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Bigg, Heather Frances
(1996).
DOI: https://doi.org/10.21954/ou.ro.0000f79e
Abstract
This project has investigated tissue inhibitor of metalloproteinases-1 (TIMP-1) gene regulation in human fibroblasts by all-trans-retinoic acid in combination with each of 4 different polypeptide growth factors - basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β(TGF-β).
Initially, TIMP-1 protein secretion in response to these agents was measured using a specific enzyme-linked immunosorbent assay (ELISA) for TIMP-1. The effect of 10-5M retinoic acid in combination with 1,10 and 100ng/ml of bFGF, EGF, PDGF-BB or TGF-β on TIMP-1 protein secretion was examined in human skin, synovial and tendon fibroblasts. Retinoic acid in combination with each of the 4 growth factors caused a potent induction of TIMP-1 protein which was greater than the additive effect of the agents by up to 4 fold. These responses occurred in all 3 types of fibroblast examined. The synergistic induction of TIMP-1 protein was dose-dependent and was not due to a general increase in cell protein synthesis or cell proliferation. The responses were also abolished by the additional presence of specific neutralizing antibodies to bFGF, EGF, PDGF-BB or TGF-β.
The effect of the same agents on collagenase (matrix metalloproteinase-1) production from the cells was also examined using a specific ELISA for this enzyme. It was found that retinoic acid potently blocked bFGF, EGF and PDGF-BB-stimulated collagenase but failed to interact in an additive manner with TGF-β to depress collagenase production.
The ability of the proinflammatory cytokine interleukin-iβ (IL-1β) to modulate further the response of fibroblasts to retinoic acid in combination with growth factors was investigated. IL-1β caused a reduction in the level of synergistic interaction between retinoic acid and bFGF, EGF and PDGF-BB. However, no consistent effect was seen with IL-1β, retinoic acid and TGF-β. IL-1β in combination with bFGF or EGF also caused a synergistic induction of collagenase protein from the cells.
The mechanism(s) of the synergistic induction of TIMP-1 protein by retinoic acid and growth factors were then addressed using skin fibroblasts. The stimulation of TIMP-1 protein production continued to increase across a 72 hour time period suggesting that these effects were secondary responses to the agents. A short incubation (up to 12 hours) with bFGF alone followed by retinoic acid treatment gave a synergistic induction of TIMP-1 protein similar to that seen with both agents together. Increasingly longer incubations with bFGF were not as effective and eventually ineffective. The transient induction of an intracellular event by bFGF is therefore involved in the mechanism. Prolonged treatment with TGF-β (72 hours) alone followed by retinoic acid treatment gave an induction of TIMP-1 similar to that seen with both agents together but induction by retinoic acid and EGF required the simultaneous presence of both agents.
The synergistic induction of TIMP-1 protein by the agents was paralleled by similar effects on steady-state levels of TIMP-1 mRNA as shown by Northern blotting. The maximum levels of TIMP-1 mRNA either preceded or concurred with the peak induction of TIMP-1 protein. TIMP-1 gene regulation in response to retinoic acid and bFGF, EGF, PDGF-BB or TGF-β therefore occurs at a pretranslational level. The induction of TIMP-1 mRNA by retinoic acid and bFGF was sensitive to cycloheximide and therefore required new protein synthesis. The changes in collagenase protein in response to the agents were also paralleled by similar changes in collagenase mRNA levels. In contrast to TIMP-1, TIMP-2 mRNA was not induced by any of the factors alone or in combination except in the case of retinoic acid and TGF-β applied together in which a modest stimulation occurred.
The tyrosine kinase inhibitor genistein but not herbimycin A abolished the inductionof TIMP-1 protein by retinoic acid and bFGF. This occurred in a dose-dependent manner without causing cytotoxicity. No inhibition was seen in response to the protein kinase C inhibitor bisindolylmaleimide (BIS) although BIS blocked known protein kinase C-dependent effects in the cells. BIS also partially blocked both the induction of TIMP-1 protein in response to retinoic acid and PDGF-BB and the induction of collagenase protein in response to PDGF-BB alone. Finally, a highly specific p38 mitogen-activated protein (MAP) kinase inhibitor was found to enhance further the synergistic stimulation of TIMP-1 protein by retinoic acid and bFGF. This inhibitor also caused a dose-dependent inhibition of IL-1β-stimulated collagenase from the cells.
CORE (COnnecting REpositories)
CORE (COnnecting REpositories)
