Insulin receptor substrate 1 antisense expression in an hepatoma cell line reduces cell proliferation and induces overexpression of the Src homology 2 domain and collagen protein (SHC).
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.
Taouis
M
M
Endocrinologie de la Croissance et du Métabolisme, Station de Recherches Avicoles, INRA, Nouzilly, France. taouis@tours.inra.fr
Dupont
J
J
Gillet
A
A
Derouet
M
M
Simon
J
J
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D002645:Chickens; D003094:Collagen / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D016373:DNA, Antisense / Q000235:genetics; D015870:Gene Expression; D007328:Insulin / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins; D008055:Lipids / Q000096:biosynthesis; D008114:Liver Neoplasms, Experimental / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics*; D011972:Receptor, Insulin / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D014162:Transfection; D014407:Tumor Cells, Cultured; D018909:src Homology Domains / Q000235:genetics*
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D017931:DNA Primers; D016373:DNA, Antisense; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D008055:Lipids; D010750:Phosphoproteins; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; D003094:Collagen; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin