title: JNK MAPK signaling contributes in vivo to injury-induced corneal epithelial migration.
abstract: OBJECTIVE
  Injury-mediated corneal epithelial wound healing in vivo is mediated through different cell signaling pathways depending on whether or not the basement membrane is removed. Given this dependence, we ascertained if c-jun N-terminal kinase (JNK/SAPK) mitogen-activated protein kinase (MAPK) cell signaling mediates this response in vivo and in vitro, irrespective of the presence or absence of the basement membrane. Furthermore, in vitro the relative contribution was determined by the JNK/SAPK pathway to that of its p38 and ERK MAPK counterparts in mediating injury-induced increases in cell migration.
  
  
  METHODS
  Corneal epithelial debridement was performed in C57BL/6 mice and their organ-cultured eyes without removal of the basement membrane. In rabbits, following basement membrane removal by keratectomy, fluorescein-staining monitored reepithelialization was performed as in the mice. Immunohistochemistry evaluated changes in JNK phosphorylation status and localization. JNK inhibitor I and its inactive analogue determined if JNK signaling activation contributes to wound healing. BrdU staining assessed cell proliferation. A scratch wound assay of healing rates in SV40-immortalized human corneal epithelial cell line (HCEC) evaluated the relative contributions by p38 and ERK and JNK MAPK signaling activation to wound healing. A TUNEL assay probed for apoptosis after wound closure of HCEC. MTT assay evaluated corneal epithelial viability.
  
  
  RESULTS
  Two hours following mice corneal epithelial debridement, phospho-JNK was transiently upregulated in the nucleus, whereas total JNK was constitutively expressed. JNK inhibitor I suppressed epithelial spreading in organ-cultured mouse eyes and rabbit corneal blocks, irrespective of the presence or absence of basement membrane. No proliferation was detected at the wound edges. In HCEC, a p38 (SB203580) and a JNK pathway inhibitor (JNK inhibitor I) inhibited migration rates more than U0126-induced ERK, whereas the JNK inhibitor I inactive analogue had no effect. JNK pathway inhibition wound closure in this region was not associated with either any TUNEL or BrdU-positive cells. Cell viability was unaffected by any of these MAPK inhibitors.
  
  
  CONCLUSIONS
  JNK/SAPK pathway activation stimulates wound healing in vitro and in vivo, irrespective of the presence or absence of the basement membrane. Therefore, studies on how wound closure is elicited in HCEC are relevant for identifying how MAPK signaling mediates this response in vivo and in organ-cultured eyes. This realization suggests that the JNK signaling system has a role in vivo that is intermediate to those of ERK and p38 in mediating increases in cell migration.
authors: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. yokada@wakayama-med.ac.jp
authors: Yuka
authors: Y
authors: Okada
authors: Shizuya
authors: S
authors: Saika
authors: Kumi
authors: K
authors: Shirai
authors: Osamu
authors: O
authors: Yamanaka
authors: Ai
authors: A
authors: Kitano
authors: Zheng
authors: Z
authors: Wang
authors: Hua
authors: H
authors: Yang
authors: Peter
authors: P
authors: Reinach
mesh_terms: D000818:Animals; D017209:Apoptosis; D001485:Basement Membrane; D002465:Cell Movement / Q000502:physiology*; D003646:Debridement; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000293:injuries* / Q000502:physiology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005131:Eye Injuries / Q000503:physiopathology*; D006801:Humans; D007124:Immunoenzyme Techniques; D020287:In Situ Nick-End Labeling; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009924:Organ Culture Techniques; D010766:Phosphorylation; D011817:Rabbits; D015398:Signal Transduction / Q000502:physiology*; D015854:Up-Regulation; D014945:Wound Healing / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases