Signaling through the Smad Pathway by Insulin-like Growth Factor-binding Protein-3 in Breast Cancer Cells: RELATIONSHIP TO TRANSFORMING GROWTH FACTOR-β1 SIGNALING

Susan Fanayan,Sue M Firth,Robert C Baxter

doi:10.1074/jbc.m108038200

Abstract

We previously demonstrated in T47D cells transfected to express the transforming growth factor-beta receptor type II (TGF-betaRII) that insulin-like growth factor binding protein-3 (IGFBP-3) could stimulate Smad2 and Smad3 phosphorylation, potentiate TGF-beta1-stimulated Smad phosphorylation, and cooperate with exogenous TGF-beta1 in cell growth inhibition (Fanayan, S., Firth, S. M., Butt, A. J., and Baxter, R. C. (2000) J. Biol. Chem. 275, 39146-39151). This study further explores IGFBP-3 signaling through the Smad pathway. Like TGF-beta1, natural and recombinant IGFBP-3 stimulated the time- and dose-dependent phosphorylation of TGF-betaR1 as well as Smad2 and Smad3. This effect required the presence of TGF-betaRII. IGFBP-3 mutated in carboxyl-terminal nuclear localization signal residues retained activity in TGF-betaR1 and Smad phosphorylation, whereas IGFBP-5 was inactive. Immunoneutralization of endogenous TGF-beta1 suggested that TGF-beta1 was not essential for IGFBP-3 stimulation of this pathway, but it increased the effect of IGFBP-3. IGFBP-3, like TGF-beta1, elicited a rapid decline in immunodetectable Smad4 and Smad4.Smad2 complexes. IGFBP-3 and nuclear localization signal mutant IGFBP-3 stimulated the activation of the plasminogen activator inhibitor-1 promoter but was not additive with TGF-beta, suggesting that this end point is not a direct marker of the IGFBP-3 effect on cell proliferation. This study defines a signaling pathway for IGFBP-3 from a cell surface receptor to nuclear transcriptional activity, requiring TGF-betaRII but not dependent on the nuclear translocation of IGFBP-3. The precise mechanism by which IGFBP-3 interacts with the TGF-beta receptor system remains to be established.

Highlights

We previously demonstrated in T47D cells transfected to express the transforming growth factor-␤ receptor type II (TGF-␤RII) that insulin-like growth factor binding protein-3 (IGFBP-3) could stimulate Smad2 and Smad3 phosphorylation, potentiate Transforming growth factor-␤ (TGF-␤)1-stimulated Smad phosphorylation, and cooperate with exogenous TGF-␤1 in cell growth inhibition
Because the activation of Smad phosphorylation by IGFBP-3 was observed in that study, we determined the role of TGF-␤RI in IGFBP-3 signaling
Our data show clearly that IGFBP-3 signaling in T47D and MCF-7 breast cancer cells can be initiated at the level of TGF-␤RI activation and that this requires the presence of TGF-␤RII

Summary

Introduction

We previously demonstrated in T47D cells transfected to express the transforming growth factor-␤ receptor type II (TGF-␤RII) that insulin-like growth factor binding protein-3 (IGFBP-3) could stimulate Smad and Smad phosphorylation, potentiate TGF-␤1-stimulated Smad phosphorylation, and cooperate with exogenous TGF-␤1 in cell growth inhibition Like TGF-␤1, natural and recombinant IGFBP-3 stimulated the time- and dose-dependent phosphorylation of TGF-␤RI as well as Smad and Smad3 This effect required the presence of TGF-␤RII. This study defines a signaling pathway for IGFBP-3 from a cell surface receptor to nuclear transcriptional activity, requiring TGF-␤RII but not dependent on the nuclear translocation of IGFBP-3. These studies did not suggest a functional interaction between IGFBP-3 and TGF-␤ signaling in their regulation of cell growth We demonstrated this interaction by showing that in T47D cells transfected to express the TGF-␤RII (T47D-RII cells), TGF-␤ and IGFBP-3 act together to inhibit cell proliferation, an effect not seen in control cells transfected with empty vector (T47D-vec cells). A mechanism for this concerted action was suggested by the observation that IGFBP-3 stimulates the phosphorylation of Smad and Smad in these cells and potentiates the stimulation of Smad phosphorylation by TGF-␤ [7]

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