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VOL. 75 (4), 901-910, 2009 OVER-EXPRESSION OF P2Y2 RECEPTOR AFTER SILENCING...

with many other agonists (for example insulin), when Ap4A binds to
its receptor P2Y2 on the cell surface, the Ap4A-P2Y2 complex undergoes
down-regulation and presumably endocytosis and is subsequently
intracellular lysosomal/proteosomal degradation (12, 13). This down-
regulatory mechanism together with the receptor rate of synthesis
permits to maintain a minimal number of P2Y2 receptor on epithelial
cell surface. This is absolutely relevant since in case that an injury
occur the cornea needs to trigger the wound healing mechanism to
keep this ocular structure perfectly transparent.

All this equilibrium between the production and degradation of
the P2Y2 receptor is altered when a selective siRNA against the
P2Y2 mRNA is tested. When the siRNA starts it effect, there are still
receptors both in their way to degradation and from the Golgi to the
membrane. This fact produces a delay between the moment the siRNA
is applied to the moment when it is possible to see a decrease in the
P2Y2 expression. There is a mechanism of repression of the protein
synthesis that the epithelial cells try to resist, possibly by increasing
the synthesis of P2Y2-mRNA, but which is destroyed by the siRNA.
Nevertheless, when the ability of the oligonucleotide decreases, it
is possible that the overproduction of P2Y2-mRNA can start to
synthesize the protein reason by which we see an over-expression of
P2Y2 receptor 96 hours after the first siRNA instillation.

It is clear that more experiments should be done to confirm this
hypothesis and also it would be interesting to see whether or not this
effect is tissue selective or if this is a general feature of siRNA.

5. ACKNOWLEDGEMENTS

This work has been supported by research grants from
Comunidad de Madrid, NEUROTRANS-CM Ref.: S-SAL-0253-2006,
RETICS RD07/006/0004 and BSCHUCM (GR58/08). AM holds a
fellowship from Universidad Complutense de Madrid. We thank
Penny Rollinson for her help in the preparation of this manuscript.

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