ARTÍCULO |
Over-expression of P2Y2 receptor after silencing in corneal wound healing.
Aránzazu Mediero#, Almudena Crooke#, Jesús Pintor*.
Departamento de Bioquímica y Biología Molecular IV. Escuela Universitaria de Óptica. Universidad Complutense de Madrid.
#Estos autores contribuyeron de la misma manera al trabajo.
The cornea is one of the most important components of the optical pathway. It is a multilayered tissue characterized by its transparency, avascularity, the ability to refract light and to filter out incoming ultraviolet radiation. Within the five layers that compound the cornea -epithelium, Bowman´s membrane, stroma, Descemet´s membrane and endothelium- the epithelium is the outer layer and the one that is easily damaged due to diverse factors. These include the entry of a foreign body, any traumatic process, a defect in contact lenses or the use of refractive surgery to correct refractive alterations.
When this happens, a process named corneal wound healing starts to regenerate normal epithelium in order to maintain the correct refraction of light. Corneal wound healing involved three consecutive phases that are part of a continuous process. Animal studies have shown that these three stages are: lag phase (from 0 hours to 10 hours after the wound), cell migration (until 24 to 36 hours after the wound) and cell proliferation (lasting from 24-36 hours after the wound to weeks) (1).
There are many substances present in tears, aqueous humour or released from corneal nerves, that modified the wound healing process after ocular surface injuries (2, 3). Within these molecules we find nucleotides and dinucleotides (3, 4). In our previous works we demonstrated that the dinucleotides can modify rate of corneal re-epithelialization in New Zealand White Rabbits both in vivo and in vitro (3, 5). We have demonstrated, both pharmacologically and with the used of the RNA interference (RNAi) technology, that Ap4A produces acceleration in the rate of corneal re-epithelialization by stimulating to P2Y2 receptors. On the contrary other dinucleotides, Ap3A and Ap5A exert the opposite effect delaying corneal re-epithelialization by binding to a P2Y6 receptor (5, 6).
The aim of this manuscript is to describe the presence of the P2Y2 receptor in the cornea and to see the effect of a siRNA against the P2Y2 receptor in the presence and in the absence of Ap4A.
Male, adult New Zealand White Rabbits were used. All the animals were kept in individual cages with free access to food and water, under controlled cycles (12 hours light:12 hours dark), and the experimental procedures were carried out in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the European Communities Council Directive (89/609/EEC).
2.2. P2Y2 silencing
To design P2Y2 receptor-specific siRNA duplexes, rabbit P2Y2 receptor coding sequence (GenBank accession number EU886321) was submitted to the Ambion siRNA target Finder website (http://www.ambion.com/techlib/misc/siRNA_finder.html) for siRNA prediction. One sequence of nine (max. GC content 60%) suggested candidates was selected (6). Nucleotide sequence of the siRNA target sites was as follows: P2Y2 siRNA #2, 5´-AACCTGTACTGCAGCATCCTC-3´. This siRNAs was obtained from Applied Biosystems, in annealed and lyophilized forms and were suspended in 0.9% NaCl before in vivo use.
2.3. In vivo delivery of P2Y2 siRNA and wounding procedure
The siRNA was applied in one single eye in 10 nmol 0.9% NaCl drops (volume instilled 40 µl) along four consecutive days. The contralateral eye received the same volume of saline solution (0.9% NaCl). Slit-lamp biomicroscopy was performed during instillation process to evaluate possible changes in the cornea.
Corneal wounds were performed 10 hours before the fourth siRNA instillation. After topical anaesthesia (0.4% oxibuprocaine and 1% tetracaine, Alcon Cusi, Barcelona, Spain), corneal wound were made to the epithelium of both eyes by applying a 5-mm disc of Whatman no. 1 paper soaked in n-heptanol (Sigma-Aldrich, St. Louis, MO) as previously described (3). Briefly, discs were place in the centre of the cornea and left there for 30 seconds (7) and after removal of the disc, the eyes were washed with isotonic saline solution.
Ap4A treatment was performed every six hours as described previously (3).
2.4. Immunohistochemistry
12, 24 and 36 hours after epithelium wounding (72, 84 and 96 hours after the first siRNA instillation), rabbits were euthanized with sodium pentothal and eyes were enucleated. Corneas were dissected and fixed with 4% paraformaldehyde in PBS 0.15 M at 4 ºC for 6 hours. After fixation, corneas were embedded in Jung Tissue Freezing Medium (Leica Microsystems, Barcelona, Spain) and 10 µm sections were done. P2Y2 immunocytochemical assay was performed as previously described for cells. Briefly, sections were permeabilized with blocking solution (PBS 1X BSA 3% Triton X-100 FBS 5%) for 1 hour to block the non-specific binding, and after washing with PBS 1X BSA 3%, sections are incubated with primary goat polyclonal anti-P2Y2 (1:50) or PBS 1X BSA 3% for negative controls overnight at 4 ºC. Sections were washed twice in PBS 1X BSA 3% and incubated with the secondary antibody donkey anti-goat IgG-FITC (1:200) for 1 hour at room temperature. Finally, after washing in PBS 1X slices were mounting with Vectashield mounting medium and observed under confocal microscope (Axiovert 200M; Carl Zeiss Meditec GmbH, Jena, Germany), equipped with a Pascal confocal module (LSM 5; Zeiss). All images were managed with the accompanying Pascal software.
3.1. P2Y2 location in the cornea
Immunocytochemical analysis for P2Y2 in the cornea reveals that this receptor is mainly localized in the outer layer of the epithelium (Figure 1), while the inner layers of the epithelium are barely marked. We have not found any P2Y2 signal in the other layers of the cornea, neither in the stroma nor in the endothelium.
3.2. Inhibition of P2Y2 receptor expression by siRNA
After performing the treatments described in Methods, corneas were wounded and the effect of the siRNA against the P2Y2 receptor was tested by immunohystochemical analysis 12, 24 and 36 hours after the healing (72, 84 and 96 hours after the first siRNA instillation). As we can observe, after the corneas were wounded, the P2Y2 receptor was still localized in the outer layer of the epithelium, being this signal higher in control corneas than in Ap4A treated corneas, both at 12 and 24 hours after wounding (Figure 2A and 2B). 36 hours after the wounds were performed, P2Y2 staining was similar in the three different treatments, including the siRNA treated corneas, revealing a full recovery of the P2Y2 receptor (Figure 2C).
These results indicate that silencing the P2Y2 receptor in our model was detected 12 hours after the wound was performed. Nevertheless, P2Y2 receptor signal was again visible 36 hours after the wound had been made.
Figure 1. P2Y2 receptor location in the cornea. Immunocytochemical analysis for the P2Y2 in the cornea revealing the presence of the P2Y2 receptor in the corneal epithelium (green fluorescence by FITC). Image managed with the Pascal software of the Axiovert 200M confocal microscope at 40X magnification.
3.3. Over-expression of P2Y2 receptor after silencing
In half of the siRNA treated corneas, and 36 hours after the wounding (96 hours after the first siRNA instillation), we have observed an increase in the expression of P2Y2 receptors compared with control and Ap4A (Figure 3). In this case, the P2Y2 signal was not constrained to the outer layer of the epithelium, and it was possible to localize P2Y2 receptors in the whole epithelium and in the stroma (Figure 3).
Figure 2. P2Y2 immunostaining of treated corneas after wound. (A) A series of micrographs showing the P2Y2 signal in corneas treated with saline 0.9%, Ap4A 100 µM and siRNA + Ap4A 100 µM, 12 hours after wound. (B) Immunostaining for P2Y2 in treated corneas 24 hours after the wound. (C) A series of micrographs showing the P2Y2 signal in corneas treated with saline 0.9%, Ap4A 100 µM and siRNA + Ap4A 100 µM, 36 hours after wound. Green fluorescence (FITC) localizes P2Y2 receptor while in blue we can observe the nuclear staining for DAPI. Images are managed at a magnification of 40X.
Figure 3. Over-expression of P2Y2 receptor after silencing. Immunostaining for P2Y2 36 hours after wound (96 hours after the first siRNA instillation) where we can observed an increase in P2Y2 expression after siRNA instillation compared with control and Ap4A treated corneas. Green fluorescence (FITC) localizes P2Y2 receptor while in blue we can observe the nuclear staining for DAPI. Images are managed at a magnification of 40X.
As we have previously mentioned, the cornea is formed by five to six different layers, including the outer one, the epithelium. The distribution of the purinergic receptors present in the cornea revealed that P2Y1, P2Y2, P2Y4 and P2Y6 receptor are present this part of the eye (8).
The present experimental work confirms the location of the P2Y2 receptor after corneal wound healing and how when using a siRNA against this receptor there is an initial disappearance of the receptor followed by an over-expression of this protein.
The presence of the P2Y2 receptor in the epithelium is related to the ability of some nucleotides to increase the rate of re-epithelialization after a corneal wound (6). The involvement of metabotropic P2 receptors in corneal wound healing has been also reported by other groups and in all the cases the different researchers report that ATP, UTP and Ap4A accelerate the rate of healing (9-11).
Our IHC results reveal that after wounded, the P2Y2 staining in Ap4A treated lessons is less intense that in control wounds. As happens 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.
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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Dr. Jesús Pintor.
Dpto. de Bioquímica y Biología Molecular IV. Escuela Universitaria de Óptica. Universidad Complutense de Madrid.
C/ Arcos de Jalón, 118. 28037, Madrid. Spain.
Tf.: +34 91 394 68 59. Fax: +34 91 394 68 85.
Email: jpintor@vet.ucm.es