Mechanisms of induction of human tissue inhibitor of metalloproteinases-1 (TIMP-1) gene expression by all-trans retinoic acid in combination with basic fibroblast growth factor

The addition of all-trans retinoic acid (ATRA) in combination with basic fi broblast growth factor (bFGF) to human fibroblasts results in a synergistic induction of tissue inhibitor of metalloproteinases-1 (TIMP-1) protein pro duction. The synergistic stimulation of TIMP-1 protein by ATRA and bFGF inc reased across 72 h. An incubation of 10 min to 12 h with bFGF alone followe d by ATRA gave a similar synergistic induction of TIMP-1 protein to that se en with both agents together. Treatment of cells with ATRA first followed b y bFGF was ineffective. Expression of RAR beta mRNA was induced by ATRA alo ne, but not further induced by ATRA and bFGF; expression of RAR gamma mRNA was induced by both ATRA or bFGF alone, and further induced by both reagent s together; expression of RXR gamma was repressed by ATRA alone, but not by ATRA in combination with bFGF. Steady-state levels of TIMP-1 mRNA were ind uced 14 to 40-fold above control by ATRA and bFGF. Treatment with ATRA and bFGF did not alter the stability of TIMP-1 mRNA. The induction of TIMP-1 mR NA by ATRA and bFGF was greatly diminished by cycloheximide and therefore r equired new protein synthesis. The tyrosine kinase inhibitor genistein caus ed a dose-dependent inhibition of TIMP-1 protein induction by ATRA and bFGF . A MEK1 inhibitor (PD98059) inhibited both basal and induced levels of TIM P-1. At high concentrations, p38 MAP kinase inhibitors further enhanced the synergistic stimulation of TIMP-1 protein by ATRA and bFGF, but at these c oncentrations, p42/44 MAP kinase was strongly activated. These data begin t o elucidate the mechanisms by which TIMP-1 gene expression can be upregulat ed.