LY 294002, an inhibitor of phosphatidylinositol 3-kinase, inhibits GH-mediated expression of the IGF-I gene in rat hepatocytes.
The signal transduction pathways that mediate GH-dependent regulation of IGF-I gene expression remain poorly defined. To establish a GH-responsive in vitro model system to study the effect of GH on the expression of the endogenous IGF-I gene, primary hepatocytes from adult male rats were prepared. These cells expressed both the GH receptor and the IGF-I gene, as demonstrated using a ribonuclease protection assay. Western blot analyses using antibodies directed against the phosphorylated forms of the ERKs, signal transducer and activator of transcription-5, and Akt/protein kinase B, a protein kinase that is downstream of PI3K, demonstrated GH-dependent phosphorylation of these signaling molecules. These signaling molecules are components of the major signal transduction pathways that are activated by GH. To determine whether GH-responsive IGF-I gene expression could be demonstrated in these cells, hepatocytes were treated without or with 50 ng/ml GH for 3--48 h. IGF-I mRNA levels increased within 3 h, and a maximal 2.2-fold increase was observed after 24 h of GH treatment. To determine whether ERK activation contributes to GH-induced IGF-I gene expression, hepatocytes were treated for 12 h without or with 50 ng/ml GH and 50 microM PD98059, an inhibitor of MAPK kinase-1 and -2. Treatment with PD98059 did not have a significant effect on GH-induced IGF-I gene expression. Similar studies were performed using 50 microM LY 294002, an inhibitor of PI3K. These studies demonstrated that treatment with LY 294002 completely abrogated GH-induced IGF-I gene expression. In contrast, PI3K-specific doses of another inhibitor of PI3K, wortmannin, failed to inhibit the GH-induced increase in IGF-I mRNA levels. In summary, rat hepatocytes in primary culture provide a good model system to study GH-induced IGF-I gene expression, and the results of these studies suggest that a signaling pathway inhibited by LY 294002, possibly a PI3K-dependent pathway, is important for GH-stimulated IGF-I gene expression in hepatocytes.
Shoba
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LN
Department of Medicine, Veterans Affairs Chicago Healthcare System, Lakeside Division, and Northwestern University Medical School, Chicago, Illinois 60611, USA.
Newman
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Liu
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Lowe
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WL
D000818:Animals; D002867:Chromones / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects* / Q000502:physiology*; D013006:Growth Hormone / Q000502:physiology*; D022781:Hepatocytes / Q000502:physiology*; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D009025:Morpholines / Q000494:pharmacology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D011986:Receptors, Somatotropin / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology
D002867:Chromones; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011986:Receptors, Somatotropin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D007334:Insulin-Like Growth Factor I; D013006:Growth Hormone; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one