Abstract
Insulin and insulin-like growth factor-1 (IGF-1) act through highly homologous receptors that engage similar intracellular signaling pathways, yet these hormones serve largely distinct physiological roles in the control of metabolism and growth, respectively. In an attempt to uncover the molecular mechanisms underlying their divergent functions, we compared insulin receptor (IR) and IGF-1 receptor (IGF-1R) regulation of gene expression by microarray analysis, using 3T3-L1 cells expressing either TrkC/IR or TrkC/IGF-1R chimeric receptors to ensure the highly selective activation of each receptor tyrosine kinase. Following stimulation of the chimeric receptors for 4 h, we detected 11 genes to be differentially regulated, of which 10 were up-regulated to a greater extent by the IGF-1R. These included genes involved in adhesion, transcription, transport, and proliferation. The expression of mRNA encoding heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen, was markedly increased by IGF-1R but not IR activation. This effect was dependent on MAPK, but not phosphatidylinositol 3-kinase, and did not require an autocrine loop through the epidermal growth factor receptor. HB-EGF mitogenic activity was detectable in the medium of 3T3-L1 preadipocytes expressing activated IGF-1R but not IR, indicating that the transcriptional response is accompanied by a parallel increase in mature HB-EGF protein. The differential abilities of the IR and IGF-1R tyrosine kinases to stimulate the synthesis and release of a growth factor may provide, at least in part, an explanation for the greater role of the IGF-1R in the control of cellular proliferation.
Highlights
The insulin receptor (IR)1 and the insulin-like growth factor-1 (IGF-1) receptor (IGF-1R) are highly homologous, both being class II receptor tyrosine kinases with a disulfide-linked tetrameric structure
the IGF-1R chimera (TIGR) Chimera Signaling Stimulates Transcription to a Greater Extent Than the IR chimera (TIR) Signaling in 3T3-L1 Preadipocytes—We first compared transcript levels in NT-3-stimulated TIR/TIGR cells with levels of the same transcripts in untreated cells
We chose to compare the effects of signaling from IR and IGF-1 receptor (IGF-1R) intracellular domains to gene transcription using a chimeric receptor system previously developed in our group [9]
Summary
Materials—Insulin (Actrapid) was supplied by Novo-Nordisk, and IGF-1 was supplied by GroPep. Total RNA from stimulated and unstimulated cells was extracted using the RNeasy mini kit (Qiagen). Western Blotting—Confluent cells were serum-starved overnight, before being treated as indicated in the text. They were rinsed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1 mM Na3VO4, 30 mM NaF, 10 mM Na4P2O7, 2.5 mM benzamidine, 1 g/ml pepstatin A, 1 g/ml leupeptin, 1 g/ml antipain, 0.5 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100). Thymidine Incorporation Assay—HaCat cells were seeded into 12well plates at equal density and allowed to grow to ϳ80% confluency They were serum-starved for 24 h, followed by treatment for 16 h as described in the text.
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