Abstract
purpose. Insulin-like growth factors (IGFs) and either substance P (SP) or an SP-derived peptide (FGLM-amide) synergistically facilitate corneal epithelial wound healing in vitro and in vivo. The mechanism of this synergism and the clinical potential of these agents were further investigated by determination of the relevant functional domain of IGFs.
methods. The effects of IGF-derived peptides on corneal epithelial cell migration were evaluated with the rabbit cornea in an organ culture system. Corneal epithelial wound closure in vivo was also evaluated in rabbits after epithelial debridement with n-heptanol.
results. In the presence of FGLM-amide, peptides corresponding to the C domain of IGF-1 or -2 significantly promoted corneal epithelial migration in vitro to an extent similar to that apparent with the full-length molecules. In contrast, peptides corresponding to the D domain of these growth factors had no such effect. Mutation of serine-34 in the C domain of IGF-1 to alanine abolished the synergistic effect with FGLM-amide on corneal epithelial migration. The C peptide of proinsulin did not affect corneal epithelial migration in the absence or presence of FGLM-amide. The administration of eye drops containing both the C-domain peptide of IGF-1 and FGLM-amide significantly promoted corneal epithelial wound closure in vivo.
conclusions. The C domain of IGF-1 or -2, for which no biological function has previously been identified, is essential for the synergistic effect of these growth factors with SP on corneal epithelial migration.
Wound healing is fundamental to the survival of an organism. The healing process is complex and comprises a series of events in which humoral and neural factors, extracellular matrix, and various cell types participate at the appropriate time.
1 Epithelialization is one of the most critical steps of this process. We have studied the regulation of corneal epithelial wound healing in animals, both in vitro
2 3 4 5 6 7 and in vivo
8 9 as well as in humans clinically.
10 11 The cornea possesses a relatively simple structure and is avascular. It is one of the most sensitive tissues in the body, with the density of nerve endings in the cornea being 300 to 400 times that in the skin. These characteristics render the cornea amenable to the study of epithelial wound healing or of epithelial migration per se. We have developed an organ culture system to study the cornea, with which it is possible to evaluate epithelial migration quantitatively.
2 With this system, we have previously shown that insulin-like growth factor (IGF)-1 acts synergistically with substance P (SP) to promote corneal epithelial migration in vitro.
12 The combination of IGF-1 and SP also synergistically facilitates the closure of rabbit corneal epithelial wounds in vivo.
13 Both in vitro and in vivo studies demonstrated that SP exerts its synergistic action with IGF-1 through interaction with the neurokinin receptor NK-1.
14 The carboxyl terminal four amino acids of SP (Phe-Gly-Leu-Met-amide, or FGLM-amide) are sufficient for this effect.
15 Furthermore, we showed that the persistent corneal epithelial defects of patients with neurotrophic keratopathy are successfully treated by the administration of eye drops containing IGF-1 and either SP or FGLM-amide.
16 17 18
IGF-1 and -2 each comprise four domains: the A, B, C, and D domains
(Fig. 1) . These growth factors exert various biological effects as a result of their interaction with IGF and insulin receptors.
19 IGFs and insulin share many structural similarities, especially in the A and B domains.
20 However, the C domain of IGFs does not share sequence homology with the C peptide of proinsulin, which is not retained in mature insulin. In addition, IGFs possess the D domain at their carboxyl termini, whereas insulin has no counterpart sequence
(Fig. 1) . IGF-1 binds with highest affinity to the IGF type 1 receptor, which is structurally homologous to the insulin receptor. IGF-2 also binds the IGF type 1 receptor but with lower affinity. In contrast, the IGF type 2 receptor binds IGF-2 with higher affinity and IGF-1 with lower affinity. Both IGF-1 and -2 also bind to the insulin receptor with low affinity.
21 No biological function has been attributed to the C domain of IGFs, although this domain modulates the interaction of the A and B domains with insulin and IGF receptors.
22 23 24
The synergistic effect of IGF-1 with SP on corneal epithelial migration is mimicked by IGF-2 but not by insulin,
25 suggesting that the C or D domain of IGFs plays an important role in this action. To provide greater insight into the mechanism underlying the synergism of IGFs and SP, we have now investigated which domain of IGF-1 or -2 is responsible for the effects of these growth factors with FGLM-amide on rabbit corneal epithelial migration both in organ culture and in vivo. Our results demonstrate that the C domain of IGF-1 or -2, but not the D domain of these molecules, is responsible for the synergism with SP in the promotion of corneal epithelial migration.
Human corneal fibroblasts (2 × 106) were cultured in 35-mm dishes first for 48 hours in culture medium containing 0.5% fetal bovine serum and then for 1.5 hours in serum-free medium. They were then incubated for 30 minutes at 37°C in serum-free medium containing test agents, washed with ice-cold PBS, and lysed in 100 μL of a solution containing 2% SDS, 150 mM NaCl, 2 mM NaF, 2 mM Na3VO4, 5 mM EDTA, and 20 mM Tris-HCl (pH 7.5). Protein concentration was determined with a protein assay kit (Dc; Bio-Rad, Hercules, CA). Cell lysates (38 μg of protein) were fractionated by SDS–polyacrylamide gel electrophoresis and subjected to immunoblot analysis with antibodies to Akt or to phospho-Akt (Cell Signaling, Beverly, MA). Immune complexes were detected with horseradish-peroxidase–conjugated secondary antibodies and enhanced chemiluminescence reagents (Amersham).
Elimination of the IGF Type 1 Receptor as a Mediator of Signaling by the C Domain of IGF-1
Lack of Effect of Insulin or the C Peptide of Proinsulin on Corneal Epithelial Migration
Promotion of Corneal Wound Healing In Vivo by the C Domain of IGF-1 and FGLM-Amide