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Molecular Regulation of the IGF-Binding Protein-4 Protease System in Human Fibroblasts: Identification of a Novel Inducible Inhibitor

Endocrine Research Unit, Division of Endocrinology, Metabolism, and Nutrition, Mayo Clinic and Mayo Foundation (B.-K.C., L.K.B., Z.T.R., C.A.C.), Rochester, Minnesota 55905; University of Aarhus, Department of Molecular and Structural Biology (M.T.O., C.O.), DK-8000 Aarhus C, Denmark; and Statens Serum Institute (M.C.), 2300 Copenhagen S, Denmark

Address all correspondence and requests for reprints to: Cheryl A. Conover, Ph.D., Mayo Clinic and Mayo Foundation, 200 First Street SW, 5-194 Joseph, Rochester, Minnesota 55905. E-mail: . conover.cheryl{at}mayo.edu

The IGF-binding protein-4 (IGFBP-4) protease system is an important regulator of local IGF bioavailability and cell growth. Recently, the IGF-dependent IGFBP-4 protease secreted by cultured human fibroblasts was identified as pregnancy-associated plasma protein A (PAPP-A). In pregnancy serum, PAPP-A circulates as a disulfide-bound complex with the precursor form of major basic protein (pro-MBP), and in this complex PAPP-A’s proteolytic activity is not evident. In this study we analyzed the IGFBP-4 protease system in normal human fibroblasts to determine regulation outside of pregnancy. Treatment with the phorbol ester tumor promoter, ß-phorbol 12,13-didecanoate (ß-PDD), resulted in time-dependent inhibition of the IGF-dependent IGFBP-4 protease activity in cell-conditioned medium, which was evident at 6 h and complete by 24 h. PAPP-A mRNA was constitutively expressed in control cells, and levels were decreased only after 24 h of ß-PDD treatment. Secretion of PAPP-A protein into conditioned medium did not change with ß-PDD treatment. On the other hand, pro-MBP mRNA was undetectable in control human fibroblasts, and treatment with ß-PDD induced pro-MBP mRNA and protein expression within 6 h. ß-PDD-induced pro-MBP mRNA expression and protease inhibition were blocked with an inhibitor of RNA synthesis, actinomycin D. Actinomycin D had no effect on PAPP-A mRNA levels in the absence or presence of ß-PDD. Similarly, transformation of human fibroblasts with simian virus 40 large T antigen resulted in the synthesis of pro-MBP mRNA and protein and inhibition of IGFBP-4 protease activity. Coculture of fibroblasts with cells transfected with pro-MBP cDNA resulted in inhibition of IGFBP-4 proteolytic activity without having any effect on PAPP-A synthesis. In summary, phorbol ester tumor promoters and simian virus 40 transformation regulate IGFBP-4 proteolysis in human fibroblasts through induction of a novel inhibitor of PAPP-A, pro-MBP. These findings expand our understanding of the IGFBP-4 protease system and suggest an additional level of local cell growth control.

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