Insulin-induced egr-1 expression in Chinese hamster ovary cells is insulin receptor and insulin receptor substrate-1 phosphorylation-independent. Evidence of an alternative signal transduction pathway.
Insulin's effects primarily are initiated by insulin binding to its plasma membrane receptor and the sequential tyrosine phosphorylation of the insulin receptor and intracellular substrates, such as insulin receptor substrate-1 (IRS-1). However, studies suggest some insulin effects, including those at the nucleus, may not be regulated by this pathway. The present study compared the levels of insulin binding, insulin receptor and IRS-1 tyrosine phosphorylation, and phosphatidylinositol 3'-kinase activity to immediate early gene c-fos and egr-1 mRNA expression in Chinese hamster ovary (CHO) cells expressing only neomycin-resistant plasmid (CHONEO), overexpressing wild type human insulin receptor (CHOHIRc) or ATP binding site-mutated insulin receptors (CHOA1018K). Insulin binding in CHONEO cells was markedly lower than that in other cell types. 10 nM insulin significantly increased tyrosine phosphorylation of insulin receptor and IRS-1 in CHOHIRc cells. Phosphorylation of insulin receptor and IRS-1 in CHONEO and CHOA1018K cells was not detected in the presence or absence of insulin. Similarly, insulin increased phosphatidylinositol 3-kinase activity only in CHOHIRc cells. As determined by Northern blot, nuclear run-on analysis, and in situ hybridization, insulin induced c-fos mRNA expression, through transcription, in CHOHIRc cells but not in CHONEO and CHOA1018K cells, consistent with previous reports. In contrast, all three cell types showed a similar insulin dose-dependent increase of egr-1 mRNA expression through transcription. These data indicated that insulin-induced egr-1 mRNA expression did not correlate with the levels of insulin binding to insulin receptor or phosphorylation of insulin receptor and IRS-1. These results suggest that different mechanisms are involved in induction of c-fos and egr-1 mRNA expression by insulin, the former by the more classic insulin receptor tyrosine kinase pathway and the latter by a yet to be determined alternative signal transduction pathway.
Harada
S
S
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Smith
R M
RM
Smith
J A
JA
Shah
N
N
Hu
D Q
DQ
Jarett
L
L
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D051766:Early Growth Response Protein 1; D015870:Gene Expression / Q000187:drug effects*; D017781:Genes, Immediate-Early; D006801:Humans; D017874:Immediate-Early Proteins; D017403:In Situ Hybridization; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D053590:Interleukin 1 Receptor Antagonist Protein; D007700:Kinetics; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D012333:RNA, Messenger / Q000096:biosynthesis; D011972:Receptor, Insulin / Q000378:metabolism*; D012795:Sialoglycoproteins / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C507763:IL1RN protein, human; D017874:Immediate-Early Proteins; D007328:Insulin; D053590:Interleukin 1 Receptor Antagonist Protein; D012333:RNA, Messenger; D012795:Sialoglycoproteins; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D011972:Receptor, Insulin