IGFBP‐3 and TGF‐β inhibit growth in epithelial cells by stimulating type V TGF‐β receptor (TβR‐V)‐mediated tumor suppressor signaling

The TGF-β type V receptor (TβR-V) mediates growth inhibition by IGFBP-3 and TGF-β in epithelial cells and loss of TβR-V expression in these cells leads to development of carcinoma. The mechanisms by which TβR-V mediates growth inhibition (tumor suppressor) signaling remain elusive. Previous studies revealed that IGFBP-3 and TGF-β inhibit growth in epithelial cells by stimulating TβR-V-mediated IRS-1/2-dependent activation and cytoplasm-to-nucleus translocation of IGFBP-3- or TGF-β-stimulated protein phosphatase (PPase), resulting in dephosphorylation of pRb-related proteins (p107, p130) or pRb, and growth arrest. To define the signaling, we characterized/identified the IGFBP-3- and TGF-β-stimulated PPases in cell lysates and nucleus fractions in Mv1Lu cells treated with IGFBP-3 and TGF-β, using a cell-free assay with 32P-labeled casein as a substrate. Both IGFBP-3- and TGF-β-stimulated PPase activities in cell lysates are abolished when cells are co-treated with TGF-β/IGFBP-3 antagonist or RAP (LRP-1/TβR-V antagonist). However, the IGFBP-3-stimulated PPase activity, but not TGF-β-stimulated PPase activity, is sensitive to inhibition by okadaic acid (OA). In addition, OA or PP2Ac siRNA reverses IGFBP-3 growth inhibition, but not TGF-β growth inhibition, in Mv1Lu and 32D cells. These suggest that IGFBP-3- and TGF-β-stimulated PPases are identical to PP2A and PP1, respectively. By Western blot/phosphorimager/immunofluorescence-microscopy analyses, IGFBP-3 and TGF-β stimulate TβR-V-mediated IRS-2-dependent activation and cytoplasm-to-nucleus translocation of PP2Ac and PP1c, resulting in dephosphorylation of p130/p107 and pRb, respectively, and growth arrest. Small molecule TGF-β enhancers, which potentiate TGF-β growth inhibition by enhancing TβR-I-TβR-II-mediated canonical signaling and thus activating TβR-V-mediated tumor suppressor signaling cascade (TβR-V/IRS-2/PP1/pRb), could be used to prevent and treat carcinoma.