March 2005
Volume 46, Issue 3
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Cornea  |   March 2005
Sensitizing Effect of Substance P on Corneal Epithelial Migration Induced by IGF-1, Fibronectin, or Interleukin-6
Author Affiliations
  • Naoyuki Yamada
    From the Departments of Biomolecular Recognition and Ophthalmology and
    Pharmacology, Yamaguchi University School of Medicine, Yamaguchi, Japan.
  • Ryoji Yanai
    From the Departments of Biomolecular Recognition and Ophthalmology and
    Pharmacology, Yamaguchi University School of Medicine, Yamaguchi, Japan.
  • Makoto Inui
    Pharmacology, Yamaguchi University School of Medicine, Yamaguchi, Japan.
  • Teruo Nishida
    From the Departments of Biomolecular Recognition and Ophthalmology and
Investigative Ophthalmology & Visual Science March 2005, Vol.46, 833-839. doi:10.1167/iovs.04-0775
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      Naoyuki Yamada, Ryoji Yanai, Makoto Inui, Teruo Nishida; Sensitizing Effect of Substance P on Corneal Epithelial Migration Induced by IGF-1, Fibronectin, or Interleukin-6. Invest. Ophthalmol. Vis. Sci. 2005;46(3):833-839. doi: 10.1167/iovs.04-0775.

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

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Abstract

purpose. Substance P (SP) is present in the sensory nerve fibers of the corneal epithelium. Various biological agents, including epidermal growth factor, fibronectin, interleukin-6, and the combination of SP and insulin-like growth factor (IGF)-1, promote the healing of corneal epithelial wounds. The role of SP in corneal epithelial migration was examined.

methods. The effects of various agents on corneal epithelial migration were investigated with the rabbit cornea in an organ culture system.

results. An SP-derived tetrapeptide, FGLM-amide, shifted the dose–response relations for the induction of corneal epithelial migration not only by an IGF-1–derived peptide (C-domain peptide) but also by fibronectin or interleukin-6 to lower concentrations. This action of SP was prevented by inhibitors of phospholipase C, of the inositol 1,4,5-trisphosphate receptor–mediated release of Ca2+ from intracellular stores, and of Ca2+- and calmodulin-dependent protein kinase II (CaM-PK II).

conclusions. These results indicate that SP, acting at the neurokinin type 1 receptor, functions as an important modulator of corneal epithelial wound healing by activating CaM-PK II in epithelial cells and thereby sensitizing them to the induction of migration by various biological agents. They also provide important insight into a new strategy for the treatment of corneal wounds.

Epithelia are important barriers that protect the internal structures and functions of the body from external insults. Rapid and active resurfacing of epithelial defects, or re-epithelialization, is thus necessary to maintain the internal environment after damage to epithelia by infection, trauma, or surgery. The cornea, which constitutes the transparent outermost surface of the eyeball, shares an embryonic origin with the skin. It possesses a relatively simple structure, consisting only of the epithelium, stroma, and endothelium and lacking a vascular system. The cornea thus provides a useful model for investigations into the regulatory mechanisms of epithelial wound healing. Various biological agents have been shown to regulate epithelial wound healing. 1 2 3 With the use of an organ culture system for the cornea, we have shown that several factors, including epidermal growth factor, 4 fibronectin, 5 and interleukin (IL)-6, 6 promote corneal epithelial migration. These factors also facilitate corneal epithelial wound closure in vivo. 7 8 9 10 11 12 13  
Another characteristic of the cornea is that it contains the highest density of sensory nerve fibers of any tissue in the body, and this innervation plays an important role in maintenance of the structure and function of the corneal epithelium. Indeed, corneal denervation results in a variety of abnormalities, including delayed wound healing. 14 The sensory neurotransmitter substance P (SP) has been detected in the sensory nerve fibers of the cornea. 15 16 17 SP is thought to contribute to diverse physiological functions in various tissues, including neurogenic inflammation, 18 vasodilation, 19 contraction of smooth muscle, 20 modulation of immune cell activity, 21 and induction of E-cadherin in corneal epithelial cells. 22 SP exerts its biological effects through binding to G protein–coupled receptors, among which the neurokinin type 1 (NK-1) receptor shows the highest affinity for SP. 23 The binding of ligand to NK receptors results in the activation of phospholipase C (PLC) and adenylate cyclase and the consequent production of the second messengers inositol 1,4,5-trisphosphate (IP3) and cAMP, respectively. 24  
We have shown that SP and insulin-like growth factor (IGF)-1 synergistically promote both corneal epithelial migration in vitro 25 as well as the closure of corneal epithelial wounds in vivo, both in animals 12 and in humans. 13 The organ culture system is a simple and reliable model with which to assess epithelial migration quantitatively and to study the mechanisms of wound healing. The tetrapeptide sequence at the COOH terminus of SP, Phe-Gly-Leu-Met-amide (FGLM-amide), is sufficient for the synergistic effect with IGF-1 on corneal epithelial migration. 12 The effect of this peptide, like that of the full-length molecule, is mediated through interaction with the NK-1 receptor. 26 We have also recently shown that the effect of IGF-1 in this system is mimicked by a synthetic peptide of 12 amino acids corresponding to the C domain of this growth factor. The effect of IGF-1 or the C-domain peptide on epithelial migration, however, is not mediated by the known IGF-1 or insulin receptors. 27  
We have now investigated the role of SP in corneal epithelial migration. We found that SP sensitizes epithelial cells to the induction of migration by IGF-1 and that this sensitizing effect of SP is also apparent with epithelial migration induced by other biological agents, including fibronectin and IL-6. We also determined that the intracellular signaling pathway responsible for this action of SP includes the IP3-induced release of Ca2+ from intracellular stores and the consequent activation of Ca2+- and calmodulin-dependent protein kinase II (CaM-PK II). 
Methods
Materials
Recombinant human IGF-1, IL-6, and human plasma fibronectin were obtained from BD Biosciences (Franklin Lakes, NJ), Genzyme (Cambridge, MA), and Roche Diagnostics (Indianapolis, IN), respectively. U73122, U73343, and Xestospongin C were from Calbiochem (La Jolla, CA); H-89 and KN-04 were from Seikagaku (Tokyo, Japan); and calphostin C, thapsigargin, W-7, W-5, and KN-62 were from Wako (Osaka, Japan). Plastic multiwell culture dishes and medium-199 were from Corning-Costar (Corning, NY) and Invitrogen-Gibco (Gaithersburg, MD), respectively. The C-domain peptide of IGF-1 (GYGSSSRRAPQT) and FGLM-amide were synthesized by the Peptide Institute (Osaka, Japan). 
Animals
Albino Japanese rabbits (body mass, 2–3 kg) were obtained from KBT Oriental (Saga, Japan). Their care and treatment conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Rabbit Corneal Epithelial Migration Assay
The rabbit corneal epithelial migration assay was performed as described previously. 5 27 Rabbits were killed with an overdose of pentobarbital sodium injected intravenously, and both eyes were enucleated. The sclerocorneal rim was cut, and the cornea was excised and washed several times with sterile phosphate-buffered saline. Six tissue blocks were cut from each cornea with a razor blade, and each corneal block was placed with the epithelial side down in the wells of a 24-well tissue culture plate containing medium-199 in the absence (control) or presence of test agents. After incubation for 24 hours at 37°C in a humidified atmosphere of 5% CO2 in air, the blocks were fixed overnight at 4°C with a mixture of glacial acetic acid and absolute ethanol (5:95, vol/vol). They were then dehydrated by exposure to a graded series of ethanol solutions, immersed in chloroform, and embedded in paraffin. Four thin (7 μm) sections were cut at 200-μm intervals from each block and, after the removal of paraffin, were stained with hematoxylin-eosin. The sections were examined with a light microscope and photographed, and the length of the path of corneal epithelial migration down both sides of each section (toward the endothelial side) was measured on the photographs with a computer-assisted digitizer. 
Statistical Analysis
Data are expressed as the mean ± SE and were analyzed by the Dunnett multiple comparison test. P < 0.05 was considered statistically significant. 
Results
Sensitizing Effect of FGLM-Amide on Corneal Epithelial Migration Induced by the C-Domain Peptide of IGF-1
Given that the synergistic effect of the COOH-terminal tetrapeptide of SP (FGLM-amide) and IGF-1 on corneal epithelial migration is identical with that of SP and IGF-1, 12 we used FGLM-amide to elucidate the role of SP in this process in the present study. This synergistic effect is dependent on the concentration of FGLM-amide up to 20 μM and then reaches a plateau in the presence of IGF-1 (1 nM). 12 FGLM-amide (20 μM) alone has no effect on epithelial migration. 12 Even at higher concentrations up to 1 mM, it did not affect this process (data not shown). FGLM-amide thus does not possess an intrinsic ability to induce corneal epithelial migration by itself. As we demonstrated previously, 27 although incubation of full-thickness cut blocks of the rabbit cornea with the C-domain peptide of IGF-1 alone at a concentration of 1 nM also failed to affect epithelial migration, the C-domain peptide or IGF-1 (1 nM) exhibited a significant stimulatory effect on this process in the presence of 20 μM FGLM-amide (Fig. 1) . In the absence of FGLM-amide, the C-domain peptide of IGF-1 alone had no significant effect on corneal epithelial migration at concentrations up to 10 nM (Fig. 1A) . However, the C-domain peptide significantly promoted epithelial migration at a concentration of 100 μM in the absence of FGLM-amide (Fig. 1) , indicating that it possesses an intrinsic ability to stimulate this process. We were not able to achieve such a high concentration of IGF-1 with the amount of growth factor available to determine whether the full-length molecule is also able to induce corneal epithelial migration by itself. Incubation of corneal blocks with various concentrations of the C-domain peptide in the absence or presence of FGLM-amide (20 μM) revealed that the latter peptide induced a marked shift in the dose–response curve of the C-domain peptide to lower concentrations, reducing the median effective concentration (EC50) from 5.60 μM to 0.187 nM without affecting the magnitude of the maximum response (Fig. 1A) . These results thus indicate that FGLM-amide sensitizes the corneal epithelium to the induction of migration by the C domain of IGF-1. 
Characterization of Intracellular Signaling Triggered by FGLM-Amide
To investigate the mechanism by which FGLM-amide modulates the stimulatory effect of IGF-1 (or the C-domain peptide) on corneal epithelial migration, we characterized the intracellular signaling pathway activated by FGLM-amide. We had shown that the effect of SP or FGLM-amide in synergistic action with IGF-1 on corneal epithelial migration is mediated by the NK-1 receptor, 26 a G protein–coupled receptor that activates PLC in response to ligand binding. 24 28 29 30 Incubation of rabbit corneal blocks with the PLC inhibitor U73122 (see Fig. 7for signaling pathways and targets of inhibitors examined in this study) alone at a concentration of 5 μM had no significant effect on basal epithelial migration. However, U73122, but not its inactive analogue U73343, at this concentration completely inhibited the induction of migration by IGF-1 (1 nM) in the presence of 20 μM FGLM-amide (Fig. 2) . In contrast, 1 μM H-89, an inhibitor of cAMP-dependent protein kinase, had no effect on epithelial migration in the absence or presence of IGF-1 plus FGLM-amide (Fig. 2) . These results thus suggest that activation of PLC, but not that of adenylate cyclase, is necessary for the action of FGLM-amide. 
PLC catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to yield diacylglycerol and IP3, the former of which activates protein kinase C (PKC). Although the PKC inhibitor calphostin C (3 μM) reduced the extent of basal epithelial migration by 17%, it did not impair the stimulation of epithelial migration by IGF-1 and FGLM-amide (Fig. 2) , suggesting that PKC does not mediate this latter effect. 
We therefore next examined the possible role of IP3, which interacts with the intracellular IP3 receptor and thereby triggers the release of Ca2+ from intracellular stores, in the stimulation of corneal epithelial migration. Incubation of corneal blocks with Xestospongin C (25 μM), an inhibitor of the IP3 receptor, prevented the stimulatory effect of IGF-1 and FGLM-amide on epithelial migration without affecting basal migration (Fig. 3) , thus implicating intracellular Ca2+ mobilization through the IP3 receptor in the action of FGLM-amide. At a concentration of 20 nM, thapsigargin, a specific inhibitor of the Ca2+-dependent ATPase of the endoplasmic reticulum (ER), had no significant effect on basal migration but prevented the stimulatory effect of IGF-1 and FGLM-amide (Fig. 3)
We also examined signaling downstream of Ca2+ release from the ER. Incubation of corneal blocks with W-7 (50 μM), an antagonist of the Ca2+-binding protein calmodulin, completely inhibited the stimulation of epithelial migration by IGF-1 and FGLM-amide (Fig. 3) . W-5 (50 μM), an inactive analogue of W-7, did not exhibit such an inhibitory effect on corneal epithelial migration. The CaM-PK II inhibitor KN-62 (Fig. 3) , but not its inactive analogue KN-04 (data not shown), also inhibited the stimulation of corneal epithelial migration by IGF-1 and FGLM-amide, without affecting basal migration, at a concentration of 10 μM, indicating that CaM-PK II mediates the action of FGLM-amide on IGF-1–induced corneal epithelial migration. Essentially identical results were obtained when epithelial migration was stimulated with the C-domain peptide instead of IGF-1. 
Relation between Signaling Triggered by SP and That Induced by IGF-1
To determine the relation between the SP signaling pathway and that activated by IGF-1, we examined the effect of the inhibitor of CaM-PK II on epithelial migration induced by the C-domain peptide of IGF-1 in the absence of FGLM-amide. Whereas KN-62 (10 μM) completely inhibited the epithelial migration induced by a low concentration (1 nM) of the C-domain peptide in the presence of FGLM-amide, it had no significant effect on that elicited by a high concentration (100 μM) of this peptide alone (Fig. 4) , indicating that the activation of CaM-PK II does not directly mediate IGF-1–induced epithelial migration but rather modulates it. 
Sensitizing Effect of FGLM-Amide on Corneal Epithelial Migration Induced by Fibronectin or IL-6
We next examined the possible effects of FGLM-amide on the stimulation of epithelial migration by agents other than IGF-1 such as fibronectin and IL-6. Consistent with our previous observations, 5 6 fibronectin or IL-6 alone at concentrations of 230 nM and 500 pM, respectively, induced corneal epithelial migration, whereas these same agents at concentrations of 1 nM and 15 pM, respectively, were without effect (Figs. 5A 6A) . In the presence of 20 μM FGLM-amide, however, these lower concentrations of fibronectin and IL-6 significantly increased the extent of epithelial migration. 
Analysis of the effect of FGLM-amide in the presence of various concentrations of fibronectin or IL-6 revealed that this peptide also induced a leftward shift in the dose–response curves for the stimulation of corneal epithelial migration by these agents, reducing the EC50 value for fibronectin from 3.60 nM to 43.8 pM (Fig. 5A)and that for IL-6 from 35.8 to 3.77 pM (Fig. 6A) . The SP-derived peptide thus exhibits a common effect on corneal epithelial migration induced by various agents. 
KN-62 blocked the facilitation of epithelial migration by low concentrations of fibronectin (1 nM) or IL-6 (15 pM) in the presence of FGLM-amide (Figs. 5B 6B) , indicating that the sensitizing effect of FGLM-amide on fibronectin- or IL-6-induced epithelial migration is also mediated by CaM-PK II. KN-62 had no effect on the epithelial migration induced by high concentrations of fibronectin (230 nM) or IL-6 (500 pM) in the absence of FGLM-amide (Figs. 5B 6B) . These results also indicate that the activation CaM-PK II does not directly contribute to the induction of epithelial migration by fibronectin or IL-6 but rather modulates it. The modulatory effects of FGLM-amide on corneal epithelial migration induced by IGF-1, fibronectin, or IL-6 thus appear to be mediated by a common mechanism. 
Discussion
We have shown that corneal epithelial migration is stimulated by the combination of IGF-1 (or the C-domain peptide) and SP (or FGLM-amide), but not by either agent alone. 12 25 27 Our present study has revealed, however, that high concentrations of the C-domain peptide induce epithelial migration in the absence of SP or FGLM-amide. We further showed that SP or FGLM-amide plays a modulatory role in the stimulation of epithelial migration by IGF-1 or the C-domain peptide by sensitizing epithelial cells to the effects of these agents. We have also now shown that this modulatory effect of SP or FGLM-amide is mediated by a signaling pathway triggered by the NK-1 receptor that leads to activation of CaM-PK II. Furthermore, the sensitizing effect of SP or FGLM-amide was found to be common to the induction of epithelial migration by other biological agents, including fibronectin and IL-6. 
The stimulatory effect of the C-domain peptide alone was apparent in the present study only at concentrations about four orders of magnitude greater than those at which synergism with FGLM-amide is apparent (EC50 = 5.60 μM and 0.187 nM, respectively). Given that concentrations of IGF-1 of ∼100 μM are not physiological, an action of endogenous IGF-1 on epithelial migration is probably manifested only in vivo in the presence of SP. FGLM-amide also reduced the EC50 of fibronectin and IL-6 for stimulation of corneal epithelial migration from 3.60 nM to 43.8 pM and from 35.8 to 3.77 pM, respectively. The sensory neurotransmitter SP thus functions to reduce the amounts of various biologically active factors, including growth factors, extracellular matrix proteins, and cytokines, that are necessary for the promotion of corneal epithelial migration and may therefore be essential for epithelial wound healing in vivo. The sensitizing effect of FGLM-amide was greater for epithelial migration induced by the C domain of IGF-1 than for that induced by fibronectin or IL-6. The EC50 for the C-domain peptide differed by a factor of ∼30,000 between the presence and absence of FGLM-amide, whereas the corresponding factors were between 10 and 100 for fibronectin and IL-6. The reason for this difference remains to be determined. 
We have shown that the synergistic effect of SP with IGF-1 on corneal epithelial migration is mediated by the NK-1 receptor. 26 We have now shown that the activation of PLC and consequent generation of IP3 triggered by ligand binding to this receptor are essential for the modulation of epithelial migration by FGLM-amide. The importance of IP3 in SP signal transduction has been demonstrated in smooth muscle contraction. 31 32 Our results indicate that the activation of PKC by diacylglycerol, the other product of PIP2 hydrolysis catalyzed by PLC, is not necessary for the modulatory effect of FGLM-amide on corneal epithelial migration, although PKC may contribute to basal epithelial migration. The interaction of IP3 with its specific intracellular receptor results in the release of Ca2+ from the ER. With the use of the IP3 receptor blocker Xestospongin C as well as thapsigargin, a specific inhibitor of the ER Ca2+ pump, we showed that the modulatory action of FGLM-amide in corneal epithelial migration is mediated by Ca2+ release from the ER. Furthermore, we demonstrated that the Ca2+ signal is then propagated by CaM-PK II (Fig. 7)
The sensitizing effect of SP or FGLM-amide on epithelial migration in the organ culture system is apparent at micromolar concentrations. 12 25 The affinity of SP for the NK-1 receptor measured in cultured rabbit corneal epithelial cells is in the nanomolar range, however. 26 The reason for this discrepancy in SP concentrations is unclear, but it is possible either that another signaling pathway triggered by SP in addition to NK-1 receptor signaling may be necessary for the sensitizing effect of the neurotransmitter or that SP and FGLM-amide are degraded by peptidases to a greater extent in the organ culture system than in cell cultures. 
Our results indicate that the activation of CaM-PK II does not directly contribute to the signaling pathways that underlie the stimulatory effects of IGF-1, fibronectin, or IL-6 on corneal epithelial migration but rather participates in the modulation of these pathways by SP. The target protein (or proteins) of CaM-PK II in the SP signaling pathway thus probably contributes to cross talk with the signaling pathways triggered by IGF-1, fibronectin, or IL-6 (Fig. 7) . The mechanisms by which these latter agents promote corneal epithelial migration have been unclear. The interaction of fibronectin with integrins is important for the facilitation of epithelial migration by this extracellular matrix protein in the skin. 33 Integrins are also implicated in the facilitation of corneal epithelial migration by IL-6. 6 Known IGF receptors are not responsible for the stimulatory effect of IGF-1 or the C-domain peptide on corneal epithelial migration, which suggests the involvement of a novel signaling pathway for IGF-1. 27 Our demonstration that CaM-PK II modulates the facilitation of corneal epithelial migration by IGF-1, fibronectin, or IL-6 indicates that the signaling pathways triggered by these three agents converge at some point. Potential targets of CaM-PK II thus include proteins that contribute to the attachment of epithelial cells to the substrate. 
We have shown that SP sensitizes the corneal epithelium to the migration-promoting effects of various agents, suggesting that this sensory neurotransmitter plays an important protective role in the cornea by facilitating epithelial wound healing. Such a role may be especially essential in pathologic conditions such as those associated with persistent epithelial defects or ulcers of the cornea. Indeed, the persistent corneal epithelial defects in individuals with neurotrophic keratopathy are successfully treated by the administration of eyedrops containing IGF-1 and SP. 13 14 34 35 Our study thus provides important insight into a new strategy for the treatment of corneal wounds. 
 
Figure 1.
 
Sensitization of corneal epithelial migration by FGLM-amide to the stimulatory effect of the C-domain peptide of IGF-1. (A) Rabbit corneal blocks were incubated at 37°C for 24 hours with the indicated concentrations of the C-domain peptide of IGF-1 in the absence (○) or presence (•) of 20 μM FGLM-amide, after which the extent of epithelial migration was determined. Data are expressed as percentage facilitation of epithelial migration relative to control levels (those obtained for corneal blocks incubated in the absence of agents) and are the mean ± SE of six determinations. *P < 0.05 compared with the control. (B) Corneal blocks were incubated at 37°C for 24 hours in the absence (control) or presence either of 1 nM of the C-domain peptide of IGF-1 plus 20 μM FGLM-amide or of 100 μM of the C-domain peptide alone. Sections of the corneal blocks were then prepared and stained with hematoxylin-eosin. Arrows: edge of the epithelial surface of each corneal block; arrowheads: edge of the migrating epithelial cells. Scale bar, 100 μm.
Figure 1.
 
Sensitization of corneal epithelial migration by FGLM-amide to the stimulatory effect of the C-domain peptide of IGF-1. (A) Rabbit corneal blocks were incubated at 37°C for 24 hours with the indicated concentrations of the C-domain peptide of IGF-1 in the absence (○) or presence (•) of 20 μM FGLM-amide, after which the extent of epithelial migration was determined. Data are expressed as percentage facilitation of epithelial migration relative to control levels (those obtained for corneal blocks incubated in the absence of agents) and are the mean ± SE of six determinations. *P < 0.05 compared with the control. (B) Corneal blocks were incubated at 37°C for 24 hours in the absence (control) or presence either of 1 nM of the C-domain peptide of IGF-1 plus 20 μM FGLM-amide or of 100 μM of the C-domain peptide alone. Sections of the corneal blocks were then prepared and stained with hematoxylin-eosin. Arrows: edge of the epithelial surface of each corneal block; arrowheads: edge of the migrating epithelial cells. Scale bar, 100 μm.
Figure 7.
 
Intracellular signaling responsible for the modulatory action of SP on corneal epithelial migration induced by IGF-1, fibronectin (FN), or IL-6. Binding of SP (or FGLM-amide) to the NK-1 receptor results in the activation of PLC, the IP3-mediated release of Ca2+ from the ER, and the activation of CaM-PK II. The target protein (or proteins) of CaM-PK II may contribute to cross talk with the signaling pathways triggered by IGF-1 (or its C domain), fibronectin, or IL-6. The targets of inhibitors used in the present study are also indicated.
Figure 7.
 
Intracellular signaling responsible for the modulatory action of SP on corneal epithelial migration induced by IGF-1, fibronectin (FN), or IL-6. Binding of SP (or FGLM-amide) to the NK-1 receptor results in the activation of PLC, the IP3-mediated release of Ca2+ from the ER, and the activation of CaM-PK II. The target protein (or proteins) of CaM-PK II may contribute to cross talk with the signaling pathways triggered by IGF-1 (or its C domain), fibronectin, or IL-6. The targets of inhibitors used in the present study are also indicated.
Figure 2.
 
Effects of inhibitors of PLC, cAMP-dependent protein kinase, and PKC on the stimulation of corneal epithelial migration by IGF-1 and FGLM-amide. Corneal blocks were incubated at 37°C for 24 hours in the absence (control) or presence of 5 μM U73122, 5 μM U73343 (an inactive analogue of U73122), 1 μM H-89, or 3 μM calphostin C with a combination of 20 μM FGLM-amide plus 1 nM IGF-1, as indicated. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05 versus control levels or for the indicated comparisons.
Figure 2.
 
Effects of inhibitors of PLC, cAMP-dependent protein kinase, and PKC on the stimulation of corneal epithelial migration by IGF-1 and FGLM-amide. Corneal blocks were incubated at 37°C for 24 hours in the absence (control) or presence of 5 μM U73122, 5 μM U73343 (an inactive analogue of U73122), 1 μM H-89, or 3 μM calphostin C with a combination of 20 μM FGLM-amide plus 1 nM IGF-1, as indicated. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05 versus control levels or for the indicated comparisons.
Figure 3.
 
Role of intracellular Ca2+ signaling in the induction of corneal epithelial migration by IGF-1 and FGLM-amide. Corneal blocks were incubated at 37°C for 24 hours in the absence (control) or presence of 25 μM Xestospongin C, 20 nM thapsigargin, 50 μM W-7, 50 μM W-5 (an inactive analogue of W-7), or 10 μM KN-62 and of the combination of 20 μM FGLM-amide plus 1 nM IGF-1. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05 compared with the control.
Figure 3.
 
Role of intracellular Ca2+ signaling in the induction of corneal epithelial migration by IGF-1 and FGLM-amide. Corneal blocks were incubated at 37°C for 24 hours in the absence (control) or presence of 25 μM Xestospongin C, 20 nM thapsigargin, 50 μM W-7, 50 μM W-5 (an inactive analogue of W-7), or 10 μM KN-62 and of the combination of 20 μM FGLM-amide plus 1 nM IGF-1. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05 compared with the control.
Figure 4.
 
Effects of a CaM-PK II inhibitor on the stimulation of corneal epithelial migration by the C-domain peptide of IGF-1. Corneal blocks were incubated at 37°C for 24 hours with the indicated combinations of 1 nM (L) or 100 μM (H) of the C-domain peptide of IGF-1, 20 μM FGLM-amide, and 10 μM KN-62. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05, **P < 0.01 compared with the control.
Figure 4.
 
Effects of a CaM-PK II inhibitor on the stimulation of corneal epithelial migration by the C-domain peptide of IGF-1. Corneal blocks were incubated at 37°C for 24 hours with the indicated combinations of 1 nM (L) or 100 μM (H) of the C-domain peptide of IGF-1, 20 μM FGLM-amide, and 10 μM KN-62. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05, **P < 0.01 compared with the control.
Figure 5.
 
Sensitization of corneal epithelial migration by FGLM-amide to the stimulatory effect of fibronectin. (A) Rabbit corneal blocks were incubated at 37°C for 24 hours with the indicated concentrations of fibronectin in the absence (□) or presence (▪) of 20 μM FGLM-amide, after which the extent of epithelial migration was determined. Data are expressed as percentage facilitation of epithelial migration relative to control levels (those obtained for corneal blocks incubated in the absence of agents) and are the mean ± SE of six determinations. *P < 0.05 compared with the control. (B) Corneal blocks were incubated at 37°C for 24 hours with the indicated combinations of 1 nM (L) or 230 nM (H) fibronectin (FN), 20 μM FGLM-amide, and 10 μM KN-62. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05, **P < 0.01 compared with the control.
Figure 5.
 
Sensitization of corneal epithelial migration by FGLM-amide to the stimulatory effect of fibronectin. (A) Rabbit corneal blocks were incubated at 37°C for 24 hours with the indicated concentrations of fibronectin in the absence (□) or presence (▪) of 20 μM FGLM-amide, after which the extent of epithelial migration was determined. Data are expressed as percentage facilitation of epithelial migration relative to control levels (those obtained for corneal blocks incubated in the absence of agents) and are the mean ± SE of six determinations. *P < 0.05 compared with the control. (B) Corneal blocks were incubated at 37°C for 24 hours with the indicated combinations of 1 nM (L) or 230 nM (H) fibronectin (FN), 20 μM FGLM-amide, and 10 μM KN-62. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05, **P < 0.01 compared with the control.
Figure 6.
 
Sensitization of corneal epithelial migration by FGLM-amide to the stimulatory effect of IL-6. (A) Rabbit corneal blocks were incubated at 37°C for 24 hours with the indicated concentrations of IL-6 in the absence (▵) or presence (▴) of 20 μM FGLM-amide, after which the extent of epithelial migration was determined. Data are expressed as percentage facilitation of epithelial migration relative to control levels (those obtained for corneal blocks incubated in the absence of agents) and are the mean ± SE of six determinations. *P < 0.05 compared with the control. (B) Corneal blocks were incubated at 37°C for 24 hours with the indicated combinations of 15 pM (L) or 500 pM (H) IL-6, 20 μM FGLM-amide, and 10 μM KN-62. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05 compared with the control.
Figure 6.
 
Sensitization of corneal epithelial migration by FGLM-amide to the stimulatory effect of IL-6. (A) Rabbit corneal blocks were incubated at 37°C for 24 hours with the indicated concentrations of IL-6 in the absence (▵) or presence (▴) of 20 μM FGLM-amide, after which the extent of epithelial migration was determined. Data are expressed as percentage facilitation of epithelial migration relative to control levels (those obtained for corneal blocks incubated in the absence of agents) and are the mean ± SE of six determinations. *P < 0.05 compared with the control. (B) Corneal blocks were incubated at 37°C for 24 hours with the indicated combinations of 15 pM (L) or 500 pM (H) IL-6, 20 μM FGLM-amide, and 10 μM KN-62. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05 compared with the control.
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Figure 1.
 
Sensitization of corneal epithelial migration by FGLM-amide to the stimulatory effect of the C-domain peptide of IGF-1. (A) Rabbit corneal blocks were incubated at 37°C for 24 hours with the indicated concentrations of the C-domain peptide of IGF-1 in the absence (○) or presence (•) of 20 μM FGLM-amide, after which the extent of epithelial migration was determined. Data are expressed as percentage facilitation of epithelial migration relative to control levels (those obtained for corneal blocks incubated in the absence of agents) and are the mean ± SE of six determinations. *P < 0.05 compared with the control. (B) Corneal blocks were incubated at 37°C for 24 hours in the absence (control) or presence either of 1 nM of the C-domain peptide of IGF-1 plus 20 μM FGLM-amide or of 100 μM of the C-domain peptide alone. Sections of the corneal blocks were then prepared and stained with hematoxylin-eosin. Arrows: edge of the epithelial surface of each corneal block; arrowheads: edge of the migrating epithelial cells. Scale bar, 100 μm.
Figure 1.
 
Sensitization of corneal epithelial migration by FGLM-amide to the stimulatory effect of the C-domain peptide of IGF-1. (A) Rabbit corneal blocks were incubated at 37°C for 24 hours with the indicated concentrations of the C-domain peptide of IGF-1 in the absence (○) or presence (•) of 20 μM FGLM-amide, after which the extent of epithelial migration was determined. Data are expressed as percentage facilitation of epithelial migration relative to control levels (those obtained for corneal blocks incubated in the absence of agents) and are the mean ± SE of six determinations. *P < 0.05 compared with the control. (B) Corneal blocks were incubated at 37°C for 24 hours in the absence (control) or presence either of 1 nM of the C-domain peptide of IGF-1 plus 20 μM FGLM-amide or of 100 μM of the C-domain peptide alone. Sections of the corneal blocks were then prepared and stained with hematoxylin-eosin. Arrows: edge of the epithelial surface of each corneal block; arrowheads: edge of the migrating epithelial cells. Scale bar, 100 μm.
Figure 7.
 
Intracellular signaling responsible for the modulatory action of SP on corneal epithelial migration induced by IGF-1, fibronectin (FN), or IL-6. Binding of SP (or FGLM-amide) to the NK-1 receptor results in the activation of PLC, the IP3-mediated release of Ca2+ from the ER, and the activation of CaM-PK II. The target protein (or proteins) of CaM-PK II may contribute to cross talk with the signaling pathways triggered by IGF-1 (or its C domain), fibronectin, or IL-6. The targets of inhibitors used in the present study are also indicated.
Figure 7.
 
Intracellular signaling responsible for the modulatory action of SP on corneal epithelial migration induced by IGF-1, fibronectin (FN), or IL-6. Binding of SP (or FGLM-amide) to the NK-1 receptor results in the activation of PLC, the IP3-mediated release of Ca2+ from the ER, and the activation of CaM-PK II. The target protein (or proteins) of CaM-PK II may contribute to cross talk with the signaling pathways triggered by IGF-1 (or its C domain), fibronectin, or IL-6. The targets of inhibitors used in the present study are also indicated.
Figure 2.
 
Effects of inhibitors of PLC, cAMP-dependent protein kinase, and PKC on the stimulation of corneal epithelial migration by IGF-1 and FGLM-amide. Corneal blocks were incubated at 37°C for 24 hours in the absence (control) or presence of 5 μM U73122, 5 μM U73343 (an inactive analogue of U73122), 1 μM H-89, or 3 μM calphostin C with a combination of 20 μM FGLM-amide plus 1 nM IGF-1, as indicated. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05 versus control levels or for the indicated comparisons.
Figure 2.
 
Effects of inhibitors of PLC, cAMP-dependent protein kinase, and PKC on the stimulation of corneal epithelial migration by IGF-1 and FGLM-amide. Corneal blocks were incubated at 37°C for 24 hours in the absence (control) or presence of 5 μM U73122, 5 μM U73343 (an inactive analogue of U73122), 1 μM H-89, or 3 μM calphostin C with a combination of 20 μM FGLM-amide plus 1 nM IGF-1, as indicated. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05 versus control levels or for the indicated comparisons.
Figure 3.
 
Role of intracellular Ca2+ signaling in the induction of corneal epithelial migration by IGF-1 and FGLM-amide. Corneal blocks were incubated at 37°C for 24 hours in the absence (control) or presence of 25 μM Xestospongin C, 20 nM thapsigargin, 50 μM W-7, 50 μM W-5 (an inactive analogue of W-7), or 10 μM KN-62 and of the combination of 20 μM FGLM-amide plus 1 nM IGF-1. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05 compared with the control.
Figure 3.
 
Role of intracellular Ca2+ signaling in the induction of corneal epithelial migration by IGF-1 and FGLM-amide. Corneal blocks were incubated at 37°C for 24 hours in the absence (control) or presence of 25 μM Xestospongin C, 20 nM thapsigargin, 50 μM W-7, 50 μM W-5 (an inactive analogue of W-7), or 10 μM KN-62 and of the combination of 20 μM FGLM-amide plus 1 nM IGF-1. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05 compared with the control.
Figure 4.
 
Effects of a CaM-PK II inhibitor on the stimulation of corneal epithelial migration by the C-domain peptide of IGF-1. Corneal blocks were incubated at 37°C for 24 hours with the indicated combinations of 1 nM (L) or 100 μM (H) of the C-domain peptide of IGF-1, 20 μM FGLM-amide, and 10 μM KN-62. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05, **P < 0.01 compared with the control.
Figure 4.
 
Effects of a CaM-PK II inhibitor on the stimulation of corneal epithelial migration by the C-domain peptide of IGF-1. Corneal blocks were incubated at 37°C for 24 hours with the indicated combinations of 1 nM (L) or 100 μM (H) of the C-domain peptide of IGF-1, 20 μM FGLM-amide, and 10 μM KN-62. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05, **P < 0.01 compared with the control.
Figure 5.
 
Sensitization of corneal epithelial migration by FGLM-amide to the stimulatory effect of fibronectin. (A) Rabbit corneal blocks were incubated at 37°C for 24 hours with the indicated concentrations of fibronectin in the absence (□) or presence (▪) of 20 μM FGLM-amide, after which the extent of epithelial migration was determined. Data are expressed as percentage facilitation of epithelial migration relative to control levels (those obtained for corneal blocks incubated in the absence of agents) and are the mean ± SE of six determinations. *P < 0.05 compared with the control. (B) Corneal blocks were incubated at 37°C for 24 hours with the indicated combinations of 1 nM (L) or 230 nM (H) fibronectin (FN), 20 μM FGLM-amide, and 10 μM KN-62. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05, **P < 0.01 compared with the control.
Figure 5.
 
Sensitization of corneal epithelial migration by FGLM-amide to the stimulatory effect of fibronectin. (A) Rabbit corneal blocks were incubated at 37°C for 24 hours with the indicated concentrations of fibronectin in the absence (□) or presence (▪) of 20 μM FGLM-amide, after which the extent of epithelial migration was determined. Data are expressed as percentage facilitation of epithelial migration relative to control levels (those obtained for corneal blocks incubated in the absence of agents) and are the mean ± SE of six determinations. *P < 0.05 compared with the control. (B) Corneal blocks were incubated at 37°C for 24 hours with the indicated combinations of 1 nM (L) or 230 nM (H) fibronectin (FN), 20 μM FGLM-amide, and 10 μM KN-62. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05, **P < 0.01 compared with the control.
Figure 6.
 
Sensitization of corneal epithelial migration by FGLM-amide to the stimulatory effect of IL-6. (A) Rabbit corneal blocks were incubated at 37°C for 24 hours with the indicated concentrations of IL-6 in the absence (▵) or presence (▴) of 20 μM FGLM-amide, after which the extent of epithelial migration was determined. Data are expressed as percentage facilitation of epithelial migration relative to control levels (those obtained for corneal blocks incubated in the absence of agents) and are the mean ± SE of six determinations. *P < 0.05 compared with the control. (B) Corneal blocks were incubated at 37°C for 24 hours with the indicated combinations of 15 pM (L) or 500 pM (H) IL-6, 20 μM FGLM-amide, and 10 μM KN-62. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05 compared with the control.
Figure 6.
 
Sensitization of corneal epithelial migration by FGLM-amide to the stimulatory effect of IL-6. (A) Rabbit corneal blocks were incubated at 37°C for 24 hours with the indicated concentrations of IL-6 in the absence (▵) or presence (▴) of 20 μM FGLM-amide, after which the extent of epithelial migration was determined. Data are expressed as percentage facilitation of epithelial migration relative to control levels (those obtained for corneal blocks incubated in the absence of agents) and are the mean ± SE of six determinations. *P < 0.05 compared with the control. (B) Corneal blocks were incubated at 37°C for 24 hours with the indicated combinations of 15 pM (L) or 500 pM (H) IL-6, 20 μM FGLM-amide, and 10 μM KN-62. The length of the path of epithelial migration was then measured. Data are expressed as a percentage of control levels and are the mean ± SE of six determinations. *P < 0.05 compared with the control.
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