Effect of wound type on Smad 2 and 4 translocation.
OBJECTIVE
In a prior study, it was reported that both TGF-beta receptors type-I and -II are upregulated after wounding, suggesting that TGF-beta signaling may play a role in corneal epithelial repair. The Smad proteins, which translocate into the nucleus after activation of the TGF-beta receptors, are key factors in the major TGF-beta signaling pathway. The present study was undertaken to examine whether Smads 2 and 4 translocate into the nucleus during wound repair and whether the wound type affects the extent of translocation.


METHODS
Either a 3-mm superficial keratectomy or epithelial debridement was performed on adult Sprague-Dawley rats. The eyes were allowed to heal from 4 hours to 2 weeks. Indirect immunofluorescence was performed with anti-Smads 2 and 4, anti-laminin, a marker of basement membrane, and anti-alphavbeta6 integrin, which has been implicated in TGF-beta activation. In addition, the effect of the p38MAPK inhibitor SB202190 on healing rates of debridement and keratectomy wounds was determined in organ culture.


RESULTS
In unwounded tissue, Smad 2 was cytoplasmic. By 4 hours after keratectomy, nuclear localization was visible in a few epithelial basal cells at the leading edge of the wound. The number of basal cells expressing nuclear Smad 2 in the wound area increased with time, peaking at 48 hours (95%). However, in the debridement model, Smad 2 localization remained primarily cytoplasmic. Smad 4 showed similar localization. In both wound models, p38MAPK inhibitor slowed epithelial migration, and alphavbeta6 integrin appeared to be upregulated with localization primarily observed in the basal cells migrating over the wound area.


CONCLUSIONS
The presence of the basement membrane appears to have an effect on the extent and duration of translocation of the Smad 2 and 4 proteins during corneal epithelial wound repair. The Smad pathway does not appear to be essential for migration; rather, it may play a role in resynthesis of the basement membrane.
Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
Audrey E K
AE
Hutcheon
Xiaoqing Q
XQ
Guo
Mary Ann
MA
Stepp
Kenneth J
KJ
Simon
Paul H
PH
Weinreb
Shelia M
SM
Violette
James D
JD
Zieske
D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism; D001485:Basement Membrane / Q000502:physiology; D018929:Cell Culture Techniques; D002465:Cell Movement; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003646:Debridement; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000187:drug effects / Q000293:injuries* / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D016023:Integrins / Q000378:metabolism; D008297:Male; D009924:Organ Culture Techniques; D021381:Protein Transport; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D051899:Smad2 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000378:metabolism*; D015854:Up-Regulation; D014945:Wound Healing; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007093:Imidazoles; D016023:Integrins; D011725:Pyridines; C497782:SMAD2 protein, human; D051899:Smad2 Protein; C497784:Smad2 protein, rat; D051901:Smad4 Protein; C490811:Smad4 protein, rat; D015534:Trans-Activators; C073878:integrin alphavbeta6; D048051:p38 Mitogen-Activated Protein Kinases; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole