Novel laminin 5 gamma 2-chain fragments potentiating the limbal epithelial cell outgrowth on amniotic membrane.
OBJECTIVE
Matrix metalloproteases (MMPs)-mediated extracellular matrix (ECM) degradation potentially releases cryptic motility factors involved in somatic stem cell migration and epithelial outgrowth. The authors previously demonstrated that MMP-9 is upregulated in limbal epithelial cells cultivated on amniotic membrane (AM). Here, the authors further investigated whether plasminogen activator (PA)/plasmin regulates MMP-9 activity in this model implicated in the processing of laminin 5 (Ln5), a component of amniotic basement membrane.


METHODS
Limbal epithelial cells migrated from limbal explants were expanded on intact AM. The activities and proteins of uPA and MMP-9 in limbal epithelial cells were determined by fibrin and gelatin zymography, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescent staining. Specific pharmacological inhibitors including MMPs inhibitor GM6001, MMP-2/-9 inhibitor, and uPA inhibitor B428 were used to determine whether the PA/plasmin/MMP-9 axis induces cell growth via Ln5 in this model.


RESULTS
These data showed that MMP-9 activity was attenuated by a selective uPA inhibitor, B428. Furthermore, MMP-9 activity was enhanced by exogenous addition or pre-incubation with plasmin. These results demonstrated that PA/plasmin regulates MMP-9 expression. An interesting proteolytic fragment of Ln5 gamma 2-chain was suppressed by pretreatment with GM6001, B428, or neutralizing antibodies of MMP-9 and uPA, indicating that Ln5 gamma 2-chain is processed by uPA/MMP-9. Moreover, the extent of limbal outgrowth was also retarded by B428.


CONCLUSIONS
This study suggested that MMP-9 activity was upregulated by PA/plasmin, which in turn processed Ln5 gamma 2-chain to facilitate limbal outgrowth on intact AM.
Cheng
Ching-Yi
CY
Department of Physiology, Chang Gung University, Tao-Yuan, Taiwan.
Sun
Chi-Chin
CC
Yu
Wei-Hsuan
WH
Hsieh
Hsi-Lung
HL
Ma
David Hui-Kang
DH
Pang
Jong-Hwei Su
JH
Yang
Chuen-Mao
CM
D000578:Amidines / Q000494:pharmacology; D000650:Amnion; D015153:Blotting, Western; D002465:Cell Movement; D049109:Cell Proliferation; D018920:Coculture Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005341:Fibrinolysin; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007797:Laminin / Q000378:metabolism*; D016850:Limbus Corneae / Q000166:cytology*; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013876:Thiophenes / Q000494:pharmacology; D015854:Up-Regulation; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000502:physiology*
C097398:4-iodine-benzo(b)thiophene-2-carboxamidine; D000578:Amidines; C400567:LAMC2 protein, human; D007797:Laminin; D012333:RNA, Messenger; D013876:Thiophenes; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; D020780:Matrix Metalloproteinase 9