Garrouste F L, Remacle-Bonnet M M, Lehmann M M, Marvaldi J L, Pommier G J
Unité Interactions entre Systèmes Protéiques et Différenciation dans la Cellule Tumorale, CNRS URA 1924, Faculté de Médecine, Marseille, France.
Endocrinology. 1997 May;138(5):2021-32. doi: 10.1210/endo.138.5.5100.
To assess the autocrine function of insulin-like growth factor II (IGF-II) in the balance of proliferation and differentiation in HT29-D4 human colonic cancer cells, we studied the expression of IGF-I receptors (IGF-IR) and insulin receptors (IR) in relation to the state of cell differentiation. IGF-IR and IR were expressed in both undifferentiated and enterocyte-like differentiated HT29-D4 cells. IGF-IR had two isoforms with a 97-kDa and a 102-kDa beta-subunit. In addition, HT29-D4 cells expressed hybrid receptors (HR) formed by the association of two alphabeta heterodimers from both IR and IGF-IR. HR were evidenced through 1) inhibition of IGF-I binding by the B6 anti-IR antibody and 2) immunoprecipitation with the alpha-IR3 anti-IGF-IR antibody, which revealed an additional 95-kDa IR beta-subunit that disappeared when the heterotetrameric receptor was dissociated by disulfide reduction into alphabeta heterodimers before immunoprecipitation. Like IGF-IR, HR had a high affinity for IGF-I (Kd, approximately 1.5 nM), but did not bind insulin significantly; the latter interacted with the native IR only (Kd, approximately 4 nM). In the differentiated HT29-D4 cell monolayer, all receptor species were strongly polarized (>97%) toward the basolateral membrane. Moreover, HT29-D4 cell differentiation was accompanied by an approximately 2-fold increase in the number of IR, whereas the number of IGF-I-binding sites was unaltered. However, in differentiated HT29-D4 cells, approximately 55% of the latter were involved in HR vs. approximately 20% in undifferentiated HT29-D4 cells. Thus, HT29-D4 cell differentiation is characterized by an up-regulation (approximately 3-fold) of the level of HR coupled to a down-regulation (approximately 40%) of the level of native tetrameric IGF-IR. Alterations were induced early during the cell differentiation process, i.e. 5 days postconfluence, and remained unchanged for at least 21 days. Taken together, these results suggest that the IGF-II autocrine loop in HT29-D4 cells may trigger distinct signaling pathways if it activates native IGF-IR, which predominate in undifferentiated cells, or if it activates HR, which are up-regulated in differentiated cells.
为了评估胰岛素样生长因子II（IGF-II）在HT29-D4人结肠癌细胞增殖与分化平衡中的自分泌功能，我们研究了IGF-I受体（IGF-IR）和胰岛素受体（IR）的表达与细胞分化状态的关系。IGF-IR和IR在未分化的以及肠上皮样分化的HT29-D4细胞中均有表达。IGF-IR有两种亚型，其β亚基分子量分别为97 kDa和102 kDa。此外，HT29-D4细胞表达由IR和IGF-IR的两个αβ异二聚体缔合形成的杂合受体（HR）。HR可通过以下方式证实：1）B6抗IR抗体抑制IGF-I结合；2）用α-IR3抗IGF-IR抗体进行免疫沉淀，结果显示存在一个额外的95 kDa IRβ亚基，当在免疫沉淀前通过二硫键还原将异四聚体受体解离为αβ异二聚体时，该亚基消失。与IGF-IR一样，HR对IGF-I具有高亲和力（Kd约为1.5 nM），但对胰岛素无明显结合；胰岛素仅与天然IR相互作用（Kd约为4 nM）。在分化的HT29-D4细胞单层中，所有受体种类均强烈极化（>97%）至基底外侧膜。此外，HT29-D4细胞分化伴随着IR数量增加约2倍，而IGF-I结合位点数量未改变。然而，在分化的HT29-D4细胞中，约55%的IGF-I结合位点参与形成HR，而在未分化的HT29-D4细胞中这一比例约为20%。因此，HT29-D4细胞分化的特征是HR水平上调（约3倍），同时天然四聚体IGF-IR水平下调（约40%）。这些变化在细胞分化过程早期即汇合后5天就已诱导产生，并且至少21天保持不变。综上所述，这些结果表明，如果HT29-D4细胞中的IGF-II自分泌环激活未分化细胞中占主导的天然IGF-IR，或者激活分化细胞中上调的HR，可能会触发不同的信号通路。
