title: Molecular regulation of the IGF-binding protein-4 protease system in human fibroblasts: identification of a novel inducible inhibitor.
abstract: 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, beta-phorbol 12,13-didecanoate (beta-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 beta-PDD treatment. Secretion of PAPP-A protein into conditioned medium did not change with beta-PDD treatment. On the other hand, pro-MBP mRNA was undetectable in control human fibroblasts, and treatment with beta-PDD induced pro-MBP mRNA and protein expression within 6 h. beta-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 beta-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.
authors.affiliation: Endocrine Research Unit, Division of Endocrinology, Metabolism, and Nutrition, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
authors.forename: Bing-Kun
authors.initials: BK
authors.lastname: Chen
authors.forename: Michael T
authors.initials: MT
authors.lastname: Overgaard
authors.forename: Laurie K
authors.initials: LK
authors.lastname: Bale
authors.forename: Zachary T
authors.initials: ZT
authors.lastname: Resch
authors.forename: Michael
authors.initials: M
authors.lastname: Christiansen
authors.forename: Claus
authors.initials: C
authors.lastname: Oxvig
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Conover
mesh_terms: D001798:Blood Proteins / Q000096:biosynthesis; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004797:Enzyme-Linked Immunosorbent Assay; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011266:Pregnancy-Associated Plasma Protein-A / Q000235:genetics; D011480:Protease Inhibitors / Q000378:metabolism*; D015347:RNA Probes; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D001798:Blood Proteins; D017077:Culture Media, Conditioned; D047091:Eosinophil Granule Proteins; D011480:Protease Inhibitors; D015347:RNA Probes; D012333:RNA, Messenger; D012260:Ribonucleases; D008666:Metalloendopeptidases; D011266:Pregnancy-Associated Plasma Protein-A