Chen Chun-Lin, Huang Franklin W, Huang Shuan Shian, Huang Jung San
Department of Biological Science National Sun Yat-sen University Kaohsiung Taiwan.
Departments of Biochemistry and Molecular Biology Saint Louis University School of Medicine St. Louis MO USA.
FASEB Bioadv. 2021 Jun 16;3(9):709-729. doi: 10.1096/fba.2021-00016. eCollection 2021 Sep.
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 P-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 PP2A 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 PP2A and PP1, 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.
转化生长因子-β V型受体（TβR-V）介导胰岛素样生长因子结合蛋白-3（IGFBP-3）和转化生长因子-β在上皮细胞中的生长抑制作用，这些细胞中TβR-V表达的缺失会导致癌的发生。TβR-V介导生长抑制（肿瘤抑制）信号传导的机制仍不清楚。先前的研究表明，IGFBP-3和转化生长因子-β通过刺激TβR-V介导的胰岛素受体底物-1/2（IRS-1/2）依赖性激活以及IGFBP-3或转化生长因子-β刺激的蛋白磷酸酶（PPase）从细胞质到细胞核的转位来抑制上皮细胞的生长，导致视网膜母细胞瘤相关蛋白（p107、p130）或视网膜母细胞瘤蛋白（pRb）去磷酸化，并使生长停滞。为了确定信号传导途径，我们使用以P标记的酪蛋白为底物的无细胞检测法，对用IGFBP-3和转化生长因子-β处理的Mv1Lu细胞的细胞裂解物和细胞核组分中的IGFBP-3和转化生长因子-β刺激的PPase进行了表征/鉴定。当细胞用转化生长因子-β/IGFBP-3拮抗剂或RAP（低密度脂蛋白受体相关蛋白-1/TβR-V拮抗剂）共同处理时，细胞裂解物中IGFBP-3和转化生长因子-β刺激的PPase活性均被消除。然而，IGFBP-3刺激的PPase活性对冈田酸（OA）敏感，而转化生长因子-β刺激的PPase活性则不敏感。此外，OA或蛋白磷酸酶2A（PP2A）的小干扰RNA（siRNA）可逆转Mv1Lu和32D细胞中IGFBP-3的生长抑制作用，但不能逆转转化生长因子-β的生长抑制作用。这些结果表明，IGFBP-3和转化生长因子-β刺激的PPase分别与PP2A和蛋白磷酸酶1（PP1）相同。通过蛋白质印迹/磷光成像/免疫荧光显微镜分析，IGFBP-3和转化生长因子-β刺激TβR-V介导的IRS-2依赖性激活以及PP2A和PP1从细胞质到细胞核的转位，分别导致p130/p107和pRb去磷酸化，并使生长停滞。小分子转化生长因子-β增强剂可用于预防和治疗癌症，它们通过增强转化生长因子-β I型受体（TβR-I）-转化生长因子-β II型受体（TβR-II）介导的经典信号传导，从而激活TβR-V介导的肿瘤抑制信号级联反应（TβR-V/IRS-2/PP1/pRb）来增强转化生长因子-β的生长抑制作用。
