Dong Feng, Wu Hai-Bin, Hong Jiang, Rechler Matthew M
Growth and Development Section, Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases/NIH, Bldg. 10, Room 8D12, Bethesda, MD 20892, USA.
J Cell Physiol. 2002 Jan;190(1):63-73. doi: 10.1002/jcp.10034.
Insulin-like growth factor binding protein-3 (IGFBP-3) has been proposed to mediate the growth inhibitory effects of transforming growth factor (TGF)-beta in breast and prostate cancer cells. Both TGF-beta and exogenous IGFBP-3 inhibit DNA synthesis in Mv1 mink lung epithelial cells (CCL64). The present study asks whether IGFBPs synthesized by CCL64 cells mediate growth inhibition by TGF-beta. CCL64 cells synthesize and secrete a single 34-kDa IGFBP that was identified as IGFBP-2 by immunoprecipitation and immunodepletion. Recombinant bovine IGFBP-2 inhibited CCL64 DNA synthesis in serum-free media in an IGF-independent manner. Coincubation with Leu(60)-IGF-I, an IGF-I analog that binds to IGFBPs with higher affinity than to IGF-I receptors, decreased the inhibition by bIGFBP-2. Leu(60)-IGF-I also decreased the inhibition of CCL64 DNA synthesis by TGF-beta by up to 70%, whereas Long-R3-IGF-I, an IGF-I analog with higher affinity for IGF-I receptors than for IGFBPs, did not decrease inhibition, suggesting that the effect of Leu(60)-IGF-I resulted from its forming complexes with endogenous IGFBPs. Leu(60)-IGF-I did not decrease TGF-beta stimulation of a Smad3-dependent reporter gene. Following incubation of intact CCL64 cells with bIGFBP-2 at 0 degrees C, bIGFBP-2 was recovered in membrane fractions; membrane association was abolished by coincubation with Leu(60)-IGF-I. If exogenous and secreted IGFBP-2 must bind to CCL64 cells to inhibit DNA synthesis, Leu(60)-IGF-I might reduce the inhibition of DNA synthesis by bIGFBP-2 or TGF-beta by inhibiting the association of IGFBP-2 in the media with CCL64 cells. Since TGF-beta does not increase IGFBP-2 abundance, we propose that TGF-beta sensitizes CCL64 cells to the latent growth inhibitory activity of endogenous IGFBP-2 by potentiating an intracellular IGFBP-2 signaling pathway or by promoting the association of secreted IGFBP-2 with the plasma membrane.
胰岛素样生长因子结合蛋白-3(IGFBP-3)被认为可介导转化生长因子(TGF)-β对乳腺癌和前列腺癌细胞的生长抑制作用。TGF-β和外源性IGFBP-3均抑制Mv1貂肺上皮细胞(CCL64)中的DNA合成。本研究探讨CCL64细胞合成的IGFBPs是否介导TGF-β的生长抑制作用。CCL64细胞合成并分泌一种单一的34 kDa IGFBP,通过免疫沉淀和免疫去除鉴定为IGFBP-2。重组牛IGFBP-2在无血清培养基中以不依赖IGF的方式抑制CCL64细胞的DNA合成。与Leu(60)-IGF-I(一种与IGFBP结合的亲和力高于与IGF-I受体结合的亲和力的IGF-I类似物)共同孵育,可降低bIGFBP-2的抑制作用。Leu(60)-IGF-I还可将TGF-β对CCL64细胞DNA合成的抑制作用降低多达70%,而Long-R3-IGF-I(一种对IGF-I受体的亲和力高于对IGFBP的亲和力的IGF-I类似物)则不会降低抑制作用,这表明Leu(60)-IGF-I的作用是由于其与内源性IGFBPs形成复合物。Leu(60)-IGF-I不会降低TGF-β对Smad3依赖性报告基因的刺激作用。在0℃下将完整的CCL64细胞与bIGFBP-2孵育后,bIGFBP-2在膜组分中被回收;与Leu(60)-IGF-I共同孵育可消除膜结合。如果外源性和分泌的IGFBP-2必须与CCL-64细胞结合才能抑制DNA合成,那么Leu(60)-IGF-I可能通过抑制培养基中的IGFBP-2与CCL64细胞的结合来降低bIGFBP-2或TGF-β对DNA合成的抑制作用。由于TGF-β不会增加IGFBP-2的丰度,我们提出TGF-β通过增强细胞内IGFBP-2信号通路或促进分泌的IGFBP-2与质膜的结合,使CCL64细胞对内源性IGFBP-2的潜在生长抑制活性敏感。