Involvement of insulin-like growth factor-I and insulin-like growth factor binding protein-3 in corneal fibroblasts during corneal wound healing.
OBJECTIVE
The involvement of downstream messengers of transforming growth factor (TGF)-beta in the differentiation of corneal fibroblasts into myofibroblasts was investigated. The effects of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-3 upregulated by TGF-beta were examined in human corneal fibroblasts, and the possible involvement of IGF axis components in corneal wound healing was assessed in a mouse model.


METHODS
Human corneal fibroblasts were incubated with TGF-beta2 or IGF-I, to investigate IGF-I, IGF-II, IGFBP-3, type I collagen, and alpha-smooth muscle actin (alpha-SMA) mRNA, as well as IGFBP-3 protein expression, during myofibroblast differentiation. DNA synthesis was evaluated with a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. IGFBP-3 mRNA expression, protein expression, and immunolocalization were investigated in mouse corneas after photorefractive keratectomy (PRK).


RESULTS
TGF-beta2 treatment induced expression of IGF-I and IGFBP-3 mRNA and of IGFBP-3 protein in human corneal fibroblasts. TGF-beta2 and IGF-I both stimulated expression of type I collagen. TGF-beta2 but not IGF-I potently stimulated alpha-SMA mRNA expression. IGF-I potently stimulated basal DNA synthesis, whereas IGFBP-3 inhibited it. IGF-I potently stimulated proliferation of TGF-beta2-activated myofibroblasts without reversing the activated fibrogenic phenotype, whereas IGFBP-3 suppressed IGF-I-induced proliferation of corneal fibroblasts. IGFBP-3 mRNA and protein increased in mouse corneas soon after PRK, when in vivo immunostaining of the corneas showed expression of IGFBP-3 in the deep layer of the corneal stroma.


CONCLUSIONS
These results suggest that during corneal wound healing, TGF-beta stimulates IGF axis components, whereas IGFBP-3 may modulate IGF-I-induced myofibroblast proliferation to suppress corneal mesenchymal overgrowth.
Izumi
Kanako
K
Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
Kurosaka
Daijiro
D
Iwata
Takeshi
T
Oguchi
Yoshihisa
Y
Tanaka
Yasuhiko
Y
Mashima
Yukihiko
Y
Tsubota
Kazuo
K
D000199:Actins / Q000235:genetics; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics; D003315:Cornea / Q000187:drug effects* / Q000378:metabolism / Q000601:surgery; D004247:DNA / Q000096:biosynthesis; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000235:genetics* / Q000378:metabolism; D054018:Lasers, Excimer; D018903:Photorefractive Keratectomy; D012333:RNA, Messenger / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053781:Transforming Growth Factor beta2; D015854:Up-Regulation; D014945:Wound Healing / Q000502:physiology*
D000199:Actins; D024042:Collagen Type I; D018972:Insulin-Like Growth Factor Binding Protein 3; D012333:RNA, Messenger; C509041:TGFB2 protein, human; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D007334:Insulin-Like Growth Factor I; D004247:DNA