Phorbol 12-myristate 13-acetate-activated protein kinase C increased migratory activity of subconjunctival fibroblasts via stress-activated protein kinase pathways.
OBJECTIVE
The aim of this study was to clarify the mechanism of subconjunctival fibroblast migration, focusing on the effect of protein kinase C (PKC).


METHODS
Subconjunctival fibroblasts were isolated from rats and cultured in 10% fetal bovine serum-containing culture medium. The fibroblasts were treated with phorbol 12-myristate 13-acetate (PMA) to activate PKC. Migration was assessed using a wound-healing assay. Western blot analysis was employed to examine activation of stress-activated protein kinase (SAPK) pathways. Immunofluorescent analysis was performed to examine the actin cytoskeleton dynamics.


RESULTS
Seven PKC isoforms (alpha, beta, gamma, delta, epsilon, iota, and lambda) were present in rat subconjunctival fibroblasts. PMA induced lamellipodia formation and subsequent migration of the subconjunctival fibroblasts. PMA treatment in the subconjunctival fibroblasts elicited phosphorylation of SAPKs, including c-jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAPK). Treatment of the subconjunctival fibroblasts with an inhibitor of PKC abrogated the phosphorylation of these proteins. Furthermore, specific inhibitors of JNK and p38MAPK blocked PMA-induced migration of the subconjunctival fibroblasts. The phosphorylation of c-jun and heat shock protein 27, downstream effectors of JNK and p38MAPK, respectively, was upregulated by PMA treatment.


CONCLUSIONS
PMA-activated PKC increased migratory activity, and SAPK pathways were critical for migration of subconjunctival fibroblasts.
Nomura
Naoko
N
Department of Ophthalmology and Visual Science, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan. nomura413jp@yahoo.co.jp
Nomura
Motohiro
M
Takahira
Masayuki
M
Sugiyama
Kazuhisa
K
D000199:Actins / Q000378:metabolism; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D002465:Cell Movement / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D007093:Imidazoles / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000199:Actins; D000873:Anthracenes; D007093:Imidazoles; D011725:Pyridines; C432165:pyrazolanthrone; D011493:Protein Kinase C; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate; C093642:SB 203580