Characterization of a macrophage-based system for studying the activation of latent TGF-beta.
TGF-beta has been implicated in scarring and tissue fibrosis. Most cells secrete TGF-beta as a high molecular weight, latent complex that must be processed to a lower molecular weight, biologically active form. A number of molecules are involved in this activation step including the mannose 6-phosphate/insulin-like growth factor-II receptor, tissue transglutaminase, thrombospondin, plasmin, and others. Here we describe a rapid macrophage-based system for TGF-beta1 activation, which could be used for screening potential anti-fibrotic agents. The system employs transformed mouse peritoneal macrophages treated with lipopolysaccharide as a cell line capable of activating latent TGF-beta. The activation mechanism in our system involves mannose 6-phosphate/insulin-like growth factor-II receptor and transglutaminase. The activation of latent TGF-beta in this system can be prevented by the addition of mannose-6-phosphate but not mannose-1-phosphate. In addition, transglutaminase inhibitors, antibodies to thrombospondin, insulin-like growth factor-II in the presence of its binding protein IGFBP-2, but not IGFBP-1, suppressed the activation of TGF-beta. Anti-inflammatory molecules, such as hydrocortisone, when added to LPS-treated macrophages, inhibited TGF-beta activation by a mechanism, that may involve downregulation of transglutaminase expression. In summary, this new, rapid and reproducible system allows testing molecules for their ability to inhibit TGF-beta activation, thus providing a screening method for potential anti-scarring molecules.
Gosiewska
A
A
Johnson & Johnson Wound Healing Technology Resource Center, Skillman, NJ 08558, USA.
Yi
C F
CF
Blanc-Brude
O
O
Geesin
J C
JC
D000818:Animals; D015152:Blotting, Northern; D002103:Cadaverine / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D017077:Culture Media, Conditioned; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000494:pharmacology; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000037:antagonists & inhibitors / Q000494:pharmacology; D008070:Lipopolysaccharides; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000378:metabolism*; D008360:Mannosephosphates / Q000494:pharmacology; D051379:Mice; D016133:Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011503:Transglutaminases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
D017077:Culture Media, Conditioned; D018970:Insulin-Like Growth Factor Binding Protein 1; D018971:Insulin-Like Growth Factor Binding Protein 2; D008070:Lipopolysaccharides; D008360:Mannosephosphates; D016212:Transforming Growth Factor beta; C027693:mannose-6-phosphate; D007335:Insulin-Like Growth Factor II; D011503:Transglutaminases; C008542:monodansylcadaverine; D002103:Cadaverine; D006854:Hydrocortisone