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      JNK MAPK Signaling Contributes in vivo to Injury-Induced Corneal Epithelial Migration

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          Abstract

          Purpose: 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. Conclusion: 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.

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          Most cited references 27

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          Role of the pericorneal papillary structure in renewal of corneal epithelium.

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            p38 and ERK1/2 coordinate cellular migration and proliferation in epithelial wound healing: evidence of cross-talk activation between MAP kinase cascades.

            One important action of growth factors is their participation in tissue repair; however, the signaling pathways involved are poorly understood. In a model of corneal wound healing, we found that two paracrine growth factors, hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF), induced rapid and marked activation and prompt nuclear accumulation of phospho-p38 (p-p38) and -ERK1/2 (p-ERK1/2), but not of JNK (p-JNK1/2), in corneal epithelial cells. Interruption of p38 and ERK1/2 signaling pathways by pretreatment with inhibitors SB203580 and PD98059 and subsequent stimulation with HGF or KGF abolished the activation and nuclear localization. Inhibition of either one of these mitogen-activated protein kinases, p38 or ERK1/2, induced a robust cross-activation of the other. In immunofluorescence studies of wounded cornea, p-p38, unlike p-ERK1/2, was immediately detectable in epithelium after injury. Inhibition of p38 by SB203580 blocked migration of epithelial cells almost completely. In contrast, PD98059 seemed to slightly increase the migration, through concomitant activation of p38. Unlike ERK1/2, p38 did not significantly contribute to proliferation of epithelial cells. Inhibition of either the ERK1/2 or p38 pathway resulted in delayed corneal epithelial wound healing. Interruption of both signaling cascades additively inhibited the wound-healing process. These findings demonstrate that both p38 and ERK1/2 coordinate the dynamics of wound healing: while growth factor-stimulated p38 induces epithelial migration, ERK1/2 activation induces proliferation. The cross-talk between these two signal cascades and the selective action of p38 in migration appear to be important to corneal wound healing, and possibly wound healing in general, and may offer novel drug targets for tissue repair.
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              Growth factors: importance in wound healing and maintenance of transparency of the cornea

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                Author and article information

                Journal
                ORE
                Ophthalmic Res
                10.1159/issn.0030-3747
                Ophthalmic Research
                S. Karger AG
                0030-3747
                1423-0259
                2009
                November 2009
                07 August 2009
                : 42
                : 4
                : 185-192
                Affiliations
                aDepartment of Ophthalmology, Wakayama Medical University, Wakayama, Japan; bState University of New York College of Optometry, New York, N.Y., USA
                Article
                232401 Ophthalmic Res 2009;42:185–192
                10.1159/000232401
                19672126
                © 2009 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 4, References: 34, Pages: 8
                Categories
                Original Paper

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