferation 24 and 36 hours after the first incubation, being this proliferation reduced in the presence of all tested inhibitors in a percentage cover from 30 to 70 % in all cases. The pre-treatment with one dinucleotide in the presence of the other produce a high increased in the proliferation rate, being Ap4A 100 uM + Ap3A 0.1 uM the one which produced the higher increased. qRT-PCR assay for the activation of CCNE1 / CDK2 system reveal that 24 hours after corneal wound in the presence of Ap4A, mRNA expression for CCNE1 was increased while CDK2 is decreased, being both reduced at 36 hours. Pre-treatment with P2Y2 siRNA produce a significant decreased in CCNE1 mRNA expression at 24 hours after wounding and an increased at 36 hours and CDK2 mRNA expression was reverted to control levels. In the presence of Ap3A, CCNE1 and CDK2 mRNA expression after 24 hours of the wound was increased in a significant manner related to control expression, being these expressions potentiated 36 hours after the wound. The pre-treatment with P2Y2 siRNA produced a decreased in CCNE1 mRNA expression after 24 hours related to control, while 36 hours after the wound mRNA levels for CCNE1 remained increased but in levels slighter higher than those for Ap3A alone.
Conclusions: :
Ap4A produced its effect on proliferation by activation of PLC / PKC/ RhoA / ROCK-I pathway and the transactivation of a Tirosin Kinase receptor that trigged the phoshorylation of ERK1/2, by the action of a P2Y2 receptor, while Ap3A produce an increased in proliferation by activation of Erk1/2 and p38 by a P2Y6 receptor.
Keywords: cornea: epithelium • wound healing • receptors: pharmacology/physiology