Transforming growth factor-beta induces plasminogen activator inhibitor type-1 in cultured human orbital fibroblasts.
OBJECTIVE
The level of constitutive plasminogen activator inhibitor type-1 (PAI-1) expression in cultured human orbital fibroblasts is considerably lower than that found in dermal fibroblasts. This divergence in PAI-1 expression implies differences in the pericellular proteolytic environment and, therefore, in the turnover of extracellular matrix. In this article, the authors examine the effect of transforming growth factor-beta (TGF-beta) on PAI-1 expression in orbital fibroblasts.


METHODS
Human orbital and dermal fibroblasts were grown in culture. Confluent monolayers were treated with TGF-beta. PAI-1 in the extracellular matrix was quantitated by radiolabeling the cultures and electrophoresing the cellular material on SDS-PAGE. Medium content was determined by immunoprecipitation of [35S]PAI-1 with a rabbit, anti-human, polyclonal antibody. PAI-1 mRNA was determined by Northern hybridization.


RESULTS
TGF-beta increased PAI-1 levels in orbital fibroblasts in a dose-dependent manner, up to 35-fold. The induction was maximal after 16 hours of treatment. The increases in extracellular matrix PAI-1 paralleled those observed in the medium. The steady state levels of the mRNA encoding the protein were upregulated by TGF-beta up to 60-fold 8 hours after the addition of TGF-beta. The fractional increase in PAI-1 expression in orbital fibroblasts was consistently greater than that observed in dermal strains.


CONCLUSIONS
Exposure to TGF-beta consistently induces PAI-1 expression in orbital fibroblasts, cells that do not express the polypeptide constitutively at high levels. The effects are mediated at the pretranslational level and involve the upregulation of PAI-1 mRNA. These results suggest that TGF-beta may exert a profound regulatory influence on the pericellular proteolytic environment in orbital connective tissue.
Cao
H J
HJ
Department of Biochemistry and Molecular Biology, Albany Medical College, NY 12208, USA.
Hogg
M G
MG
Martino
L J
LJ
Smith
T J
TJ
D015152:Blotting, Northern; D002478:Cells, Cultured; D003238:Connective Tissue / Q000187:drug effects / Q000378:metabolism; D003239:Connective Tissue Cells; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006111:Graves Disease / Q000378:metabolism / Q000473:pathology; D006801:Humans; D009915:Orbit / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000494:pharmacology; D012867:Skin / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta