| BackgroundRetinal pigment epithelial (RPE) cells form the monolayer between photoreceptors and the choroid, which play an important role in supporting the neural retina. RPE is essential for the integrity and function of neural retina. Damages to human RPE monolayer, such as happened in age-related mocular degeneration (AMD), proliferative vitreoretinopathy (PVR), and retinitis pigmentosa, may cause serious loss of vision. Once hRPE layer was wounded, normal hRPE cells surrounding the wound area will migrate and proliferate to cover the area. This process is crucial to recover the structure and function of the retina.During the wound healing process, many factors might play a role. And an endogenous electric field may yield. Moreover, the wound healing of the cornea and the skin can be accelerated by additional EFs. Our research group previously observed that EFs induced directed migration of hRPE cells and that limited period of exposure to EFs under 10 V/cm did not affect the normal viability of human RPE. But EFs exposure alone seems not sufficient to enhance migration of cells to restitute hRPE cell layer. We therefore tried to find some factors which can enhance the cell migration under exposure of EFs.Migration and wound healing process of RPE cells are a complex process. Many cytokines, growth factors take part in this process. Formyl Peptide Receptor (FPR) was first defined biochemically in 1976. Upon ligand binding, FPR undergoes a conformation change enabling interactions with G family proteins. Agonist-induced signaling also results in phosphorylation of protein kinases (PI3K,MAPK and NF-κB) and regulation of celluar activations and migration.Based on our previous study and understanding of FPR, we supposed that activation of FPR may enhance hRPE cell restitution under EFs.Aims1. To identify the expression of functional FPR in RPE cells and then observe that the activation of FPR can enhance migration of wounded RPE monolayer under EFs. And to demonstrate the cell sheet as a monolayer with CX-43, E-cadherin, ZO-1, and cell junctions.2. To evaluate the increase in cell migration to be associated with activation of PI3K/Akt, Cdc42, Rac1 and RhoA.3. To investigate the increase in cell migration to be associated with expressions and secretions of epidermal growth factor (EGF), interleukin 8 (IL-8),monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-β1 (TGF-β1 ) in the cells.Methods1. The expression of functional FPR in hRPE cells was analyzed with RT-PCR. Based on the previous study, the wound hRPE cells were exposed to EFs at 6 V/cm. FPR and F-actin were stained by immunofluoresence lableling assay and observed the location of FPR in hRPE cells and the change of F-actin of the cells. F-actin and Vimentin of hRPE cells were stained by immunity fluorescence labled assay and observed with a confocal microscope before and 1 h, 2 h and 3 h after exposure to EFs. Images of the monolayer migration were obtained at regular intervals under a Zeiss Axiovert microscope every 1 h. Cell junction, including CX-43, E-cadherin and ZO-1, were measured with RT-PCR. And the cell junctions in the hRPE monolayer were observed with transmission electron microscopy (TEM).2. In order to observe FPR activation enhancing the activation of Cdc42, Rac1 and RhoA through PI3K/Akt, the expression of Cdc42, Rac1 and RhoA was determined by RT-PCR and Western blotting. The expression of PI3K/Akt and pAkt was also determined by Western blotting.3. To observe FPR activation enhancing the expressions and secretions of EGF, IL-8, MCP-1 and TGF-β1 under EFs through PI3K/Akt or Rho GTP ases, the mRNA expressions of these cytokines were measured with RT-PCR. ELISA was also used to observe the secretions of them in supernatants.Results1. The expression of functional FPR was observed and localized along actin filaments in the lamellipodia and filopodia of hRPE cells. After treated with Boc and fMLF, the fluorescence value of FPR was decreased. EFs and fMLF significantly increased the migration rate of the wound hRPE monolayer. The migrating distances of hRPE monolayers were measured as 24.262±6.82μm,40.243±5.069μm (1.7-fold) and 56.926±7.821μm (2.4-fold) in hRPE cells cultured with free serum, 20% serum,fMLF under EFs at 3 h, respectively (P<0.01). The mRNA expression of CX-43 and ZO-1 were detected in hRPE cells cultured with free serum, 20% serum and fMLF respectively; but the mRNA expression of E-cadherin was detectable until 2 h, 30 min and 1 h in hRPE cells cultured with free serum, 20% serum and fMLF under EFs respectively. TEM revealed that cell junctions formed. After exposed to EFs, the polyarity and spatial structure of RPE cells was changed, and the lamellipodial and filopodial of hRPE cells were getting along with the direction of the cathode. HRPE cells were migrated to the cathode. After exposure to an EFs for 1 h, the fluorescence value of cytoskeleton was 106.49±8.21, which was higher in the culture with fMLF than that of the control cultures (P<0.05).2. After exposed to EFs, western blotting reached a peak in pAkt of hRPE cells at 30 min. The level of pAkt in hRPE cells were measured as 0.6±0.012, 1.36±0.093 (1.8-fold), 2.44±0.089 (4-fold) in cells cultured with free serum, 20% serum and fMLF under EFs. The inhibitor of FPR, Boc abrogated the fMLF-induced increase in the level of pAkt in hRPE cells. The results of RT-PCR showed that a significant increase in Cdc42, Rac1 and RhoA mRNA at 30 min after exposure to EFs. Western blotting showed that a peak in Cdc42, Rac1 and RhoA protein expression of hRPE cells after exposure to EFs at 1 h. The Cdc42, Rac1 and RhoA of hRPE cells were measured as 3.5±0.2 (5 - fold, 1.6 - fold),5.5±0.21 (1.92 - fold, 1.34 - fold),4.78±0.22 (1.9 - fold, 1.9 - fold) in cell cultured with fMLF compared with the cells cultured with free serum and 20% serum, respectively. This up-regulation of Cdc42, Rac1 and RhoA was blocked by Boc and LY294002.3. After EFs exposure, the mRNA expression of EGF, MCP-1, IL-8 and TGF-β1 reached a peak at 1 h., the secretion of EGF, MCP-1, IL-8 and TGF-β1 reached a peak at 2 h. After activation of FPR, the level of expression and secretion of these cytokines were higher than control culture. This up-regulation of them was blocked by Boc and LY294002.Conclusion1. These results firstly showed that functional FPR express in hRPE cells. FPR localized along actin filaments in lamellipodial and dilopodial extrusions, which were getting along with the direction of the cathode. The FPR activation enhances hRPE cell migration and wound healing. Activation of FPR in hRPE cells and the distribution of the cytoskeleton in hRPE cells were changed after exposure to EFs. The expression of cell junction mRNA and cellular junctions shown by TEM further demonstrated the RPE sheet as a monolayer migrating under EFs.2. EFs induce directed hRPE cell migration and wound healing which can be enhanced by fMLF. The activation of FPR is associated with activation of PI3K/Akt and through PI3K/Akt-dependent activation of Rho GTP ases family members (Cdc42, Rac1, RhoA).3. Activation of FPR could induce expression and secretion of EGF, MCP-1, IL-8 and TGF-β1 in hRPE cells, which could be inhibited by Boc, LY294002 and Y 27632. |
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