title: Involvement of P38MAPK in human corneal endothelial cell migration induced by TGF-β(2).
abstract: Because human corneal endothelial cells do not proliferate once the endothelial monolayer is formed, corneal wound healing is thought to be mediated by cell enlargement or migration rather than proliferation. However, the cellular mechanisms involved in corneal wound healing have not been fully determined. Because transforming growth factor-β(2) (TGF-β(2)) isoform is present in high concentrations in normal human aqueous humor, it may play a role in human corneal endothelial cell wound healing. The purpose of this study was to determine the effect of TGF-β(2) on the proliferation and migration of cultured human corneal endothelial cells (HCECs). To achieve this, we first examined the effect of TGF-β(2) on the wound closure rate in an in vitro HCEC wound healing model. However, unexpectedly TGF-β(2) had no effect on the wound closure rate in this model. Therefore, a real-time cell electronic sensing (RT-CES) system and the BrdU incorporation assay were used to determine the effect of TGF-β(2) (0.1-10 ng/ml) on cultured HCEC proliferation during in vitro wound healing. The specificity of this effect was confirmed by adding the TGF-β receptor I kinase inhibitor. TGF-β(2) inhibited the proliferation of HCECs in a dose dependent way and was blocked by TGF-β receptor I kinase inhibitor. Next, the Boyden chamber assay was used to determine how TGF-β(2) (10 ng/ml) affect HCEC migration. Exposure to TGF-β(2) increased cell migration, and a synergistic effect was observed when FGF-2 was added. To determine whether the mitogen-activated protein kinase (MAPK) signaling pathway is involved in the migration of HCECs, western blot analysis and Bio-Plex™ suspension array were used to detect phosphorylation of Erk1/2, p38, and JNK in HCECs stimulated by TGF-β(2) and/or FGF-2. The effect of the p38 MAPK inhibitor, SB239063 (10 μM), on TGF-β(2) and/or FGF-2-induced cellular migration was determined by the Boyden chamber assay. Both TGF-β(2) and FGF-2-induced p38 phosphorylation, and a synergistic effect was observed with exposure to both growth factors. SB 239063 inhibited TGF-β(2) and FGF-2-induced migration of HCECs. These results indicate that TGF-β(2) reduces proliferation but stimulates migration of cultured HCECs. In addition, TGF-β(2) and FGF-2 may have synergistic effects on the migration of HCECs mediated by p38 MAPK phosphorylation.
authors.affiliation: Department of Ophthalmology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Joko
authors.forename: Atsushi
authors.initials: A
authors.lastname: Shiraishi
authors.forename: Yoko
authors.initials: Y
authors.lastname: Akune
authors.forename: Sho
authors.initials: S
authors.lastname: Tokumaru
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Kazunori
authors.initials: K
authors.lastname: Miyata
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Ohashi
mesh_terms: D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004728:Endothelium, Corneal / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D053781:Transforming Growth Factor beta2 / Q000378:metabolism*; D014945:Wound Healing / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; C509041:TGFB2 protein, human; D053781:Transforming Growth Factor beta2; D016222:Fibroblast Growth Factor 2; D017346:Protein Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I