January 2004
Volume 45, Issue 1
Free
Cornea  |   January 2004
Role of p38 MAP Kinase in Regulation of Cell Migration and Proliferation in Healing Corneal Epithelium
Author Affiliations
  • Shizuya Saika
    From the Departments of Ophthalmology and
  • Yuka Okada
    From the Departments of Ophthalmology and
  • Takeshi Miyamoto
    From the Departments of Ophthalmology and
  • Osamu Yamanaka
    From the Departments of Ophthalmology and
  • Yoshitaka Ohnishi
    From the Departments of Ophthalmology and
  • Akira Ooshima
    Pathology, Wakayama Medical University, Wakayama, Japan; the
  • Chia-Yang Liu
    Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida; and the
  • Daniel Weng
    Department of Ophthalmology, University of Cincinnati Medical Center, Cincinnati, Ohio.
  • Winston W.-Y. Kao
    Department of Ophthalmology, University of Cincinnati Medical Center, Cincinnati, Ohio.
Investigative Ophthalmology & Visual Science January 2004, Vol.45, 100-109. doi:10.1167/iovs.03-0700
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Shizuya Saika, Yuka Okada, Takeshi Miyamoto, Osamu Yamanaka, Yoshitaka Ohnishi, Akira Ooshima, Chia-Yang Liu, Daniel Weng, Winston W.-Y. Kao; Role of p38 MAP Kinase in Regulation of Cell Migration and Proliferation in Healing Corneal Epithelium. Invest. Ophthalmol. Vis. Sci. 2004;45(1):100-109. doi: 10.1167/iovs.03-0700.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

purpose. The purpose of the present study was to examine the roles of signaling pathways potentially activated by TGFβ (i.e., Smad and p38 mitogen-activated kinase [MAPK]) in regulation of cell migration and proliferation of healing mouse corneal epithelium.

methods. Activation of Smads or p38MAPK was evaluated by immunohistochemistry in healing mouse corneal epithelium after debridement. The role of endogenous TGFβ or p38MAPK in epithelial healing was determined in organ-cultured mouse corneas with an epithelial defect, in the presence or absence of a TGFβ-neutralizing antibody or p38MAPK inhibitors, respectively. Cell proliferation was evaluated by incorporation of bromodeoxyuridine.

results. Migrating mouse corneal epithelium had minimal cell proliferation. Smad3 and -4 were found in nuclei of normal corneal epithelium, whereas they were absent in nuclei of migrating cells in association with Smad7 upregulation on epithelial debridement. Administration of TGFβ-neutralizing antibody reduced the protein expression of Smad7 in vivo after a corneal injury. In contrast, phosphorylation and nuclear translocation of p38MAPK were markedly evident in migrating epithelium during healing, but not in uninjured epithelium. In organ culture, addition of p38MAPK inhibitors blocked cell migration more markedly than neutralizing TGFβ-antibody and enhanced cell proliferation in the injured corneal epithelium, in association with phosphorylation of Erk.

conclusions. Endogenous TGFβ enhances migration of corneal epithelium during wound healing in mice. The p38MAPK, but not the Smad, cascade plays a major role in promoting cell migration and in suppressing cell proliferation in migrating epithelium.

Corneal epithelial defects must be rapidly resurfaced to avoid microbial infection and further damage to the underlying stroma. Epithelial healing is achieved by migration of the epithelial sheet to cover the denuded surface and enhanced cell proliferation to reestablish the epithelial stratification quickly after resurfacing. It is of interest to note that in the early phase of healing only one of the two cellular responses, cell migration, takes place, whereas cell proliferation is suppressed. 1 2 Although cell migration promotes rapid reepithelialization, the cessation of cell proliferation may impede healing if such cessation is prolonged. 
Various growth factors, including transforming growth factor-β (TGFβ), are believed to orchestrate the behavior of healing corneal epithelium: for example, cell migration and proliferation, cell death, and protein synthesis. 3 4 5 6 In mammals, three isoforms of transforming growth factor-β, (β1, -2, and -3) are known. Members of TGFβ family are multifunctional cytokines involved in development, tissue repair, and other physiological or pathologic processes. 7 8 9 It has been demonstrated that the TGFβ isoforms and their receptors are present in corneal and limbal epithelia and other supporting tissues (e.g., conjunctiva and tear fluid). 10 Therefore, it has long been speculated that the TGFβ isoforms play pivotal roles in maintaining corneal homeostasis in a paracrine and autocrine fashion. 11 12 13 14 15 TGFβ is believed to inhibit corneal epithelial cell proliferation in vivo, because it reportedly inhibits cell proliferation of cultured keratinocytes and corneal epithelial cells in vitro. 2 16 17 18 This notion is further supported by the observation in which the administration of anti-TGFβ–neutralizing antibodies reduces scar tissue formation in injured corneas 19 and enhances epithelial cell proliferation after a penetrating injury of corneas in mice (Saika S, unpublished observation, 2002). Recently, Zieske et al. 20 reported that epithelial debridement causes an upregulation of TGFβ receptor expression on migrating corneal epithelial cells, suggesting that this ligand may have a pivotal role in modulation of functions of migrating corneal epithelial cells during wound healing. However, the exact mechanism by which TGFβ modulates epithelial cell behavior (cell migration and proliferation) in wound healing remains to be clarified. 
In the present study, we examined the roles of signal transduction pathways that can be activated on endogenous TGFβ stimulation and may regulate epithelial cell migration and proliferation in healing corneal epithelium. To examine whether cell migration may be associated with the suppression of cell proliferation during wound healing, cell proliferation after wounding was determined by incorporation of bromodeoxyuridine (BrdU). Immunohistochemistry was used to determine whether the signaling cascades of Smads and p38MAPK are activated in migrating corneal epithelium after injury. Our data indicate that there was a cessation of cell proliferation and an absence of nuclear Smad3 and -4 in migrating epithelium. In contrast, phosphorylated p38MAPK translocated to nuclei of migrating epithelial cells within 1 hour after debridement. Neutralization of the endogenous TGFβ isoforms resulted in delay in epithelial resurfacing and reduction in p38MAPK activation in organ-cultured epithelium. Finally, specific p38MAPK inhibitors suppressed cell migration but induced cell proliferation, as well as MAPK-Erk activation, in healing corneal epithelium after debridement in organ culture. In our study, p38MAPK signal, but not Smad, played a major role in promoting cell migration and suppression of cell proliferation, possibly by blocking the MAPK-Erk cascade in healing epithelium after corneal epithelium debridement. 
Materials and Methods
Animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committees of Wakayama Medical University and the University of Cincinnati Medical Center. 
Epithelial Defect in Mouse Cornea
Adult male C57BL/6J mice (n = 42) were anesthetized by intraperitoneal injection of pentobarbital sodium (70 mg/kg) or combined xylazine (13 mg/kg) and ketamine (87 mg/kg) and topical oxybuprocaine (Santen, Osaka, Japan), and a corneal epithelial debridement (2 mm in diameter) was performed in one eye, as previously reported. 21 The other eye served as the control. Our previous studies have shown that a 2-mm central corneal epithelial debridement is resurfaced in 24 hours. 21 In the current studies, after different periods of injury (1–22 hours), the animals were given BrdU and killed 2 hours later by CO2 asphyxiation and cervical dislocation, as previously reported. 22 For specimens obtained immediately after epithelial debridement, central corneal epithelium debridement was performed 2 hours after intraperitoneal administration of BrdU, and the experimental animals were killed. Affected eyes from four animals at each time point were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 48 hours. Specimens were dehydrated and embedded in paraffin. 22 Affected eyes from three experimental animals were embedded in optimal cutting temperature (OCT) compound (Miles Inc., Elkhart, IN) and subjected to cryosection. 
Effect of TGFβ-Neutralizing Antibody on Expression of Smad7 In Vivo
In adult male C57BL/6J mice (n = 34) anesthetized as described earlier, the central cornea of one eye was perforated with a 26-gauge hypodermic needle attached to a micropipette. On puncture, 3 μL of neutralizing antibodies against individual and all TGFβ isoforms in PBS was injected into an eye at the same concentration as previously reported. 21 Nonimmune host IgG at a concentration of 10 μg/mL (rabbit or goat; Cappel, ICN Pharmaceuticals, Aurora, OH) was used as the control. The animals were killed at 24 hours, and the enucleated eyes were fixed in 4% paraformaldehyde and subjected to morphologic examination. Paraffin sections from each eye were cut through the central corneal perforation and were immunostained for Smad7, as described later. 
Organ Culture of Injured Eyes in the Presence of TGFβ-Neutralizing Antibody or p38MAPK Inhibitors
We examined the effect of a neutralizing anti-TGFβ antibody or the p38MAPK inhibitors SB202190 and SB203580 on the cell migration and proliferation of corneal epithelial cells. An organ-culture system of debrided corneas was established as previously reported, with a minor modification. 21 An epithelial defect (2 mm in diameter) was created in the center of corneas of 4-week-old C57/BL6 mice under general anesthesia by intraperitoneal injection of pentobarbital sodium. The animals were killed without reawakening, and individual enucleated eyes were then cultured in 1.0 mL of DMEM supplemented with 2.0% fetal bovine serum with either mouse monoclonal anti-TGFβ (β1, -2 and -3)–neutralizing antibody (20 μg/mL; R & D Systems, Minneapolis, MN) or p38MAPK inhibitors (SB202190 and SB203580; 10 μM in 0.5% dimethylsulfoxide; Calbiochem, San Diego, CA), respectively. The control culture contained nonimmune IgG at the same concentration as the neutralizing antibody or 0.5% dimethylsulfoxide, respectively. The closure of the epithelial defect was determined by fluorescein staining after a 2-hour labeling with BrdU after 12, 18, 24, 36, and 48 hours of culture. Four or five eyes were prepared and analyzed for each experimental condition at each time point of the TGFβ neutralization and the p38 MAPK inhibitors, respectively. Cell proliferation of enucleated eyes was determined with BrdU incubation for 2 hours after the surgery (n = 6 at each time point). Eyes were then fixed in 4% paraformaldehyde after the BrdU-labeling and embedded in paraffin. Deparaffinized sections (5 μm thick) were processed for histology and immunostaining for BrdU and phospho-p38MAPK with a mouse monoclonal IgM and IgG anti-phospho-p38MAPK antibody (1:200 or 1:100 dilution in PBS; Santa Cruz Biotechnology, Santa Cruz, CA, and Cell Signaling Technology, Beverly, MA, respectively) in the TGFβ neutralization experiment and p38MAPK inhibitor experiments, respectively. 
Immunohistochemistry
Deparaffinized sections 5 μm thick were processed for immunohistochemistry for Smad3, -4, and -7; phosphorylated p38MAPK; and BrdU. After blocking with 3% dry milk and 5% bovine serum in PBS, the sections were incubated with rabbit polyclonal antibody against Smad3 (1:100 dilution in phosphate-buffered saline [PBS]; Zymed, South San Francisco, CA), goat polyclonal antibody against Smad4 (1:200 dilution in PBS; Santa Cruz Biotechnology), Smad7 (1:400 dilution in PBS; Santa Cruz Biotechnology), or mouse monoclonal IgM anti-phospho-p38MAPK antibody (1:200 dilution in PBS) for 12 hours at 4°C. After washes in PBS, the specimens were treated with peroxidase-conjugated antibodies against each immunoglobulin for 4 hours at 4°C. After another wash in PBS, a peroxidase reaction was performed with 3,3′-diaminobenzidine (DAB), as previously reported. 21 23 After they were counterstained with methyl green, the sections were mounted in balsam and observed under light microscopy. 
Phospho-p38MAPK and phospho-Erk were also detected by immunofluorescent staining. Mouse monoclonal IgG anti-phospho-p38MAPK antibody (1:100 dilution in PBS) and rabbit polyclonal anti-phospho-Erk (1:100 in PBS; Cell Signaling Technology) were allowed to react with specimens overnight at 4°C. After a wash with PBS, they were treated with FITC-conjugated antibodies against each immunoglobulin. FITC was detected under a fluorescence microscope after embedding in a medium containing 4′,6′-diamino-2-phenylindole (DAPI) nuclear staining dye (VectaShield H-1200; Vector Laboratories, Burlingame, CA). 
For BrdU immunostaining, paraffin sections were treated with 2N HCl for 1 hour at 37°C and then washed in PBS before the application of anti-BrdU antibody (11× in PBS; Roche Diagnostics, Mannheim, Germany). Cryosections 7 μm thick were processed for immunostaining with goat polyclonal anti-Smad2 antibody (1:200 dilution in PBS; Santa Cruz Biotechnology). These slides were then processed for secondary antibody reaction, DAB reaction and observed under light microscopy. 
Results
Cell Proliferation and Activation of the Erk-MAP Kinase Cascade in Healing Corneal Epithelium after Epithelial Debridement
Uninjured corneal epithelium and corneal epithelium immediately after epithelial ablation contained occasional BrdU-positive basal cells (Figs. 1aA 1aB 1aC 1aD 1aE 1aF 1aG 1aH 1aI 1aJ 1aK) . At 3 hours (not illustrated) and 6 hours after injury, a few BrdU-positive epithelial cells were observed in the periphery, but none at the edge of the migrating epithelium (Figs. 1aD 1aE) . At 18 hours after injury, BrdU-labeled epithelial cells were primarily found in the basal layer of the limbal-peripheral (Fig. 1aF) and midperipheral (Fig. 1aG) epithelia, whereas the central resurfacing thin epithelium was still completely lacking BrdU-labeled cells (Fig. 1aH) . Twenty-four hours after injury, many BrdU-labeled cells were present in the central regenerated epithelium (Fig. 1aK) , and there was a concomitant decrease of BrdU-labeled cells in the limbal-peripheral and midperipheral corneal epithelium (Figs. 1aI 1aJ , respectively). We divided the corneal epithelium into five zones: the central zone of 200 μm length of the cornea was marked, and the remaining area on each side was then divided into two areas, a midperipheral area and a peripheral area, on each side. The incidence of BrdU-positive cells in these areas was determined and is shown in Figure 1b
Such increased cell proliferation was concomitant with an upregulation of phosphorylation of Erk (Fig. 1c) . Phospho-Erk was faintly detected in the cytoplasm of basal epithelial cells of uninjured epithelium (Fig. 1cA) with occasional nuclear localization, but was not detected in migrating corneal epithelial cells 1 (Fig. 1cC) and 12 (not shown) hours after debridement. The upregulation of phospho-Erk in the cytoplasm of central epithelial cell was observed at 18 hours (Figs. 1cE) . Basal cells were positive for phospho-Erk in the nuclei at 24 hours after debridement (Fig. 1cG)
Subcellular Distribution of Smad Proteins during Wound Healing of Corneal Epithelium
Smad signaling has been implicated in mediating the inhibition of epithelial cell proliferation induced by TGFβ in many organs and cell types. 16 17 18 First, to examine whether the Smad cascade is activated in migrating epithelial cells, we determined the intracellular distribution of Smad family members during corneal epithelium wound healing after an epithelial debridement, using immunostaining with antibodies directed against individual Smad components. 
Figure 2a shows the results of immunofluorescent staining for Smad3 in healing corneal epithelium. Smad3 protein was detected in almost all the nuclei of epithelial cells in all layers of normal uninjured corneal epithelium, although the intensity of immunoreactivity varied considerably among the nuclei (Fig. 2aA) . These observations suggest that constitutive TGFβ signaling through the Smad pathway exists in normal corneal epithelium. This constitutive TGFβ signaling is probably required for the maintenance of corneal epithelium homeostasis. At 6 (Fig. 2aB) and 12 (not shown) hours after injury, migrating corneal epithelial cells showed no nuclear Smad3 staining, but the cytoplasm showed prominent staining for Smad3. Eighteen hours after injury, positive nuclear Smad3 staining reappeared in double-layered epithelium that had resurfaced the denuded cornea (Fig. 2aC) . At 24 hours, nuclear Smad3 staining was again observed in all cells of the regenerated stratified central corneal epithelium (Fig. 2aD) . Anti-Smad3 antibody labeling of nuclei of peripheral epithelium was present during healing intervals lasting from 0 to 24 hours (data not shown). Nonimmune control IgG did not label the corneas (data not shown). Subcellular localization of Smad2 (data not shown) and Smad3 in the corneal epithelium was similar to one another. In Figure 2b , the intracellular localization of Smad4 exhibited a similar pattern to Smad3 during corneal wound healing as shown in Figure 2a . The reappearance of Smad3 and -4 in nuclei is concomitant with BrdU incorporation by epithelial cells of injured corneas at 18 hours after injury. 
Smad7, an inhibitory Smad, inhibits the dimerization and nuclear translocation of Smad3 and -4 and blocks TGFβ signaling. 16 17 18 To determine whether the disappearance of Smad3 and -4 in the nuclei of migrating cells is associated with upregulation of Smad7, the expression pattern of Smad7 protein in healing epithelium was examined by immunohistochemistry (Fig 2c) . In uninjured corneal epithelium (Fig. 2cA) and in corneal epithelium immediately after debridement (not shown) Smad7 protein was very weakly detected in the epithelial cell cytoplasm. At 6 (Fig. 2cB) and 12 (Fig. 2cC) hours after debridement, the cytoplasm of epithelial cells around the defect exhibited marked immunoreactivity to Smad7. Eighteen (not shown) to 24 hours after injury, the immunoreactivity to Smad7 returned to its basal level, as seen in uninjured corneas (Fig. 2cD) . To elucidate further the mechanism of upregulation of Smad7 on injury and its association with the loss of Smad3 and -4 nuclear localization in migrating epithelial cells, the expression patterns of Smad7 were determined in injured epithelium in the presence or absence of neutralizing anti-TGFβ antibodies (Fig. 2d) . Smad7 protein expression was upregulated over the entire epithelium after a penetrating injury of cornea with control PBS injection (Figs. 2dA 2dB 2dC) . Administration of neutralizing antibodies against TGFβ1 (not illustrated), TGFβ2 (Figs. 2dD 2dE 2dF) , and pan anti-TGFβ antibodies (data not shown) reduced Smad7 expression in the central and midperipheral, but not in the peripheral, epithelium, indicating that both endogenous TGFβ1 and -2 are the factors responsible for Smad7 upregulation in injured epithelium. 
Loss of nuclear localization of Smad3 and -4 in migrating corneal epithelial cells implies that Smad signaling may not be directly involved in eliciting cell movement and inhibiting epithelial cell proliferation of migrating cells. This notion prompted us to hypothesize that such cellular behavior in migrating epithelium may be mediated by signaling pathways other than Smads (e.g., p38MAPK cascade). 
Phosphorylated p38MAPK in Corneal Epithelium
To examine the possibility that activation of the p38MAPK cascade may mediate the cell migration and suppression of cell proliferation during corneal epithelial wound healing, subcellular localization of phosphorylated p38MAPK was first determined by immunofluorescent staining with an anti-phospho-p38MAPK antibody. As shown in Figure 3 , nuclear translocation of phospho-p38MAPK was detected in the healing corneal epithelium weakly, but positively, as early as 1 hour (Fig. 3B) and lasted until 24 hours (Fig. 3F) after injury, whereas cells of uninjured epithelium had phospho-p38MAPK mainly in the cytoplasm (Fig. 3A) . Prominent accumulation of phospho-p38MAPK were identified in nuclei of migrating epithelial cells at 6 (Fig. 3D) and 12 (Fig. 3E) hours. At 18 (not shown) and 24 (Fig. 3F) hours after injury it was observed in both the nuclei and cytoplasm of regenerated epithelium. This finding promoted us to hypothesize that signaling mediated by p38MAPK has a major role in regulating cell behavior in healing epithelium. 
Organ-Culture with a TGFβ-Neutralizing Antibody
To determine whether corneal epithelial healing is regulated by endogenous TGFβ through p38MAPK activation, neutralizing antibodies against TGFβ1, -2, and -3 were included in an ex vivo corneal epithelial healing model of cultured eyes (Fig. 4) . The results show that the antibodies delayed the reepithelialization (Fig. 4a) . BrdU-labeled cells were absent in migrating epithelium in control cultures (Figs. 4cA 4cC) , whereas several BrdU-labeled cells were seen in migrating epithelium of anti-TGFβ-antibody–treated corneas (Figs. 4cB 4cD) . Immunofluorescent staining (Fig. 4d) showed marked positive immunoreactivity for phospho-p38MAPK, mainly in basal cells of migrating epithelium in control specimens at each culture interval (Fig. 4dA) , whereas staining for phospho-p38MAPK was very faint in corneal epithelium treated with TGFβ-neutralizing antibody (Fig. 4dB) . These observations support the notion that endogenous TGFβ is one of the cytokines mediating p38MAPK activation, resulting in increases in cell migration and suppression of cell proliferation. 
Organ Culture with p38MAPK Inhibitors
The results of organ-culture experiments revealed that endogenous TGFβ activates p38MAPK and modulates corneal epithelial wound healing. To further confirm this notion, the specific p38MAPK inhibitors SB202190 and SB203580 were added to the medium in an ex vivo wound healing model of cultured mouse eyes. The results show that SB202190 markedly delayed reepithelialization (Fig. 5) . Its presence delayed resurfacing of epithelial defects (Figs. 5aF 5aG 5aH 5aI 5b) , compared with control cultures (Figs. 5aA 5aB 5aC 5aD 5aE 5b) . In the presence of SB202190, the corneal epithelial defect persisted at 48 hours in the inhibitor-treated culture (Figs. 5aI 5b) , whereas all five corneas were resurfaced within 36 hours (Figs. 5aD 5b) in the control experiment. The addition of SB202190 produced more profound inhibitory effects on epithelial cell migration than that by the TGFβ-neutralizing antibody. 
Histology of control experiments showed that the epithelium sheet started to migrate on the denuded central cornea at 12 hours and that the defect was resurfaced by a single-layered epithelium within 36 hours (Figs. 5cF 5cH 5cJ) . In contrast, in the presence of SB202190, the edge of the epithelium remained multilayered (stratified) up to 24 hours in culture (Figs. 5cC 5cE 5cG) resembling that seen at the 0 time point (Fig. 5cA) , and the epithelial migration was noted with a single-cell–layered epithelium at the leading edge after 48 hours in culture (Figs. 5cG 5cI 5cK) . Figure 5d shows the lack of p38MAPK activation in healing epithelium in the presence of SB202190. 
Figure 6 indicates the status of Erk-MAPK and cell proliferation activity in healing, organ-cultured corneal epithelium in the presence of SB202190. As shown in Figure 1 , epithelial cell proliferation was associated with Erk activation, based on increases in its phosphorylation status, which correlated inversely with p38MAPK phosphorylation. Thus, we examined the effects of a p38MAPK inhibitor on Erk activation in epithelial cells of injured corneas. Figure 6a shows that Erk remained highly phosphorylated in the presence of SB202190 compared with the control. We therefore examined cell proliferation in these specimens by BrdU incorporation (Figs. 6b 6c) . At the 0 hour time point (incubated with BrdU 2 hours immediately after debridement) in the absence of the inhibitor, no BrdU-labeled cells were found at the edge of the debrided epithelium, whereas in inhibitor-treated specimen BrdU-labeled cells were seen at the same level found in normal uninjured cornea (data not shown). Throughout the culturing period up to 36 hours, BrdU-labeled cells were detected at the margin of the debrided epithelium in the presence of SB202190, with a peak at 18 hours (Figs. 6bB 6bD 6bF) , while in control culture no BrdU-labeled cells were observed (Fig. 6bA 6bC 6bE) . The cells of regenerated epithelium resurfacing the denuded cornea reentered the cell cycle at 48 hours in control culture as shown (Fig. 6bG) . Figure 6c summarizes the incidence of BrdU-labeled cells at individual time points. Similar results were obtained with another p38MAPK inhibitor, SB203580 (data not shown). 
Discussion
In the present study in mice, phosphorylation and nuclear translocation of p38MAPK occurred as early as 1 hour after epithelial debridement in vivo and activation of p38MAPK was involved in the stimulation of cell migration and cessation of cell proliferation in healing corneal epithelium after debridement. 
As for the role of p38MAPK in cell migration, the present study clearly showed that p38MAPK activation is essential for eliciting corneal epithelial cell migration after epithelial debridement by using specific inhibitors. The p38MAPK cascade is reportedly essential for the expression of α2 integrin for the migration of cultured keratinocytes, or for the migration-invasiveness of cancer cell lines. 24 25 26 In addition, this cascade may be involved in the modulation of cell adhesion through paxillin, which is essential for cell migration. 27 It has been recently demonstrated that TGFβ mediates p38MAPK activation followed by integrin β1 expression leading to epithelial-mesenchymal transition (EMT). 28 EMT is characterized by increased cell mobility and the expression of mesenchymal cell markers (e.g., vimentin or α-smooth muscle actin) in association with the loss of epithelial markers (e.g., cadherins) as well as the loss of cell polarity and cell–cell junction. We previously demonstrated that migrating epithelial cells of injured rabbit corneas (i.e., alkali-burn transiently expressed vimentin). 23 These observations imply that corneal epithelial cells may undergo phenotypic changes to gain migratory characteristics in a way similar to the EMT process mediated through activation of the p38MAPK cascade during wound healing. 
In our organ culture experiment, adding p38MAPK inhibitors enhanced Erk phosphorylation and induced increases in BrdU incorporation in healing epithelium, indicating that activation of p38MAPK suppresses the MAPK-Erk cascade. The effect of p38MAPK inhibitors on cell proliferation regulation is, however, reportedly cell-type dependent—either positive or negative. For example, activation of p38MAPK suppresses cell proliferation of Kaposi’s sarcoma cells, 29 chondrocytes, 30 and vascular endothelial cells. 31 This signaling suppresses cell proliferation through expression of the cdk inhibitor p27kip1 and hypophosphorylation of Rb tumor-suppressor protein in cultured vascular endothelial cells and in some neoplastic cell lines stimulated by activin. 32 33 However, p38MAPK-independent inhibition of MAPK/Erk may also be involved in cessation of cell proliferation in migrating epithelium. For example, in some pancreatic carcinoma cell lines, TGF activates a serine-threonine phosphatase that suppresses the growth-factor–dependent MAPK/Erk cascade by dephosphorylating Erk2. 34 Similarly, in macrophages it has been shown that activation of TGFβ-linked Smad signaling leads to downregulation of p38 by inducing MAPK phosphatase-1. 35 Cells in regenerated central corneal epithelium reenter the cell cycle at 24 hours with nuclear translocation of phospho-Erk after epithelial debridement, although phospho-p38MAPK is also detected in the cell nuclei at relatively higher levels at that time point than that in uninjured epithelium. This finding suggests that MAPK-Erk cascade may overcome proliferation inhibition by phosphorylated p38MAPK in regenerated corneal epithelium at this time point. 
It has been suggested that Smads may mediate TGFβ signaling resulting in inhibition of cell proliferation. 16 17 18 36 37 38 On the contrary, in our results there was a lack of Smad3 and -4 localization in nuclei of migrating corneal epithelial cells after wounding in vivo, which suggests that TGFβ-Smad signaling may not be directly involved in regulation of cell proliferation and migration in mouse corneal epithelium. In addition, Smads were localized in nuclei of uninjured corneal epithelium and restratified epithelium 24 hours after debridement, concomitant with the detection of BrdU-labeled epithelial cells, suggesting that activation of other signaling pathways (i.e., MAPK-Erk pathway) may accelerate cell proliferation at this later healing phase. Alternatively, activation and nuclear translocation of Smad members may occur in migrating epithelial cells presumed to be blocked by an immediate expression of Smad7 in these cells. Upregulation of TGFβ receptors in migrating corneal epithelial cells, reported by Zieske et al., 20 can account for our finding that Smad7 expression is upregulated in migrating epithelial cells as an immediately early gene. Indeed, this notion is further supported by our unpublished data in which the absence of Smad3 does not affect the incorporation rate of BrdU-labeled cells or the resurfacing rate in healing corneal epithelium at the time points of 6, 12, and 24 hours after debridement in Smad3-knockout mice (Saika S et al., unpublished data, 2003). Because Smad3-null mice exhibit hyperproliferation of epidermal epithelium during wound healing after an incision injury, 39 signaling mechanisms regulating cell proliferation may differ in epidermis and corneal epithelium. 
Our results in the experiment using the TGFβ-neutralizing antibody demonstrated that endogenous TGFβ is an important intrinsic factor involved in activation of p38MAPK, as well as cell migration, in injured corneal epithelium. Multiple signaling cascades are activated on TGFβ binding to its cognate receptor—that is, Smads, 40 RhoA-related signals, 41 MAPK-Erk-1/2, 38 stress kinases (i.e., JNK), 42 43 p38MAPK, 26 33 phosphatase2A, 44 or PI3-kinase/AKT. 45 46 Thus, TGFβ can lead to great variations in cellular response, depending on which of these signaling transduction pathways are within a given cell type. 7 17 For example, pathways containing Smad, Rho proteins, and PI3-kinase are involved in cell differentiation-dedifferentiation, whereas the MAPK-Erk pathway modulates cell proliferation and cell survival. 7 17 41  
Although the present study shows that TGFβ is one of the factors regulating corneal epithelial healing through p38MAPK activation, many other growth factors and cytokines are capable of modulating corneal epithelial cell behavior. Wilson et al. 5 demonstrated that epithelial debridement causes release of IL-1 by the injured cornea, which subsequently induces the expression of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) by keratocytes that serve as paracrine modulators of corneal epithelial cells. 47 48 Moreover, it has been reported recently in cultured corneal epithelial cells that p38MAPK is activated by exogenous addition of HGF or KGF, which results in stimulation of cellular migration. 49 In the same study, however, it was also reported that inhibition of p38MAPK activation enhanced EGF and KGF stimulation of Erk-1/2 activity and proliferation. 49 These in vitro results are consistent with our observation that after wounding of organ-cultured corneas, p38MAPK inhibition resulted in stimulation of cell proliferation and enhanced MAPK-Erk phosphorylation. Indeed, besides TGFβ, other growth factors (e.g., HGF and KGF), stress kinases, and inflammatory cytokines are presumably involved in the cascade of p38MAPK activation in vivo. 30 33 46 50 A recent study by other investigators showed that HGF augments migration of cultured corneal epithelial cells by using the MAPK-Erk pathway. 51 In a series of our ex vivo corneal wound-healing studies with organ-cultured eyes, we have observed that the MEK inhibitors U0126 and PD98059 do not significantly alter the resurfacing rate of a corneal epithelial defect (data not shown), an observation consistent with the suggestion that the MAPK-Erk pathway does not have a significant role in epithelial cell migration in situ. Because our ex vivo wound-healing model of organ culture mimics the in vivo condition, it is postulated that the MAPK-Erk pathway may differentially regulate migration of corneal epithelial cells between in vitro and in situ/in vivo conditions. 
 
Figure 1.
 
Distribution of BrdU-labeled cells in healing mouse corneal epithelium after induction of a central epithelial defect 2 mm in diameter. (a) Uninjured corneal epithelium contained occasional BrdU-positive cells (aAC). Immediately after the epithelial ablation (not shown) as well as 6 hours after (aD, aE), the number of BrdU-labeled epithelial cells was similar to that in normal cornea. At 18 hours after injury, BrdU-positive epithelial cells were mainly observed in the basal layer of the limbus (aF) and peripheral epithelium (aG), whereas central resurfacing and migrating monolayer epithelium (aH) lacked BrdU-labeled cells. Twenty-four hours after injury, several BrdU-positive cells appeared in the central regenerated epithelium (aK) in association with the decrease in BrdU-positive cells the limbal (aI) and midperipheral corneal epithelia (aJ). (aB, aE, aG, aJ) Corneal epithelium in the midperiphery, at the wound edge (arrowhead; edge of the remaining epithelium); (aC, aH, aK) Epithelium in the central area. (b) Number of BrdU-positive cells in each zone of the epithelium. Rapid induction of cell proliferation in peripheral epithelium was followed by induction in the midperipheral and then the central epithelium. (c) Phosphorylation of Erk in healing epithelium. Phospho-Erk was faintly detectable in the cytoplasm of basal epithelial cells of uninjured epithelium (cA) with occasional nuclear localization and was not detected in migrating-edge epithelial cells from 1 (cC) to 12 (not shown) hours. The cells then started to upregulate phospho-Erk in the central epithelial cell cytoplasm at 18 hours (cE). Basal cells were positive for phospho-Erk in the nuclei at 24 hours (cG). (cB, cD, cF, cH) The DAPI nuclear staining of the identical area shown in (cA, cC, cE, cG), respectively.
Figure 1.
 
Distribution of BrdU-labeled cells in healing mouse corneal epithelium after induction of a central epithelial defect 2 mm in diameter. (a) Uninjured corneal epithelium contained occasional BrdU-positive cells (aAC). Immediately after the epithelial ablation (not shown) as well as 6 hours after (aD, aE), the number of BrdU-labeled epithelial cells was similar to that in normal cornea. At 18 hours after injury, BrdU-positive epithelial cells were mainly observed in the basal layer of the limbus (aF) and peripheral epithelium (aG), whereas central resurfacing and migrating monolayer epithelium (aH) lacked BrdU-labeled cells. Twenty-four hours after injury, several BrdU-positive cells appeared in the central regenerated epithelium (aK) in association with the decrease in BrdU-positive cells the limbal (aI) and midperipheral corneal epithelia (aJ). (aB, aE, aG, aJ) Corneal epithelium in the midperiphery, at the wound edge (arrowhead; edge of the remaining epithelium); (aC, aH, aK) Epithelium in the central area. (b) Number of BrdU-positive cells in each zone of the epithelium. Rapid induction of cell proliferation in peripheral epithelium was followed by induction in the midperipheral and then the central epithelium. (c) Phosphorylation of Erk in healing epithelium. Phospho-Erk was faintly detectable in the cytoplasm of basal epithelial cells of uninjured epithelium (cA) with occasional nuclear localization and was not detected in migrating-edge epithelial cells from 1 (cC) to 12 (not shown) hours. The cells then started to upregulate phospho-Erk in the central epithelial cell cytoplasm at 18 hours (cE). Basal cells were positive for phospho-Erk in the nuclei at 24 hours (cG). (cB, cD, cF, cH) The DAPI nuclear staining of the identical area shown in (cA, cC, cE, cG), respectively.
Figure 2.
 
Immunohistochemical detection of Smads in healing corneal epithelium. (a) Intracellular localization of Smad3 protein in mouse corneal epithelium. Cells in the uninjured corneal epithelium exhibited both nuclear (yellow arrows) and cytoplasmic Smad3 immunoreactivity (aA). Nuclei of migrating epithelial cells were not labeled by anti-Smad3 antibody at 6 (aB) and 12 (not shown) hours after epithelial debridement (white arrows). At 18 hours after injury, several cell nuclei were labeled by an anti-Smad3 antibody (aC, yellow arrows). Twenty-four hours after injury, central regenerated epithelium (aD) contained cells showing nuclear immunoreactivity for Smad3 with an increase in cytoplasmic labeling. (b) Intracellular localization of Smad4 protein in mouse corneal epithelium. Localization pattern of Smad4 was similar to that of Smad3 shown in (a). In normal uninjured cornea, most of the epithelial cells exhibited a nuclear immunoreactivity for Smad4 (bA, arrowheads). Epithelial cells at the edge of the epithelial defect lacked nuclear Smad4 at 6 (bC, arrows) and 12 (bD, arrows) hours after injury, whereas cytoplasm was positive for Smad4 protein. Cells with nuclei positive (bE, arrowheads) or negative (bE, arrow) for Smad4 were both observed in the double-layered epithelium that resurfaced the central corneal epithelial defect at 18 hours after injury. At 24 hours, central regenerated stratified epithelium (bF, arrowheads) contained many nuclear Smad4-positive cells. No specific immunoreactivity was observed in negative control staining (bB). (c) Protein expression pattern of Smad7 in the healing corneal epithelium. Uninjured corneal epithelium (cA) and that immediately after the epithelial debridement (not shown) exhibited very faint immunoreactivity for Smad7 in the cytoplasm. Epithelial cells around the defect ( Image not available ) were strongly labeled by the anti-Smad7 at 6 (cB) and 12 (cC) hours after debridement. Eighteen (not shown) to 24 (cD) hours after injury, Smad7 immunoreactivity returned to the normal level of an uninjured cornea. (d) Downregulation of Smad7 expression by administration of TGFβ-neutralizing antibody. Smad7 protein expression was found to be upregulated in the entire epithelium at 24 hours after puncture injury of the cornea (dAC). Administration of neutralizing antibodies against TGFβ1 (not illustrated), TGFβ2 (dDF), and pan anti-TGFβ antibodies (not illustrated) reduced Smad7 protein expression in the central and midperipheral, but not in the peripheral, epithelium at the same time point. (dB, dC) High-magnification images of the left- or right-boxed areas in (dA), respectively; (dE, dF) High-magnification images of the left- or right-boxed areas in (dD), respectively.
Figure 2.
 
Immunohistochemical detection of Smads in healing corneal epithelium. (a) Intracellular localization of Smad3 protein in mouse corneal epithelium. Cells in the uninjured corneal epithelium exhibited both nuclear (yellow arrows) and cytoplasmic Smad3 immunoreactivity (aA). Nuclei of migrating epithelial cells were not labeled by anti-Smad3 antibody at 6 (aB) and 12 (not shown) hours after epithelial debridement (white arrows). At 18 hours after injury, several cell nuclei were labeled by an anti-Smad3 antibody (aC, yellow arrows). Twenty-four hours after injury, central regenerated epithelium (aD) contained cells showing nuclear immunoreactivity for Smad3 with an increase in cytoplasmic labeling. (b) Intracellular localization of Smad4 protein in mouse corneal epithelium. Localization pattern of Smad4 was similar to that of Smad3 shown in (a). In normal uninjured cornea, most of the epithelial cells exhibited a nuclear immunoreactivity for Smad4 (bA, arrowheads). Epithelial cells at the edge of the epithelial defect lacked nuclear Smad4 at 6 (bC, arrows) and 12 (bD, arrows) hours after injury, whereas cytoplasm was positive for Smad4 protein. Cells with nuclei positive (bE, arrowheads) or negative (bE, arrow) for Smad4 were both observed in the double-layered epithelium that resurfaced the central corneal epithelial defect at 18 hours after injury. At 24 hours, central regenerated stratified epithelium (bF, arrowheads) contained many nuclear Smad4-positive cells. No specific immunoreactivity was observed in negative control staining (bB). (c) Protein expression pattern of Smad7 in the healing corneal epithelium. Uninjured corneal epithelium (cA) and that immediately after the epithelial debridement (not shown) exhibited very faint immunoreactivity for Smad7 in the cytoplasm. Epithelial cells around the defect ( Image not available ) were strongly labeled by the anti-Smad7 at 6 (cB) and 12 (cC) hours after debridement. Eighteen (not shown) to 24 (cD) hours after injury, Smad7 immunoreactivity returned to the normal level of an uninjured cornea. (d) Downregulation of Smad7 expression by administration of TGFβ-neutralizing antibody. Smad7 protein expression was found to be upregulated in the entire epithelium at 24 hours after puncture injury of the cornea (dAC). Administration of neutralizing antibodies against TGFβ1 (not illustrated), TGFβ2 (dDF), and pan anti-TGFβ antibodies (not illustrated) reduced Smad7 protein expression in the central and midperipheral, but not in the peripheral, epithelium at the same time point. (dB, dC) High-magnification images of the left- or right-boxed areas in (dA), respectively; (dE, dF) High-magnification images of the left- or right-boxed areas in (dD), respectively.
Figure 3.
 
Immunofluorescence detection of nuclear translocation of phosphorylated p38MAP kinase (MAPK) protein in healing mouse corneal epithelium. (A) A very low level of phosphorylation of p38MAPK was observed in basal cell cytoplasm in uninjured epithelium. At 1 (B) and 2 (C) hours(s) after injury, along with an increase of phosphorylation in the cytoplasm, phospho-p38MAPK started to translocate to nuclei (white arrows). At 6 (D) and 12 (E) hours, marked nuclear phospho-p38MAPK was detected (white arrows). At 18 (not shown) and 24 (F) hours, there was an overall upregulation of phospho-p38MAPK in cytoplasm as well as obvious nuclear translocation. Yellow arrows: direction of epithelial migration. Bar, 10 μm.
Figure 3.
 
Immunofluorescence detection of nuclear translocation of phosphorylated p38MAP kinase (MAPK) protein in healing mouse corneal epithelium. (A) A very low level of phosphorylation of p38MAPK was observed in basal cell cytoplasm in uninjured epithelium. At 1 (B) and 2 (C) hours(s) after injury, along with an increase of phosphorylation in the cytoplasm, phospho-p38MAPK started to translocate to nuclei (white arrows). At 6 (D) and 12 (E) hours, marked nuclear phospho-p38MAPK was detected (white arrows). At 18 (not shown) and 24 (F) hours, there was an overall upregulation of phospho-p38MAPK in cytoplasm as well as obvious nuclear translocation. Yellow arrows: direction of epithelial migration. Bar, 10 μm.
Figure 4.
 
Effect of TGFβ-neutralizing antibody on epithelial wound healing and p38MAPK activation. The enucleated eyes with an epithelial defect 2 mm in diameter were organ cultured in the presence or absence of a neutralizing anti-TGFβ antibody. Epithelial defects were evaluated by fluorescein staining. The antibody delayed the reepithelialization (a, b). In control culture (aAE), the epithelial defect was resurfaced within 36 hours after debridement, whereas the defect remained, even at 48 hours with the TGFβ-neutralizing antibody. (b) The percentage of remaining defect at each time point indicated in (a). Bar of SD is less than 1.0% at each time point and therefore is obscured. •, control culture; ○, TGFβ-neutralizing antibody; **significant at P < 0.05; ***significant at P < 0.01. (c) BrdU-labeling of the specimens. Overall, BrdU-labeled cells were not or were very minimally observed in migrating epithelium of the control culture. In contrast, migrating epithelium of antibody-treated cornea contained many BrdU-labeled cells (arrows) at12 (cA, cB) and 36 (cC, cD) hours. (d) Expression of phospho-p38 MAPK in healing epithelium, with or without the TGFβ-neutralizing antibody. Positive immunoreactivity for phospho-p38MAPK was observed in migrating epithelial cells of control specimens at each culture interval (dA), whereas very weak immunoreactivity was seen in specimens treated with TGFβ-neutralizing antibody (dB). (dC, dD) Nuclear DAPI staining. Bar, 10 μm.
Figure 4.
 
Effect of TGFβ-neutralizing antibody on epithelial wound healing and p38MAPK activation. The enucleated eyes with an epithelial defect 2 mm in diameter were organ cultured in the presence or absence of a neutralizing anti-TGFβ antibody. Epithelial defects were evaluated by fluorescein staining. The antibody delayed the reepithelialization (a, b). In control culture (aAE), the epithelial defect was resurfaced within 36 hours after debridement, whereas the defect remained, even at 48 hours with the TGFβ-neutralizing antibody. (b) The percentage of remaining defect at each time point indicated in (a). Bar of SD is less than 1.0% at each time point and therefore is obscured. •, control culture; ○, TGFβ-neutralizing antibody; **significant at P < 0.05; ***significant at P < 0.01. (c) BrdU-labeling of the specimens. Overall, BrdU-labeled cells were not or were very minimally observed in migrating epithelium of the control culture. In contrast, migrating epithelium of antibody-treated cornea contained many BrdU-labeled cells (arrows) at12 (cA, cB) and 36 (cC, cD) hours. (d) Expression of phospho-p38 MAPK in healing epithelium, with or without the TGFβ-neutralizing antibody. Positive immunoreactivity for phospho-p38MAPK was observed in migrating epithelial cells of control specimens at each culture interval (dA), whereas very weak immunoreactivity was seen in specimens treated with TGFβ-neutralizing antibody (dB). (dC, dD) Nuclear DAPI staining. Bar, 10 μm.
Figure 5.
 
Effects of a p38MAPK inhibitor on epithelial wound healing and p38MAPK activation. (a) Closure of epithelial defects were examined in organ culture, with and without the p38MAPK inhibitor SB202190 (10 μM), as in the TGFβ-neutralization experiment. Defect closure was markedly delayed in the test culture (aFI) compared with the control culture (aAE). The epithelial defect was gradually resurfaced at 24 hours after injury in control (aC), but the defect remained the same as the original in the presence of the inhibitor (aG). At this time point the defect in the control culture (aC) was obviously smaller than that in the test culture with the inhibitor (aG). At 36 (aD) and 48 (aE) hours after injury, no defect was observed in all the specimens examined in the control culture, whereas more than 50% of the defect was not resurfaced at these time points in the test culture. (b) The percentage of the defect area remaining. Bar showing standard deviation is less than 1.0% at each time point and therefore is obscured. •, control culture; ○, with the inhibitor; **significant at P < 0.05; ***significant at P < 0.01. (c) Histology of healing cornea. Organ-cultured mouse eye globes as shown in (a) were subjected to histologic examination by hematoxylin and eosin staining. Immediately after epithelial debridement, disruption of the stratified epithelium was observed (cA). At 12 hours after, the epithelial cells had begun migrating in a single cell-layered epithelial sheet toward the center of the defect in control culture (cB), but in the medium containing the inhibitor SB202190, the epithelium had not started to migrate, and the edge of the wounded epithelium was similar to the original injured epithelium immediately after the debridement (cC). The single cell-layered epithelium migrated to resurface the defect in 24 hours in control cultures (cD) and had completely resurfaced the defect at 36 hours after injury (cF), whereas in the presence of the inhibitor the wounded edge of the epithelium was still multilayered at 24 hours after injury (cE) and two-cell–layered at the leading edge at 36 hours after injury (cG). At 48 hours, the central cornea was resurfaced by a single-cell–layered epithelium (cH) similar to that at 36 hours in the control; whereas, at this time point, the defect remained in culture with the inhibitor (cI). At a higher magnification (cJ and cK), a single-cell–layered epithelium (boxed area in cH) covered the central cornea in the control, whereas single-cell–layered migrating epithelium (boxed area in cI) was observed at the wounded edge in the presence of the inhibitor. (d) Phospho-p38MAPK was not present in epithelium in the specimens organ cultured with the inhibitor. Marked immunoreactivity for phospho-p38MAPK was present in the epithelia in control cultures, but not in those with the inhibitor (dA, dB, at 18 hours). Scale bars: (cAG, cJ, cK) 30 μm; (cH, cI) 300 μm; (dA, dB) 20 μm.
Figure 5.
 
Effects of a p38MAPK inhibitor on epithelial wound healing and p38MAPK activation. (a) Closure of epithelial defects were examined in organ culture, with and without the p38MAPK inhibitor SB202190 (10 μM), as in the TGFβ-neutralization experiment. Defect closure was markedly delayed in the test culture (aFI) compared with the control culture (aAE). The epithelial defect was gradually resurfaced at 24 hours after injury in control (aC), but the defect remained the same as the original in the presence of the inhibitor (aG). At this time point the defect in the control culture (aC) was obviously smaller than that in the test culture with the inhibitor (aG). At 36 (aD) and 48 (aE) hours after injury, no defect was observed in all the specimens examined in the control culture, whereas more than 50% of the defect was not resurfaced at these time points in the test culture. (b) The percentage of the defect area remaining. Bar showing standard deviation is less than 1.0% at each time point and therefore is obscured. •, control culture; ○, with the inhibitor; **significant at P < 0.05; ***significant at P < 0.01. (c) Histology of healing cornea. Organ-cultured mouse eye globes as shown in (a) were subjected to histologic examination by hematoxylin and eosin staining. Immediately after epithelial debridement, disruption of the stratified epithelium was observed (cA). At 12 hours after, the epithelial cells had begun migrating in a single cell-layered epithelial sheet toward the center of the defect in control culture (cB), but in the medium containing the inhibitor SB202190, the epithelium had not started to migrate, and the edge of the wounded epithelium was similar to the original injured epithelium immediately after the debridement (cC). The single cell-layered epithelium migrated to resurface the defect in 24 hours in control cultures (cD) and had completely resurfaced the defect at 36 hours after injury (cF), whereas in the presence of the inhibitor the wounded edge of the epithelium was still multilayered at 24 hours after injury (cE) and two-cell–layered at the leading edge at 36 hours after injury (cG). At 48 hours, the central cornea was resurfaced by a single-cell–layered epithelium (cH) similar to that at 36 hours in the control; whereas, at this time point, the defect remained in culture with the inhibitor (cI). At a higher magnification (cJ and cK), a single-cell–layered epithelium (boxed area in cH) covered the central cornea in the control, whereas single-cell–layered migrating epithelium (boxed area in cI) was observed at the wounded edge in the presence of the inhibitor. (d) Phospho-p38MAPK was not present in epithelium in the specimens organ cultured with the inhibitor. Marked immunoreactivity for phospho-p38MAPK was present in the epithelia in control cultures, but not in those with the inhibitor (dA, dB, at 18 hours). Scale bars: (cAG, cJ, cK) 30 μm; (cH, cI) 300 μm; (dA, dB) 20 μm.
Figure 6.
 
Effect of a p38MAPK inhibitor on MAP kinase/Erk phosphorylation and epithelial cell proliferation. (a) Status of Erk phosphorylation in healing epithelium of organ-cultured globes in the presence and absence of SB202190 p38MPK inhibitor. Phospho-Erk was labeled throughout specimens in SB202190+ culture compared with control culture specimens at each time point. (b) Distribution of proliferating corneal epithelial cells in organ culture. At 12 hours, monolayer epithelial cells had started migrating toward the center of the defect, but were not proliferating (bA). In the medium containing the inhibitor (bB), the marginal epithelium keeping a stratification contained BrdU-positive cells (open arrowheads). At 18 hours, no BrdU-positive cells were detected in migrating epithelium in the control (bC), whereas many cells incorporated BrdU (open arrowheads) at the healing edge in the medium containing the p38MAPK inhibitor (bD). At 36 hours in control medium, the defect was resurfaced with a single-cell–layered epithelium without proliferating activity (bE), whereas the leading epithelial edge contained a BrdU-labeled cell (open arrowhead) in culture containing the inhibitor (bF). The same results were found at 24 hours (data not shown). At 48 hours of control culture, the single-cell–layered epithelium resurfacing the defect started to proliferate, showing positive BrdU-labels in a few cells (open arrowheads), and, in the presence of the inhibitor, the injured epithelial edge contained BrdU-positive cells (open arrowheads). Arrows: edge of the remaining injured epithelium. (c) Number of BrdU-labeled cells at each time point. Except for 48 hours, the number of proliferating epithelial cells was significantly (P < 0.01) more in the SB202190+ cultures (I) than in the control (C) cultures. Scale bars: (a, b) 10 μm.
Figure 6.
 
Effect of a p38MAPK inhibitor on MAP kinase/Erk phosphorylation and epithelial cell proliferation. (a) Status of Erk phosphorylation in healing epithelium of organ-cultured globes in the presence and absence of SB202190 p38MPK inhibitor. Phospho-Erk was labeled throughout specimens in SB202190+ culture compared with control culture specimens at each time point. (b) Distribution of proliferating corneal epithelial cells in organ culture. At 12 hours, monolayer epithelial cells had started migrating toward the center of the defect, but were not proliferating (bA). In the medium containing the inhibitor (bB), the marginal epithelium keeping a stratification contained BrdU-positive cells (open arrowheads). At 18 hours, no BrdU-positive cells were detected in migrating epithelium in the control (bC), whereas many cells incorporated BrdU (open arrowheads) at the healing edge in the medium containing the p38MAPK inhibitor (bD). At 36 hours in control medium, the defect was resurfaced with a single-cell–layered epithelium without proliferating activity (bE), whereas the leading epithelial edge contained a BrdU-labeled cell (open arrowhead) in culture containing the inhibitor (bF). The same results were found at 24 hours (data not shown). At 48 hours of control culture, the single-cell–layered epithelium resurfacing the defect started to proliferate, showing positive BrdU-labels in a few cells (open arrowheads), and, in the presence of the inhibitor, the injured epithelial edge contained BrdU-positive cells (open arrowheads). Arrows: edge of the remaining injured epithelium. (c) Number of BrdU-labeled cells at each time point. Except for 48 hours, the number of proliferating epithelial cells was significantly (P < 0.01) more in the SB202190+ cultures (I) than in the control (C) cultures. Scale bars: (a, b) 10 μm.
Katakami C, Perkins T, Dorfman N, Spaulding AG, Kao WW-Y. Polymorphonuclear leukocytes inhibit proliferation of epithelial cells in rabbit cornea (in Japanese). Nippon Ganka Gakkai Zasshi (Acta Soc Ophthalmol Jpn). 1988;92:798–805.
Lu L, Reinach PS, Kao WW-Y. Corneal epithelial wound healing. Exp Biol Med (Maywood). 2001;226:653–664. [PubMed]
Schultz G, Rotatori DS, Clark W. EGF and TGF-α in wound healing and repair. J Cell Biochem. 1991;45:346–352. [CrossRef] [PubMed]
Imanishi J, Kamiyama K, Iguchi I, Kita M, Sotozono C, Kinoshita S. Growth factors: importance in wound healing and maintenance of transparency of the cornea. Prog Retin Eye Res. 2000;19:113–129. [CrossRef] [PubMed]
Wilson SE, Chen L, Mohan RR, Liang Q, Liu J. Expression of HGF, KGF, EGF and receptor messenger RNAs following corneal epithelial wounding. Exp Eye Res. 1999;68:377–397. [CrossRef] [PubMed]
Wilson SE, Liu JJ, Mohan RR. Stromal-epithelial interactions in the cornea. Prog Retin Eye Res. 1999;18:293–309. [CrossRef] [PubMed]
Piek E, Heldin CH, Ten Dijke P. Specificity, diversity, and regulation in TGF-β superfamily signaling. FASEB J. 1999;13:2105–2124. [PubMed]
Ducy P, Karsenty G. The family of bone morphogenetic proteins. Kidney Int. 2000;57:2207–2214. [CrossRef] [PubMed]
Dunker N, Krieglstein K. Targeted mutations of transforming growth factor-β genes reveal important roles in mouse development and adult homeostasis. Eur J Biochem. 2000;267:6982–6988. [CrossRef] [PubMed]
Li DQ, Tseng SC. Differential regulation of cytokine and receptor transcript expression in human corneal and limbal fibroblasts by epidermal growth factor, transforming growth factor-alpha, platelet-derived growth factor B, and interleukin-1beta. Invest Ophthalmol Vis Sci. 1996;37:2068–2080. [PubMed]
Pelton RW, Saxena B, Jones M, Moses HL, Gold LI. Immunohistochemical localization of TGFβ1, TGFβ2, and TGFβ3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development. J Cell Biol. 1991;115:1091–1105. [CrossRef] [PubMed]
Li DQ, Tseng SC. Three patterns of cytokine expression potentially involved in epithelial-fibroblast interactions of human ocular surface. J Cell Physiol. 1995;163:61–79. [CrossRef] [PubMed]
Wilson SE, Schultz GS, Chegini N, Weng J, He YG. Epidermal growth factor, transforming growth factor α, transforming growth factor β, acidic fibroblast growth factor, basic fibroblast growth factor, and interleukin-1 proteins in the cornea. Exp Eye Res. 1994;59:63–71. [CrossRef] [PubMed]
Nishida K, Sotozono C, Adachi W, Yamamoto S, Yokoi N, Kinoshita S. Transforming growth factor-β1, -β2 and -β3 mRNA expression in human cornea. Curr Eye Res. 1995;14:235–241. [CrossRef] [PubMed]
Joyce NC, Zieske JD. Transforming growth factor-β receptor expression in human cornea. Invest Ophthalmol Vis Sci. 1997;38:1922–1928. [PubMed]
Derynck R, Zhang Y, Feng XH. Smads: transcriptional activators of TGF-β responses. Cell. 1998;95:737–740. [CrossRef] [PubMed]
Massague J. How cells read TGF-β signals. Nat Rev Mol Cell Biol. 2000;1:169–178. [CrossRef] [PubMed]
Zhu HJ, Burgess AW. Regulation of transforming growth factor-β signaling. Mol Cell Biol Res Commun. 2001;4:321–330. [CrossRef] [PubMed]
Mita T, Yamashita H, Kaji Y, et al. Effects of transforming growth factor β on corneal epithelial and stromal cell function in a rat wound healing model after excimer laser keratectomy. Graefes Arch Clin Exp Ophthalmol. 1998;236:834–843. [CrossRef] [PubMed]
Zieske JD, Hutcheon AEK, Guo X, Chung EH, Joyce NC. TGF-β receptor types I and II are differentially expressed during corneal epithelial wound repair. Invest Ophthalmol Vis Sci. 2001;42:1465–1471. [PubMed]
Saika S, Shiraishi A, Saika S, et al. Role of lumican in the corneal epithelium during wound healing. J Biol Chem. 2000;275:2607–2612. [CrossRef] [PubMed]
Lovicu FJ, Kao WW-Y, Overbeek PA. Ectopic gland induction by lens-specific expression of keratinocyte growth factor (FGF-7) in transgenic mice. Mech Dev. 1999;88:43–53. [CrossRef] [PubMed]
Ishizaki M, Zhu G, Haseba T, Shafer SS, Kao WW-Y. Expression of collagen I, smooth muscle alpha-actin, and vimentin during the healing of alkali-burned and lacerated corneas. Invest Ophthalmol Vis Sci. 1993;34:3320–3328. [PubMed]
Klekotka PA, Santoro SA, Zutter MM. Alpha 2 Integrin subunit cytoplasmic domain-dependent cellular migration requires p38 MAPK. J Biol Chem. 2001;276:9503–9511. [CrossRef] [PubMed]
Li W, Nadelman C, Henry G, et al. The p38-MAPK/SAPK pathway is required for human keratinocyte migration on dermal collagen. J Invest Dermatol. 2001;117:1601–1611. [CrossRef] [PubMed]
Dumon N, Bakin AV, Arteaga CL. Autocrine transforming growth factor-β signaling mediates Smad independent motility in human cancer cells. J Biol Chem. 2003;278:3275–3285. [CrossRef] [PubMed]
Vadlamudi R, Adam L, Talukder A, Mendelsohn J, Kumar R. Serine phosphorylation of paxillin by heregulin-β1: role of p38 mitogen activated protein kinase. Oncogene. 1999;18:7253–7264. [CrossRef] [PubMed]
Bhowmick NA, Zent R, Ghiassi M, McDonnell M, Moses HL. Integrin β1 signaling is necessary for transforming growth factor-β activation of p38MAPK and epithelial plasticity. J Biol Chem. 2001;276:46707–46713. [CrossRef] [PubMed]
Murakami-Mori K, Mori S, Nakamura S. p38MAP kinase is a negative regulator for ERK1/2-mediated growth of AIDS-associated Kaposi’s sarcoma cells. Biochem Biophys Res Commun. 1999;264:676–682. [CrossRef] [PubMed]
Yosimichi G, Nakanishi T, Nishida T, Hattori T, Takano-Yamamoto T, Takigawa M. CTGF/Hcs24 induces chondrocyte differentiation through a p38 mitogen-activated protein kinase (p38MAPK), and proliferation through a p44/42 MAPK/extracellular-signal regulated kinase (ERK). Eur J Biochem. 2001;268:6058–6065. [CrossRef] [PubMed]
Matsumoto T, Turesson I, Book M, Gerwins P, Claesson-Welsh L. p38 MAP kinase negatively regulates endothelial cell survival, proliferation, and differentiation in FGF-2-stimulated angiogenesis. J Cell Biol. 2002;156:149–160. [CrossRef] [PubMed]
Yu Y, Sato JD. MAP kinases, phosphatidylinositol 3-kinase, and p70 S6 kinase mediate the mitogenic response of human endothelial cells to vascular endothelial growth factor. J Cell Physiol. 1999;178:235–246. [CrossRef] [PubMed]
Cocolakis E, Lemay S, Ali S, Lebrun JJ. The p38 MAPK pathway is required for cell growth inhibition of human breast cancer cells in response to activin. J Biol Chem. 2001;276:18430–18436. [CrossRef] [PubMed]
Giehl K, Sedel B, Gierschik P, Adler G, Menke A. TGFβ1 represses proliferation of pancreatic carcinoma cells which correlates with Smad4-independent inhibition of ERK activation. Oncogene. 2000;14:4531–4541.
Jono H, Xu H, Kai H, et al. Transforming growth factor-β-Smad signaling pathway negatively regulates nontypable Haemophilis influenzae-induced MUC5AC mucin transcription via mitogen-activated protein kinase (MAPK) phosphatase-1-dependent inhibition of p38 MAPK. J Biol Chem. 2003;278:27811–27819. [CrossRef] [PubMed]
Datto MB, Frederick JP, Pan L, Borton AJ, Zhuang Y, Wang XF. Targeted disruption of Smad3 reveals an essential role in transforming growth factor β-mediated signal transduction. Mol Cell Biol. 1999;19:2495–2504. [PubMed]
Lee S, Cho YS, Shim C, et al. Aberrant expression of Smad4 results in resistance against the growth-inhibitory effect of transforming growth factor-beta in the SiHa human cervical carcinoma cell line. Int J Cancer. 2001;94:500–507. [CrossRef] [PubMed]
ten Dijke P, Goumans M-J, Itoh F, Itih S. Regulation of cell proliferation by Smad proteins (review). J Cell Physiol. 2002;191:1–16. [CrossRef] [PubMed]
Ashcroft GS, Yang X, Glick AB, et al. Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response. Nat Cell Biol. 1999;1:260–266. [CrossRef] [PubMed]
Sano Y, Harada J, Tashiro S, Gotoh-Mandeville R, Maekawa T, Ishii S. ATF-2 is a common nuclear target of Smad and TAK1 pathways in transforming growth factor-β signaling. J Biol Chem. 1999;274:8949–8957. [CrossRef] [PubMed]
Bhowmick NA, Ghiassi M, Bakin A, et al. Transforming growth factor-β1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism. Mol Biol Cell. 2001;12:27–36. [CrossRef] [PubMed]
Wang W, Zhou G, Hu MC, Yao Z, Tan TH. Activation of the hematopoietic progenitor kinase-1 (HPK1)-dependent, stress-activated c-Jun N-terminal kinase (JNK) pathway by transforming growth factor β (TGF-β)-activated kinase (TAK1), a kinase mediator of TGF β signal transduction. J Biol Chem. 1997;272:22771–22775. [CrossRef] [PubMed]
Engel ME, McDonnell MA, Law BK, Moses HL. Interdependent SMAD and JNK signaling in transforming growth factor-β-mediated transcription. J Biol Chem. 1999;274:37413–37420. [CrossRef] [PubMed]
Petritsch C, Beug H, Balmain A, Oft M. TGF-β inhibits p70 S6 kinase via protein phosphatase 2A to induce G(1) arrest. Genes Dev. 2000;14:3093–3101. [CrossRef] [PubMed]
Gotzmann J, Huber H, Thallinger C, et al. Hepatocytes convert to a fibroblastoid phenotype through the cooperation of TGF-β1 and Ha-Ras: steps towards invasiveness. J Cell Sci. 2002;115:1189–1202. [PubMed]
Peron P, Rahmani M, Zagar Y, Durand-Schneider AM, Lardeux B, Bernuau D. Potentiation of Smad transactivation by Jun proteins during a combined treatment with epidermal growth factor and transforming growth factor-β in rat hepatocytes. role of phosphatidylinositol 3-kinase-induced AP-1 activation. J Biol Chem. 2001;276:10524–10531. [CrossRef] [PubMed]
Weng J, Mohan RR, Li Q, Wilson SE. IL-1 upregulates keratinocyte growth factor and hepatocyte growth factor mRNA and protein production by cultured stromal fibroblast cells: interleukin-1 β expression in the cornea. Cornea. 1997;16:465–471. [PubMed]
Saklatvala J, Dean J, Finch A. Protein kinase cascades in intracellular signalling by interleukin-1 and tumour necrosis factor. Biochem Soc Symp. 1999;64:63–77. [PubMed]
Sharma GD, He J, Bazan HE. p38 and ERK1/2 coordinate cellular migration and proliferation in epithelial wound healing: evidence of cross-talk activation between MAP kinase cascades. J Biol Chem. 2003;278:21989–21997. [CrossRef] [PubMed]
Kim JY, Choi JA, Kim TH, et al. Involvement of p38 mitogen-activated protein kinase in the cell growth inhibition by sodium arsenite. J Cell Physiol. 2002;190:29–37. [CrossRef] [PubMed]
McBain VA, Forrester JV, McCaig CD. HGF, MAPK, and a small physiological electric field interact during corneal epithelial cell migration. Invest Ophthalmol Vis Sci. 2003;44:540–547. [CrossRef] [PubMed]
Figure 1.
 
Distribution of BrdU-labeled cells in healing mouse corneal epithelium after induction of a central epithelial defect 2 mm in diameter. (a) Uninjured corneal epithelium contained occasional BrdU-positive cells (aAC). Immediately after the epithelial ablation (not shown) as well as 6 hours after (aD, aE), the number of BrdU-labeled epithelial cells was similar to that in normal cornea. At 18 hours after injury, BrdU-positive epithelial cells were mainly observed in the basal layer of the limbus (aF) and peripheral epithelium (aG), whereas central resurfacing and migrating monolayer epithelium (aH) lacked BrdU-labeled cells. Twenty-four hours after injury, several BrdU-positive cells appeared in the central regenerated epithelium (aK) in association with the decrease in BrdU-positive cells the limbal (aI) and midperipheral corneal epithelia (aJ). (aB, aE, aG, aJ) Corneal epithelium in the midperiphery, at the wound edge (arrowhead; edge of the remaining epithelium); (aC, aH, aK) Epithelium in the central area. (b) Number of BrdU-positive cells in each zone of the epithelium. Rapid induction of cell proliferation in peripheral epithelium was followed by induction in the midperipheral and then the central epithelium. (c) Phosphorylation of Erk in healing epithelium. Phospho-Erk was faintly detectable in the cytoplasm of basal epithelial cells of uninjured epithelium (cA) with occasional nuclear localization and was not detected in migrating-edge epithelial cells from 1 (cC) to 12 (not shown) hours. The cells then started to upregulate phospho-Erk in the central epithelial cell cytoplasm at 18 hours (cE). Basal cells were positive for phospho-Erk in the nuclei at 24 hours (cG). (cB, cD, cF, cH) The DAPI nuclear staining of the identical area shown in (cA, cC, cE, cG), respectively.
Figure 1.
 
Distribution of BrdU-labeled cells in healing mouse corneal epithelium after induction of a central epithelial defect 2 mm in diameter. (a) Uninjured corneal epithelium contained occasional BrdU-positive cells (aAC). Immediately after the epithelial ablation (not shown) as well as 6 hours after (aD, aE), the number of BrdU-labeled epithelial cells was similar to that in normal cornea. At 18 hours after injury, BrdU-positive epithelial cells were mainly observed in the basal layer of the limbus (aF) and peripheral epithelium (aG), whereas central resurfacing and migrating monolayer epithelium (aH) lacked BrdU-labeled cells. Twenty-four hours after injury, several BrdU-positive cells appeared in the central regenerated epithelium (aK) in association with the decrease in BrdU-positive cells the limbal (aI) and midperipheral corneal epithelia (aJ). (aB, aE, aG, aJ) Corneal epithelium in the midperiphery, at the wound edge (arrowhead; edge of the remaining epithelium); (aC, aH, aK) Epithelium in the central area. (b) Number of BrdU-positive cells in each zone of the epithelium. Rapid induction of cell proliferation in peripheral epithelium was followed by induction in the midperipheral and then the central epithelium. (c) Phosphorylation of Erk in healing epithelium. Phospho-Erk was faintly detectable in the cytoplasm of basal epithelial cells of uninjured epithelium (cA) with occasional nuclear localization and was not detected in migrating-edge epithelial cells from 1 (cC) to 12 (not shown) hours. The cells then started to upregulate phospho-Erk in the central epithelial cell cytoplasm at 18 hours (cE). Basal cells were positive for phospho-Erk in the nuclei at 24 hours (cG). (cB, cD, cF, cH) The DAPI nuclear staining of the identical area shown in (cA, cC, cE, cG), respectively.
Figure 2.
 
Immunohistochemical detection of Smads in healing corneal epithelium. (a) Intracellular localization of Smad3 protein in mouse corneal epithelium. Cells in the uninjured corneal epithelium exhibited both nuclear (yellow arrows) and cytoplasmic Smad3 immunoreactivity (aA). Nuclei of migrating epithelial cells were not labeled by anti-Smad3 antibody at 6 (aB) and 12 (not shown) hours after epithelial debridement (white arrows). At 18 hours after injury, several cell nuclei were labeled by an anti-Smad3 antibody (aC, yellow arrows). Twenty-four hours after injury, central regenerated epithelium (aD) contained cells showing nuclear immunoreactivity for Smad3 with an increase in cytoplasmic labeling. (b) Intracellular localization of Smad4 protein in mouse corneal epithelium. Localization pattern of Smad4 was similar to that of Smad3 shown in (a). In normal uninjured cornea, most of the epithelial cells exhibited a nuclear immunoreactivity for Smad4 (bA, arrowheads). Epithelial cells at the edge of the epithelial defect lacked nuclear Smad4 at 6 (bC, arrows) and 12 (bD, arrows) hours after injury, whereas cytoplasm was positive for Smad4 protein. Cells with nuclei positive (bE, arrowheads) or negative (bE, arrow) for Smad4 were both observed in the double-layered epithelium that resurfaced the central corneal epithelial defect at 18 hours after injury. At 24 hours, central regenerated stratified epithelium (bF, arrowheads) contained many nuclear Smad4-positive cells. No specific immunoreactivity was observed in negative control staining (bB). (c) Protein expression pattern of Smad7 in the healing corneal epithelium. Uninjured corneal epithelium (cA) and that immediately after the epithelial debridement (not shown) exhibited very faint immunoreactivity for Smad7 in the cytoplasm. Epithelial cells around the defect ( Image not available ) were strongly labeled by the anti-Smad7 at 6 (cB) and 12 (cC) hours after debridement. Eighteen (not shown) to 24 (cD) hours after injury, Smad7 immunoreactivity returned to the normal level of an uninjured cornea. (d) Downregulation of Smad7 expression by administration of TGFβ-neutralizing antibody. Smad7 protein expression was found to be upregulated in the entire epithelium at 24 hours after puncture injury of the cornea (dAC). Administration of neutralizing antibodies against TGFβ1 (not illustrated), TGFβ2 (dDF), and pan anti-TGFβ antibodies (not illustrated) reduced Smad7 protein expression in the central and midperipheral, but not in the peripheral, epithelium at the same time point. (dB, dC) High-magnification images of the left- or right-boxed areas in (dA), respectively; (dE, dF) High-magnification images of the left- or right-boxed areas in (dD), respectively.
Figure 2.
 
Immunohistochemical detection of Smads in healing corneal epithelium. (a) Intracellular localization of Smad3 protein in mouse corneal epithelium. Cells in the uninjured corneal epithelium exhibited both nuclear (yellow arrows) and cytoplasmic Smad3 immunoreactivity (aA). Nuclei of migrating epithelial cells were not labeled by anti-Smad3 antibody at 6 (aB) and 12 (not shown) hours after epithelial debridement (white arrows). At 18 hours after injury, several cell nuclei were labeled by an anti-Smad3 antibody (aC, yellow arrows). Twenty-four hours after injury, central regenerated epithelium (aD) contained cells showing nuclear immunoreactivity for Smad3 with an increase in cytoplasmic labeling. (b) Intracellular localization of Smad4 protein in mouse corneal epithelium. Localization pattern of Smad4 was similar to that of Smad3 shown in (a). In normal uninjured cornea, most of the epithelial cells exhibited a nuclear immunoreactivity for Smad4 (bA, arrowheads). Epithelial cells at the edge of the epithelial defect lacked nuclear Smad4 at 6 (bC, arrows) and 12 (bD, arrows) hours after injury, whereas cytoplasm was positive for Smad4 protein. Cells with nuclei positive (bE, arrowheads) or negative (bE, arrow) for Smad4 were both observed in the double-layered epithelium that resurfaced the central corneal epithelial defect at 18 hours after injury. At 24 hours, central regenerated stratified epithelium (bF, arrowheads) contained many nuclear Smad4-positive cells. No specific immunoreactivity was observed in negative control staining (bB). (c) Protein expression pattern of Smad7 in the healing corneal epithelium. Uninjured corneal epithelium (cA) and that immediately after the epithelial debridement (not shown) exhibited very faint immunoreactivity for Smad7 in the cytoplasm. Epithelial cells around the defect ( Image not available ) were strongly labeled by the anti-Smad7 at 6 (cB) and 12 (cC) hours after debridement. Eighteen (not shown) to 24 (cD) hours after injury, Smad7 immunoreactivity returned to the normal level of an uninjured cornea. (d) Downregulation of Smad7 expression by administration of TGFβ-neutralizing antibody. Smad7 protein expression was found to be upregulated in the entire epithelium at 24 hours after puncture injury of the cornea (dAC). Administration of neutralizing antibodies against TGFβ1 (not illustrated), TGFβ2 (dDF), and pan anti-TGFβ antibodies (not illustrated) reduced Smad7 protein expression in the central and midperipheral, but not in the peripheral, epithelium at the same time point. (dB, dC) High-magnification images of the left- or right-boxed areas in (dA), respectively; (dE, dF) High-magnification images of the left- or right-boxed areas in (dD), respectively.
Figure 3.
 
Immunofluorescence detection of nuclear translocation of phosphorylated p38MAP kinase (MAPK) protein in healing mouse corneal epithelium. (A) A very low level of phosphorylation of p38MAPK was observed in basal cell cytoplasm in uninjured epithelium. At 1 (B) and 2 (C) hours(s) after injury, along with an increase of phosphorylation in the cytoplasm, phospho-p38MAPK started to translocate to nuclei (white arrows). At 6 (D) and 12 (E) hours, marked nuclear phospho-p38MAPK was detected (white arrows). At 18 (not shown) and 24 (F) hours, there was an overall upregulation of phospho-p38MAPK in cytoplasm as well as obvious nuclear translocation. Yellow arrows: direction of epithelial migration. Bar, 10 μm.
Figure 3.
 
Immunofluorescence detection of nuclear translocation of phosphorylated p38MAP kinase (MAPK) protein in healing mouse corneal epithelium. (A) A very low level of phosphorylation of p38MAPK was observed in basal cell cytoplasm in uninjured epithelium. At 1 (B) and 2 (C) hours(s) after injury, along with an increase of phosphorylation in the cytoplasm, phospho-p38MAPK started to translocate to nuclei (white arrows). At 6 (D) and 12 (E) hours, marked nuclear phospho-p38MAPK was detected (white arrows). At 18 (not shown) and 24 (F) hours, there was an overall upregulation of phospho-p38MAPK in cytoplasm as well as obvious nuclear translocation. Yellow arrows: direction of epithelial migration. Bar, 10 μm.
Figure 4.
 
Effect of TGFβ-neutralizing antibody on epithelial wound healing and p38MAPK activation. The enucleated eyes with an epithelial defect 2 mm in diameter were organ cultured in the presence or absence of a neutralizing anti-TGFβ antibody. Epithelial defects were evaluated by fluorescein staining. The antibody delayed the reepithelialization (a, b). In control culture (aAE), the epithelial defect was resurfaced within 36 hours after debridement, whereas the defect remained, even at 48 hours with the TGFβ-neutralizing antibody. (b) The percentage of remaining defect at each time point indicated in (a). Bar of SD is less than 1.0% at each time point and therefore is obscured. •, control culture; ○, TGFβ-neutralizing antibody; **significant at P < 0.05; ***significant at P < 0.01. (c) BrdU-labeling of the specimens. Overall, BrdU-labeled cells were not or were very minimally observed in migrating epithelium of the control culture. In contrast, migrating epithelium of antibody-treated cornea contained many BrdU-labeled cells (arrows) at12 (cA, cB) and 36 (cC, cD) hours. (d) Expression of phospho-p38 MAPK in healing epithelium, with or without the TGFβ-neutralizing antibody. Positive immunoreactivity for phospho-p38MAPK was observed in migrating epithelial cells of control specimens at each culture interval (dA), whereas very weak immunoreactivity was seen in specimens treated with TGFβ-neutralizing antibody (dB). (dC, dD) Nuclear DAPI staining. Bar, 10 μm.
Figure 4.
 
Effect of TGFβ-neutralizing antibody on epithelial wound healing and p38MAPK activation. The enucleated eyes with an epithelial defect 2 mm in diameter were organ cultured in the presence or absence of a neutralizing anti-TGFβ antibody. Epithelial defects were evaluated by fluorescein staining. The antibody delayed the reepithelialization (a, b). In control culture (aAE), the epithelial defect was resurfaced within 36 hours after debridement, whereas the defect remained, even at 48 hours with the TGFβ-neutralizing antibody. (b) The percentage of remaining defect at each time point indicated in (a). Bar of SD is less than 1.0% at each time point and therefore is obscured. •, control culture; ○, TGFβ-neutralizing antibody; **significant at P < 0.05; ***significant at P < 0.01. (c) BrdU-labeling of the specimens. Overall, BrdU-labeled cells were not or were very minimally observed in migrating epithelium of the control culture. In contrast, migrating epithelium of antibody-treated cornea contained many BrdU-labeled cells (arrows) at12 (cA, cB) and 36 (cC, cD) hours. (d) Expression of phospho-p38 MAPK in healing epithelium, with or without the TGFβ-neutralizing antibody. Positive immunoreactivity for phospho-p38MAPK was observed in migrating epithelial cells of control specimens at each culture interval (dA), whereas very weak immunoreactivity was seen in specimens treated with TGFβ-neutralizing antibody (dB). (dC, dD) Nuclear DAPI staining. Bar, 10 μm.
Figure 5.
 
Effects of a p38MAPK inhibitor on epithelial wound healing and p38MAPK activation. (a) Closure of epithelial defects were examined in organ culture, with and without the p38MAPK inhibitor SB202190 (10 μM), as in the TGFβ-neutralization experiment. Defect closure was markedly delayed in the test culture (aFI) compared with the control culture (aAE). The epithelial defect was gradually resurfaced at 24 hours after injury in control (aC), but the defect remained the same as the original in the presence of the inhibitor (aG). At this time point the defect in the control culture (aC) was obviously smaller than that in the test culture with the inhibitor (aG). At 36 (aD) and 48 (aE) hours after injury, no defect was observed in all the specimens examined in the control culture, whereas more than 50% of the defect was not resurfaced at these time points in the test culture. (b) The percentage of the defect area remaining. Bar showing standard deviation is less than 1.0% at each time point and therefore is obscured. •, control culture; ○, with the inhibitor; **significant at P < 0.05; ***significant at P < 0.01. (c) Histology of healing cornea. Organ-cultured mouse eye globes as shown in (a) were subjected to histologic examination by hematoxylin and eosin staining. Immediately after epithelial debridement, disruption of the stratified epithelium was observed (cA). At 12 hours after, the epithelial cells had begun migrating in a single cell-layered epithelial sheet toward the center of the defect in control culture (cB), but in the medium containing the inhibitor SB202190, the epithelium had not started to migrate, and the edge of the wounded epithelium was similar to the original injured epithelium immediately after the debridement (cC). The single cell-layered epithelium migrated to resurface the defect in 24 hours in control cultures (cD) and had completely resurfaced the defect at 36 hours after injury (cF), whereas in the presence of the inhibitor the wounded edge of the epithelium was still multilayered at 24 hours after injury (cE) and two-cell–layered at the leading edge at 36 hours after injury (cG). At 48 hours, the central cornea was resurfaced by a single-cell–layered epithelium (cH) similar to that at 36 hours in the control; whereas, at this time point, the defect remained in culture with the inhibitor (cI). At a higher magnification (cJ and cK), a single-cell–layered epithelium (boxed area in cH) covered the central cornea in the control, whereas single-cell–layered migrating epithelium (boxed area in cI) was observed at the wounded edge in the presence of the inhibitor. (d) Phospho-p38MAPK was not present in epithelium in the specimens organ cultured with the inhibitor. Marked immunoreactivity for phospho-p38MAPK was present in the epithelia in control cultures, but not in those with the inhibitor (dA, dB, at 18 hours). Scale bars: (cAG, cJ, cK) 30 μm; (cH, cI) 300 μm; (dA, dB) 20 μm.
Figure 5.
 
Effects of a p38MAPK inhibitor on epithelial wound healing and p38MAPK activation. (a) Closure of epithelial defects were examined in organ culture, with and without the p38MAPK inhibitor SB202190 (10 μM), as in the TGFβ-neutralization experiment. Defect closure was markedly delayed in the test culture (aFI) compared with the control culture (aAE). The epithelial defect was gradually resurfaced at 24 hours after injury in control (aC), but the defect remained the same as the original in the presence of the inhibitor (aG). At this time point the defect in the control culture (aC) was obviously smaller than that in the test culture with the inhibitor (aG). At 36 (aD) and 48 (aE) hours after injury, no defect was observed in all the specimens examined in the control culture, whereas more than 50% of the defect was not resurfaced at these time points in the test culture. (b) The percentage of the defect area remaining. Bar showing standard deviation is less than 1.0% at each time point and therefore is obscured. •, control culture; ○, with the inhibitor; **significant at P < 0.05; ***significant at P < 0.01. (c) Histology of healing cornea. Organ-cultured mouse eye globes as shown in (a) were subjected to histologic examination by hematoxylin and eosin staining. Immediately after epithelial debridement, disruption of the stratified epithelium was observed (cA). At 12 hours after, the epithelial cells had begun migrating in a single cell-layered epithelial sheet toward the center of the defect in control culture (cB), but in the medium containing the inhibitor SB202190, the epithelium had not started to migrate, and the edge of the wounded epithelium was similar to the original injured epithelium immediately after the debridement (cC). The single cell-layered epithelium migrated to resurface the defect in 24 hours in control cultures (cD) and had completely resurfaced the defect at 36 hours after injury (cF), whereas in the presence of the inhibitor the wounded edge of the epithelium was still multilayered at 24 hours after injury (cE) and two-cell–layered at the leading edge at 36 hours after injury (cG). At 48 hours, the central cornea was resurfaced by a single-cell–layered epithelium (cH) similar to that at 36 hours in the control; whereas, at this time point, the defect remained in culture with the inhibitor (cI). At a higher magnification (cJ and cK), a single-cell–layered epithelium (boxed area in cH) covered the central cornea in the control, whereas single-cell–layered migrating epithelium (boxed area in cI) was observed at the wounded edge in the presence of the inhibitor. (d) Phospho-p38MAPK was not present in epithelium in the specimens organ cultured with the inhibitor. Marked immunoreactivity for phospho-p38MAPK was present in the epithelia in control cultures, but not in those with the inhibitor (dA, dB, at 18 hours). Scale bars: (cAG, cJ, cK) 30 μm; (cH, cI) 300 μm; (dA, dB) 20 μm.
Figure 6.
 
Effect of a p38MAPK inhibitor on MAP kinase/Erk phosphorylation and epithelial cell proliferation. (a) Status of Erk phosphorylation in healing epithelium of organ-cultured globes in the presence and absence of SB202190 p38MPK inhibitor. Phospho-Erk was labeled throughout specimens in SB202190+ culture compared with control culture specimens at each time point. (b) Distribution of proliferating corneal epithelial cells in organ culture. At 12 hours, monolayer epithelial cells had started migrating toward the center of the defect, but were not proliferating (bA). In the medium containing the inhibitor (bB), the marginal epithelium keeping a stratification contained BrdU-positive cells (open arrowheads). At 18 hours, no BrdU-positive cells were detected in migrating epithelium in the control (bC), whereas many cells incorporated BrdU (open arrowheads) at the healing edge in the medium containing the p38MAPK inhibitor (bD). At 36 hours in control medium, the defect was resurfaced with a single-cell–layered epithelium without proliferating activity (bE), whereas the leading epithelial edge contained a BrdU-labeled cell (open arrowhead) in culture containing the inhibitor (bF). The same results were found at 24 hours (data not shown). At 48 hours of control culture, the single-cell–layered epithelium resurfacing the defect started to proliferate, showing positive BrdU-labels in a few cells (open arrowheads), and, in the presence of the inhibitor, the injured epithelial edge contained BrdU-positive cells (open arrowheads). Arrows: edge of the remaining injured epithelium. (c) Number of BrdU-labeled cells at each time point. Except for 48 hours, the number of proliferating epithelial cells was significantly (P < 0.01) more in the SB202190+ cultures (I) than in the control (C) cultures. Scale bars: (a, b) 10 μm.
Figure 6.
 
Effect of a p38MAPK inhibitor on MAP kinase/Erk phosphorylation and epithelial cell proliferation. (a) Status of Erk phosphorylation in healing epithelium of organ-cultured globes in the presence and absence of SB202190 p38MPK inhibitor. Phospho-Erk was labeled throughout specimens in SB202190+ culture compared with control culture specimens at each time point. (b) Distribution of proliferating corneal epithelial cells in organ culture. At 12 hours, monolayer epithelial cells had started migrating toward the center of the defect, but were not proliferating (bA). In the medium containing the inhibitor (bB), the marginal epithelium keeping a stratification contained BrdU-positive cells (open arrowheads). At 18 hours, no BrdU-positive cells were detected in migrating epithelium in the control (bC), whereas many cells incorporated BrdU (open arrowheads) at the healing edge in the medium containing the p38MAPK inhibitor (bD). At 36 hours in control medium, the defect was resurfaced with a single-cell–layered epithelium without proliferating activity (bE), whereas the leading epithelial edge contained a BrdU-labeled cell (open arrowhead) in culture containing the inhibitor (bF). The same results were found at 24 hours (data not shown). At 48 hours of control culture, the single-cell–layered epithelium resurfacing the defect started to proliferate, showing positive BrdU-labels in a few cells (open arrowheads), and, in the presence of the inhibitor, the injured epithelial edge contained BrdU-positive cells (open arrowheads). Arrows: edge of the remaining injured epithelium. (c) Number of BrdU-labeled cells at each time point. Except for 48 hours, the number of proliferating epithelial cells was significantly (P < 0.01) more in the SB202190+ cultures (I) than in the control (C) cultures. Scale bars: (a, b) 10 μm.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×