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Investigative Ophthalmology & Visual Science, Vol 36, 1411-1419, Copyright © 1995 by Association for Research in Vision and Ophthalmology

ARTICLES AND REPORTS

Transforming growth factor-beta induces plasminogen activator inhibitor type-1 in cultured human orbital fibroblasts

HJ Cao, MG Hogg, LJ Martino and TJ Smith
Department of Biochemistry and Molecular Biology, Albany Medical College, NY 12208, USA.

PURPOSE. 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.


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