Taouis M, Dupont J, Gillet A, Derouet M, Simon J
Endocrinologie de la Croissance et du Métabolisme, Station de Recherches Avicoles, INRA, Nouzilly, France.
Mol Cell Endocrinol. 1998 Feb;137(2):177-86. doi: 10.1016/s0303-7207(97)00245-1.
In mammalian cells, the insulin receptor substrate 1 protein (IRS-1) is a specific substrate for insulin and IGF-1 receptor tyrosine kinases which is involved in mediating metabolic and mitogenic actions of insulin and IGFs. In order to determine if IRS-1 is also essential in a chicken derived hepatoma cell line (LMH cells), IRS-1 gene has been invalidated in these cells. For this, we subcloned chicken IRS-1 gene in an antisense orientation into a mammalian expression vector driven by the cytomegalovirus early promoter. LMH cells were stably transfected with this construct or with the empty vector carrying only the neomycin resistance gene and selected for cIRS-1 expression. One subclone, C2, showed a complete repression of cIRS-1 expression at both protein and mRNA levels. Proliferation of C2 cells was dramatically reduced (54%) compared with Neo(r) cells. Furthermore this reduction was accompanied by a decrease in insulin-dependent [3H]thymidine incorporation, indicating a reduction in DNA synthesis. Insulin-dependent [U-14C]glucose incorporation into cellular lipids was also significantly reduced in C2 cell line suggesting an alteration in lipogenesis. In wild type LMH cells, SHC which is involved in Ras pathway, also served as a substrate for insulin receptor tyrosine kinase. In C2 cells, SHC expression, its association with the insulin receptor and its tyrosine phosphorylation were largely increased. Two forms of the regulatory subunit of PI 3-kinase were present: p85 and p55 forms. Furthermore, C2 cells displayed increased basal phosphatidylinositol (PI) 3'-kinase activity. This report demonstrates a role for cIRS-1 in the metabolic and mitogenic actions of insulin in LMH cells. However, the overexpression of cIRS-1 antisense did not completely abolish cell proliferation. This may be explained by the exacerbation of an alternative pathway that only partly compensate for the knocking out of cIRS-1 gene: the overexpression of SHC.
在哺乳动物细胞中，胰岛素受体底物1蛋白（IRS-1）是胰岛素和IGF-1受体酪氨酸激酶的特异性底物，参与介导胰岛素和IGF的代谢及促有丝分裂作用。为了确定IRS-1在鸡源肝癌细胞系（LMH细胞）中是否也必不可少，已使这些细胞中的IRS-1基因失活。为此，我们将鸡IRS-1基因以反义方向亚克隆到由巨细胞病毒早期启动子驱动的哺乳动物表达载体中。用该构建体或仅携带新霉素抗性基因的空载体稳定转染LMH细胞，并选择用于cIRS-1表达。一个亚克隆C2在蛋白质和mRNA水平上均显示cIRS-1表达完全受到抑制。与Neo(r)细胞相比，C2细胞的增殖显著降低（54%）。此外，这种降低伴随着胰岛素依赖性[3H]胸苷掺入的减少，表明DNA合成减少。C2细胞系中胰岛素依赖性[U-14C]葡萄糖掺入细胞脂质也显著减少，提示脂肪生成发生改变。在野生型LMH细胞中，参与Ras途径的SHC也是胰岛素受体酪氨酸激酶的底物。在C2细胞中，SHC表达、其与胰岛素受体的结合及其酪氨酸磷酸化均大幅增加。存在两种形式的PI 3-激酶调节亚基：p85和p55形式。此外，C2细胞显示基础磷脂酰肌醇（PI）3'-激酶活性增加。本报告证明了cIRS-1在LMH细胞中胰岛素的代谢和促有丝分裂作用中的作用。然而，cIRS-1反义的过表达并未完全消除细胞增殖。这可能是由于替代途径的加剧，该途径仅部分补偿了cIRS-1基因的敲除：SHC的过表达。
