IGFBP-3 activates TGF-beta receptors and directly inhibits growth in human intestinal smooth muscle cells

• Published in: American journal of physiology: Gastrointestinal and liver physiology 2004-10, Vol.287 (4), p.G795-G802
• Main Authors: Kuemmerle, John F, Murthy, Karnam S, Bowers, Jennifer G
• Format: Article
• Language: English
• Subjects: Activin Receptors, Type I - metabolism; Autocrine Communication - physiology; Cell Division - drug effects; Cell Division - physiology; Cells, Cultured; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Humans; Insulin-Like Growth Factor Binding Protein 3 - metabolism; Insulin-Like Growth Factor Binding Protein 3 - pharmacology; Intestines - cytology; Iodine Radioisotopes; Myocytes, Smooth Muscle - cytology; Myocytes, Smooth Muscle - drug effects; Myocytes, Smooth Muscle - metabolism; Phosphorylation - drug effects; Protein-Serine-Threonine Kinases; Receptors, Transforming Growth Factor beta - metabolism; RNA, Small Interfering; Smad2 Protein; Trans-Activators - genetics; Trans-Activators - metabolism
• ISSN: 0193-1857; 1522-1547
• DOI: 10.1152/ajpgi.00009.2004

We have shown that human intestinal smooth muscle cells produce IGF-I and IGF binding protein-3 (IGFBP-3). Endogenous IGF-I acts in autocrine fashion to stimulate growth of these cells. IGFBP-3 inhibits the binding of IGF-I to its receptor and thereby inhibits IGF-I-stimulated growth. In several carcinoma cell lines and some normal cells, IGFBP-3 regulates growth independently of IGF-I. Two mechanisms for this effect have been identified: IGFBP-3 can directly activate transforming growth factor-beta (TGF-beta) receptors or it can undergo direct nuclear translocation. The aim of the present study was to determine whether IGFBP-3 acts independently of IGF-I and to characterize the mechanisms mediating this effect in human intestinal smooth muscle cells. The direct effects of IGFBP-3 were determined in the presence of an IGF-I receptor antagonist to eliminate its IGF-I-dependent effects. Affinity labeling of TGF-beta receptors (TGF-betaRI, TGF-betaRII, and TGF-betaRV) with 125I-labeled TGF-beta1 showed that IGFBP-3 displaced binding to TGF-betaRII and TGF-betaRV in a concentration-dependent fashion. IGFBP-3 stimulated TGF-betaRII-dependent serine phosphorylation (activation) of both TGF-betaRI and of its primary substrate, Smad2(Ser465/467). IGFBP-3 also caused IGF-I-independent inhibition of basal [3H]thymidine incorporation. The effects of IGFBP-3 on Smad2 phosphorylation and on smooth muscle cell proliferation were independent of TGF-beta1 and were abolished by transfection of Smad2 siRNA. Immunoneutralization of IGFBP-3 increased basal [3H]thymidine incorporation, implying that endogenous IGFBP-3 inhibits proliferation. We conclude that endogenous IGFBP-3 directly inhibits proliferation of human intestinal smooth muscle cells by activation of TGF-betaRI and Smad2, an effect that is independent of its effect on IGF-I-stimulated growth.