Activation of Formyl Peptide Receptor-1 Enhances Restitution of Human Retinal Pigment Epithelial Cell Monolayer under Electric Fields

Abstract

Purpose.: This study aimed at identifying the expression of functional formyl peptide receptor (FPR)-1 in human retinal pigment epithelium (hRPE) cells and to evaluate the role of FPR in regulation of wound closure of the hRPE monolayer under electric fields (EFs).

Methods.: The expression of FPR in hRPE cells was analyzed with an immunofluoresence labeling assay and RT-PCR. Cultured wounded hRPE monolayers were exposed to EFs with free serum, 20%, serum, and a classical FPR agonist, N-formyl-Met-Leu-Phe (fMLF), respectively, for 3 hours. Cell monolayer migrations were traced using an image analyzer. Expressions of cell junction molecules were measured by RT-PCR, and the ultrastructure of cell junctions was observed with transmission electron microscopy (TEM).

Results.: The expression of functional FPR was observed and localized along actin filaments in cellular lamellipodia and filopodia. EFs and fMLF significantly increased the migration rates of the wounded RPE monolayer. The migrating distances of monolayers were 24.262 ± 6.82 μm, 40.243 ± 5.069 μm, and 56.926 ± 7.821 μm in cells with free serum, 20% serum, and fMLF under EFs at 3 hours, respectively (P < 0.01). The mRNA expressions of connexin 43(Cx43) and zonula occludens (ZO)-1 were detected in hRPE cells. TEM revealed that cell junctions formed between hRPE cells in the monolayer.

Conclusions.: These results showed for the first time that functional FPR expresses in hRPE cells and that activation of FPR enhances migration of the wounded hRPE monolayer. The mRNA expressions and ultrastructures of cell junctions further demonstrated the RPE sheet as a monolayer migrating under EFs.

Retinal pigment epithelium (RPE) cells form the monolayer underneath photoreceptors and play an important role in supporting the neural retina. ¹ When the RPE monolayer is wounded, such as in exudative (wet) and nonexudative (dry) age-related macular degeneration (AMD), rapid migration and proliferation of nearby RPE cells across the wound surface, a process called restitution, is desirable. Because damage to RPE cells is thought to impede the recovery of visual function, ² the wound should be covered by restituted RPE cells as soon as possible, and these RPE cells need to be fully differentiated to functionally support the photoreceptors. In response to pathologic conditions, RPE cells initiate a wound-healing process by migration. The cell migration is a complex process in which secretion of certain cytokines, growth factors, and remodeling of the extracellular matrix play important roles. ^3,4 Many receptors of tyrosine kinases have been shown to regulate RPE migratory responses. ^5,6  

Wound healing is essential for maintaining the integrity of the epithelial surface in organisms. During this process, an endogenous electric field was detected and proved to be important for the re-epithelization of the wounded epithelium. ^{7 –10} Studies have shown that many kinds of cells respond to direct current electric fields (EFs) by directed migration, known as galvanotaxis or electrotaxis. ^{11 –17} Several researchers reported that directional migration of a single cell, such as a keratinocyte or corneal epithelium, ^13,18 but not the cellular monolayer was observed in an electric field. Furthermore, many types of cells, including cell sheets, migrate in specific directions when exposed to a small applied EF, similar to those found endogenously in magnitude. ¹⁵ Wang et al. ¹⁵ investigated wound healing of a lens epithelial monolayer under applied physiological EFs. Recently our group observed the effects of EFs on a cathodal-directed migration of a human RPE (hRPE) monolayer. The distribution of F-actin and β₁ integrin in the cells was polarized to the cathode, and the expression of the molecules in both mRNA and protein levels was obviously increased. ¹⁹ Although the wounded RPE monolayer at early confluent was described in the experiments, ²⁰ the cell junctions of the RPE sheet were not proved to show it in a real monolayer. In these experiments, we expected to establish a method to accelerate directed migration of relatively healthy RPE cells near the lesion to re-epithelization of the wounded area, such as in AMD. However, the migration rate of RPE cells exposed to EFs only was not satisfactory in our previous studies. ^19,20 Therefore, we tried to find some agents to promote the migration rate more in addition to EFs. 

Formyl peptide receptor (FPR)-1, a chemoattractant receptor, is mainly expressed in phagocytic cells and plays an important role in host defense against pathogen infection. ²¹ It was recently reported that activation of FPR, by its chemotactic peptide ligand N-formyl-Met-Leu-Phe (fMLF), promotes the directional migration of human glioblastoma cells. ²² However, there have not been any reports on the expression of FPR in hRPE cells and its role in migration of wounded RPE monolayer. 

In the present experiment, we first identified the expression of functional FPR in RPE cells and then showed that activation of FPR could enhance migration of a wounded RPE monolayer under EFs. Second, we also demonstrated the cell sheet as a cell monolayer when migrating under effects of EFs and activation of FPR with Connexin 43 (Cx43), E-cadherin, zonula occludens (ZO)-1, and cell junctions by RT-PCR and transmission electron microscopy (TEM). 

Materials and Methods

Cell Culture

Human RPE cells were harvested from keratoplasty donor eyes within 24 hour post mortem; their use was approved by the Ethics Committee of the Fourth Military Medical University and followed the tenets of the Declaration of Helsinki. The isolation and cultivation of RPE cells were performed as described previously. ²³ The cells were confirmed to be stain negative for von Willebrand's factor (factor VIII) and glial fibrillary acidic protein and positive for cytokeratin (Dakopatts, Glostrup, Denmark). The cells in 25 cm² cell cultured flasks were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 U/mL penicillin G, l-glutamine (0.292 mg/mL), and 100 μg /mL streptomycin at 37°C in a 5% CO₂ and 95% air-humidified incubator. Once cells reached confluence as assessed by microscopy, they were passaged using the trypsin-EDTA solution. All experiments were performed using cells from passages 4 to 7. 

RPE Cell Monolayer Preparation and EF Exposure

The RPE cells were trypsinized after 5 to 7 days of culture, then collected and seeded in a culture chamber formed by two parallel glass coverslips (22 mm ×10 mm), and a 5 mm-length-wound was created by a straight-edged razor blade. The coverslips were fixed to the base of a 100-mm dish with silicone grease (DC4; Dow Corning, Midland, MI), as described by Han et al. ¹⁹ After an observed exposure to EFs for 3 hours, the cells were maintained away from EFs. EFs were applied also as described by Han et al. ¹⁹ Briefly, a coverglass roof was applied to the trough with silicone grease to make a dimension of 22 mm × 10 mm × 0.3 mm. Agar salt bridges of 15 cm long were used to connect Ag/AgCl electrodes in beakers of Steinberg solution to pools of the excess medium at both sides of the chamber. RPE cells were exposed to EFs of 6 V/cm. The cells were all incubated in a 5% CO₂ incubator at 37°C for 3 hours. Field strengths were measured directly throughout the experiment, and no fluctuation was observed (Fig. 1). Cells without exposure to EFs served as controls. During the experiments, images of the cells were obtained every 1 hour with an image analyzer (Axiovert 25; Zeiss, Berlin, Germany). 

Figure 1.

 

Electrical contact is made to the chamber by inserting a glass chamber filled with a salt solution gelled with 1% agar through holes in the lid of the chamber. One end of each chamber rests in a pool of culture medium continuous with the medium in which the cells are growing; the other end is placed in a beaker of saline. Ag/AgCl electrodes in each beaker are attached to a direct-current (DC) power supply. The field strength is checked periodically by measuring the voltage drop across the length of the central trough directly using a voltmeter.

Effect of Functional FPR on Migration of Wounded Monolayer

The wounded hRPE monolayers were washed to remove cellular debris and then cultured with free serum, 20% serum, and fMLF (500 nM; Merck, Darmstadt, Germany) with or without N-tert-butoxycarbonyl-Met-Leu-Phe (Boc, 100 μg/mL; Merck), which is the inhibitor of FPR, respectively. In our preliminary experiments, three dosages of 250 nM, 500 nM, and 1000 nM fMLF were chosen. However, the migration distance of the cell sheet treated with 250 nM fMLF was much shorter than that with 500 nM, while the distance with 500 nM fMLF was almost equal to that with 1000 nM fMLF. Therefore, we chose only 500 nM of fMLF in the subsequent experiments. Cells were treated with fMLF without addition of any serum. The wounded cellular layers were then exposed to EFs and imaged at 0, 1, 2, and 3 hours on a microscope (Axiovert; Carl Zeiss Meditec Berlin, Germany) with an attached charge-coupled device camera. The wounded cell monolayer cultured with fMLF without exposure to EFs served as a control. Wound widths were measured from the images using commercial software (Image-Pro Plus; Media Cybernetics, Inc, Bethesda, MD). Ten measurements along the wound length were averaged to determine wound widths and the distance (μm) of the monolayer having migrated from the wound edges into the wound space. The methods could control any minor variation in the widths of initial wounds. 

Immunostaining of FPR in hRPE Cells

Cells were washed to remove cellular debris and then treated with or without 20% serum or with fMLF (500 nM). One hour later, the culture medium in the cell chamber was aspirated, and cells were immediately fixed with 4% paraformaldehyde (Sigma, St. Louis, MO) in PBS for 10 minutes. Cells were then permeabilized by incubation in 0.1% Triton X-100 (Sigma) for 5 minutes and washed twice in PBS containing 1% bovine serum albumin. For FPR staining, the cells were incubated with anti-FPR antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. After being washed three times in PBS, the cells were treated with tetramethylrhodamine isothiocyanate–conjugated secondary antibody (1:200; Jackson Laboratory, Bar Harbor, Maine) at 37°C for 1 hour. For F-actin staining, the cells were then permeabilized by incubation with FITC-conjugated phalloidin (1 μg/mL; Sigma) for 45 minutes at room temperature. The nuclei of cells were counterstained with DAPI (1 μg/mL; Sigma) for 8 minutes at room temperature and rinsed with PBS. Mounted slides were visualized and photographed using a fluorescence microscope (Olympus FV100; Olympus, Tokyo, Japan). Omitting of primary antibodies and incubated with secondary antibody alone served as controls. 

RT-PCR

The cellular monolayer that had been exposed under EFs for 0 hours, 15 minutes, 30 minutes, 1 hour, and 2 hour were isolated as previously described by Hou et al. ²⁴ Total RNA was extracted from 5 × 10⁶ RPE cells into reagent (TRIzol; Molecular Research, Cincinnati, OH) according to the manufacturer's instructions. RNA purity was estimated by measuring optical density at 260 nm. The cDNA product was subjected to 30 to 35 cycles of amplification in a 20 μL reaction system. PCR-amplified products were shown via agarose gel containing 0.5 mg/L ethidium bromide for gel electrophoresis. GAPDH mRNA was used to ensure equal loading. Oligonucleotide sequences of human Cx43, ²⁵ E-cadherin, ²⁶ ZO-1, ²⁷ FPR, ²⁸ and GAPDH ²⁹ primers used in the RT-PCR are shown in Table 1. 

Table 1.

 

Oligonucleotide Sequences of Human Cx43, E-cadherin, ZO-1, FPR, and GAPDH Primers Used in RT-PCR

Primer Binding Sites	Primer Sequences	Amplicon (bp)	Temp (°C)
Cx43	Forward: 5′-GGGTTAAGGGAAAGAGCGACC-3′	227	55
	Reverse: 5′-CCCCATTCGATTTTGTTCTGC-3′
E-cadherin	Forward: 5′-TCGACACCCGATTCAAAGTGG-3′	194	60
	Reverse: 5′-TTCCAGAAACGGAGGCCTGAT-3′
ZO-1	Forward: 5′-CGGTCCTCTGAGCCTGTAAG-3′	435	55
	Reverse: 5′-GGATCTACATGCGACGACAA-3′
FPR	Forward: 5′-CTCCAGTTGGACTAGCCACA-3′	500	62
	Reverse: 5′-CCATCACCCAGGGCCCAATG-3′
GAPDH	Forward: 5′-GAAGTGAAGGTCGGAGTCA-3′	402	55
	Reverse: 5′-TTCACACCCATGACGAACAT-3′

Transmission Electron Microscopy

TEM to determine the cell junction ultrastructure of RPE cell monolayer under EFs was done essentially as described previously. ³⁰ Briefly, RPE cells (10⁵) were plated on the special glass coverslips and allowed to attach overnight. The cells were then treated with free serum (control), 20% serum, and fMLF (500 nM) for 1, 2, and 3 hours at 37°C. The cells were harvested by trypsinization, washed twice with PBS, and fixed with 2.5% ice-cold electron microscopy grade glutaraldehyde in 0.1 M PBS (pH 7.2). The specimens were rinsed with PBS, postfixed in 1% osmium tetroxide, dehydrated through a graded series of ethanol (30–90%), and embedded in Epon 812 resin. Ultrathin (100 nm) sections were cut using a LKB NOVA ultramicrotome, stained with 2% uranyl acetate and lead citrate, and examined on a transmission electron microscope (JEM-2000EX; JEOL, Tokyo, Japan). 

Statistic Analysis

For all assays, mean ± SD from the experiments was calculated. The data were analyzed for significance by ANOVA, or the paired Student‘s t-test. P < 0.01 was considered to be statistically significant. Each test was done in triplicate. 

Results

FPR Localization in hRPE Cells and mRNA Expression of FPR

To study the expression of active FPR in hRPE cells and observe the localization, we used RT-PCR and an immunofluorescence assay. FPR mRNA was not detected within hRPE cells cultured with normal culture condition. FPR is specifically activated with fMLF at nanomolar concentration (500 nM), but low-affinity fMLF receptor, FPRL1, is not activated with fMLF at nanomolar concentration (500 nM). ³¹ After fMLF was pretreated, the FPR mRNA was detected. The expression of FPR was 0.62 ± 0.03 at 1 hour after fMLF was pretreated, based on densitometric analysis. We found that FPR mostly expressed on the cell surface and localized along actin filaments in lamellipodia and filopodia by immunofluoresence assay (Fig. 2). The F-actin bundles accumulated at the lateral borders of hRPE cells. No staining was observed in cells incubated without primary antibodies and with secondary antibody alone as expected. 

Figure 2.

 

(A) The expression of FPR (red) in hRPE cells treated with fMLF (500 nM) for 1 hour. hRPE cell nucleus was labeled DAPI. Bar, 30 μm. (B) hRPE cells treated with fMLF (500 nM) express FPR mRNA in the cells.

FPR Activation Enhances hRPE Cell Wound Closure

To examine the role of FPR in regulation hRPE cell restitution, a single wound was created through the confluent hRPE cell monolayer using a razor blade. Cell monolayers were cultured with free serum, 20% serum, and fMLF (500 nM), and cultured with fMLF no EFs, respectively. As shown in Figure 3, EFs and fMLF significantly increased the rate of wound closure. The migrating distance of hRPE monolayers was measured as 10.15 ± 3.505 μm, 22.825 ± 8.523 μm (2.2-fold), and 36.262 ± 6.82 μm (3.6-fold) in cells cultured with free serum, 20% serum, and fMLF (500 nM) under EFs at 1 hour, respectively (P < 0.01). At 2 hours, the migration rates were generally slow. At 3 hours, the migrating distances were measured as 24.262 ± 6.82 μm, 40.243 ± 5.069 μm (1.7-fold), and 56.926 ± 7.821μm (2.4-fold) in cells cultured with free serum, 20% serum, and fMLF under EFs at 3 hours, respectively (P < 0.01). Thus, EF exposure significantly enhances hRPE cell restitution, especially with FPR activation. Otherwise, the migration distance of a cell monolayer cultured with fMLF and Boc and fMLF alone without exposure to EFs was much shorter than that of a cell monolayer cultured with fMLF (500 nM), as shown in Figure 3. 

Figure 3.

 

FPR activation enhances wounded hRPE monolayer mingration. Confluent RPE cellular monolayers were wounded and incubated with free serum, 20% serum, fMLF (500 nM), and fMLF+Boc (100 μg/mL), respectively under EFs for 1–3 hours and incubated with fMLF but no EFs served as a control. (A) Differential interference contrast microscopy images. Bar, 50 μm. (B) The presence of fMLF significantly enhances wound closure. All data shown represent mean ± SE (*P < 0.01).

mRNA Expression of Cell Junction Molecules

RT-PCR was carried out to investigate the expression of mRNA of Cx43, E-cadherin, and ZO-1 of hRPE cells. As shown in Figure 4, the mRNA expression of Cx43 and ZO-1 were observed in hRPE cells cultured with free serum, 20% serum, and fMLF exposed to EFs, respectively. The mRNA of E-cadherin was detectable at different times in cells cultured with free serum, 20% serum, and fMLF under EFs (Fig. 4). 

Figure 4.

 

The mRNA expression of cell junction molecules in hRPE cells cultured with free serum (A), 20% serum (B), and fMLF (C) under EFs. Changes in mRNA expression of CX43 and ZO1 began at 15 minutes. The mRNA of E-cadherin was detectable at 2 hours, 30 minutes, and 1 hour in the cells with free serum, 20% serum, and fMLF, respectively. All data shown in the upper figures represent mean ± SE.

Ultrastructure of Cell Junction

TEM revealed that the intercellular junctional complex, desmosome-like structures, a zonula occludens, and gap junctions existed between the cells of hRPE monolayers (Fig. 5). 

Figure 5.

 

Ultrastructures of cell junctions in hRPE cellular monolayers, including intercellular junctional complex (A), desosome-like structures (B), a zonula occludens (short arrows) and a zonula adherences (long arrow) (C), and a zonula occludens (short arrow) and two gap junctions (long arrows) (D). Bars: (A) 200 nm; (B, C) 100 nm; (D) 50 nm.

Discussion

FPR is an important chemoattractant receptor, and expression of FPR has been identified mainly in phagocytic cells, including monocytes, neutrophils, and dendritic cells. ³¹ FPR is the prototypical high-affinity formyl peptide receptor that regulates leukocyte activation and chemotactic responses. Very recently some other nonleukocytic cells, such as epithelial cells, lung epithelial cells, and hepatocytes, have been demonstrated to express FPR. ³² Furthermore, activation of FPR by its selective agonist (fMLF) has been shown to simulate migration, motility, and adhesion of these epithelial cells. ^{33 –35} Viswanathan et al. ³⁶ found that human marrow-derived mesenchymal stem cells express FPR and FPRL1. De Paulis et al. ³⁷ showed that Helicobacter pylorus–derived Helicobacter pylori (2–20) promotes migration and proliferation of gastric epithelial cells by interacting with FPR in vitro and accelerates gastric mucosal healing in vivo. Babbin et al. ³⁵ reported that FPR was specifically activated using fMLF at nanomolar concentration (500 nM). The low dosage of fMLF does not activate the low-affininty fMLF receptor, FPRL1. However, in our experiments, three dosages of 250 nM, 500 nM, and 1000 nM were chosen. As a result, the effect of the dosage of 500 nM is better than the other two. In the experiment conducted by Babbin et al. ³⁵ they also chose a single dose of 500 nM, but the best dosage of fMLF needs further investigation. In the present study, we for the first time found that hRPE cells express functional FPR and that its specific agonist (fMLF) stimulates migration of the cells. This finding suggests that FPR has a potential role in wound healing, tissue remodeling, and various functional aspects related to hRPE migration. 

An endogenous EF is created immediately after epithelial injury and may be one of the earliest extracellular cues that can induce directional cell migration. There is convincing evidence that an endogenous EF is necessary for normal wound epithelization, which does not occur at normal rates when the wound-induced EF is compromised. ³⁸ There are also several claims of enhanced wound healing by EF application to skin wounds. ³⁹ In the study, we first showed the migration of a wounded RPE monolayer under EF exposure. Previous such experiments used separate RPE cells and observed a single cell migrating into the wounded area. However, in this study, we found that FPR localized along actin filaments in the cellular lamellipodia and filopodia. The actin cytoskeleton of eukaryotic cells is responsible not only for cell morphology, but also for many cellular processes, including cell migration and cell division. We have shown that an applied direct current EF enhanced the healing of RPE monolayer wounds. Both EFs and fMLF induce peripheral assembly and cathodal distribution of F-actin in RPE cells. This could be due to the new actin structures and selective accumulation of actin in the cytoplasm. EFs also induced cathodal accumulation and colocalization of FPR and F-actin. Under exposure to EF, FPR stimulation significantly enhances cellular migration rates. There is evidence to suggest that FPR forms molecular complexes with the actin cytoskeleton that regulates its ligand affinity, plasma membrane distribution, and possibly actin polymerization. ⁴⁰  

Cell junction is a characteristic element of epithelial cells, and its assembly and maintenance are parts of the cellular differentiation and polarization process of epithelial cells and tissues. ⁴¹ The expression of cell junction indicates wound closure of cellular monolayer and tissue wound repair. Connexins (Cx's) comprise a family of transmembrane proteins, which form intercellular channels between plasma membranes of two adjoining cells, commonly known as gap junctions. Recent reports revealed that Cx proteins interact with diverse cellular components to form a multiprotein complex, which has been termed a “Nexus.” Potential interaction partners include proteins such as cytoskeletal proteins, scaffolding proteins, protein kinases, and phosphatases. These interactions allow correct subcellular localization of Cx's and functional regulation of gap junction–mediated intercellular communication. Evidence is accruing that Cx's might have channel-independent functions, which potentially include regulation of cellular migration, polarization, and growth control. ⁴² ZO-1 as a scaffold protein linked the actin cytoskeleton with proteins in the leading edge of the lamellipodia or in cell-cell junctions. ⁴³ E-cadherin is the major component of the adherens junction and is crucial for the establishment and maintenance of polarized and differentiated epithelia, in development and in adult tissues. ⁴⁴ Hage et al. ⁴¹ found that active Rac1 destabilizes E-cadherin–mediated cell-cell adhesion in pancreatic carcinoma cells by interacting IQ motif containing GTPase-activating protein, which is associated with a disassembly of E-cadherin–mediated cellular adhesion. We found expression of Cx43 and ZO-1 in hRPE monolayers, indicating that cell monolayers with cell junctions, not separate cells, migrate toward the cathode. In directed cell migration, acquiring spatial asymmetry is a necessary and early event in establishing polarized leading and trailing edges, and accumulation of F-actin is a driving force for membrane protrusion during this process. The tight junction molecules, especially ZO-1, were localized to cell-cell contact regions in the retinal pigment epithelium, both in the monolayer and in the trilayer system. ⁴⁵ Yoeruek ⁴⁶ demonstrated that the expression for ZO-1 and Cx43 was found when the HCECs were grown to confluence and formed a continuous viable monolayer. Therefore the mRNA expression of cell junction, especially ZO-1 and Cx43, indicated cells as a monolayer migrating under EF and active FPR. 

In summary, these results showed for the first time that functional FPR express in hRPE cells and that activation of FPR enhances migration of the wounded hRPE monolayer. The mRNA expressions of Cx43 and ZO-1 and ultrastructural cellular junctions shown by TEM further demonstrated the RPE sheet as a monolayer migrating under EFs. However, the molecular mechanisms that control and regulate these effects need further investigation. 

Footnotes

 Supported by the National Natural Science Foundation of China (Grant No. 30670510) and sponsored in part by the Alexander von Humboldt Foundation in Germany.

Footnotes

 Disclosure: X.-G. Zhang, None; Y.-N. Hui, None; X.-F. Huang, None; H.-J. Du, None; J. Zhou, None; J.-X. Ma, None