Regulation of IGF-I mRNA and signal transducers and activators of transcription-3 and -5 (Stat-3 and -5) by GH in C2C12 myoblasts.
GH and IGF-I are critical hormones for the regulation of longitudinal growth and the maintenance of lean body mass in humans. The regulation of IGF-I expression by GH in hepatocytes is well documented; however less is known about the regulation of IGF-I in peripheral tissues such as muscle. We have examined the regulation of IGF-I mRNA by GH and IGF-I in C2C12 myoblasts. GH stimulated the accumulation of IGF-I mRNA dose- and time-dependently. An elevation of IGF-I mRNA was observed with GH doses as low as 0.75 ng/ml and after exposure to GH for as little as 1 h, and the increase required ongoing transcription and translation. GH applied in a pulsatile fashion for 10 min followed by an 8-h interpulse interval increased IGF-I mRNA to a greater extent than continuous exposure. GH stimulated tyrosine phosphorylation of the GH receptor, signal transducer and activator of transcription-3 (Stat3), and Stat5. Stat5 was resistant to additional phosphorylation if cells were given a GH pulse within 2 h of a previous GH exposure. The refractory period lasted for 4 h, and cells could be maximally stimulated again after 6 h. Stat3 phosphorylation was also enhanced in cells that were allowed to recover from a previous application of GH. The tyrosine kinase inhibitors, genistein, PP1, and AG-490, and the MAPK kinase inhibitor, PD98059, did not block Stat3 or Stat5 phosphorylation. In contrast, WHI-P154, a Janus kinase-3 inhibitor, dose-dependently prevented Stat3, but not Stat5, phosphorylation. GH-inducible nuclear transport of Stat3 was likewise inhibited by WHI-P154. Most importantly, GH-dependent IGF-I mRNA expression was inhibited by WHI-P154. In contrast, IGF-I mRNA expression was inhibited by IGF-I peptide, and the effect of IGF-I was dominant over that of GH. IGF-I mRNA was regulated by both PI3K and MAPK signal transduction pathways, but IGF-I peptide signaled predominantly through a wortmannin-sensitive pathway to down-regulate its own mRNA. Our data suggest that Janus kinases (Jak2 or Jak3) and their downstream targets (Stat3 and Stat5) may play important roles in the expression of IGF-I mRNA and the myoblast response to GH. In addition, C2C12 cells appear to be a good model system to examine GH regulation of Janus kinase/Stat signaling and the regulation of IGF-I mRNA.
Frost
Robert A
RA
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA. rfrost@psu.edu
Nystrom
Gerald J
GJ
Lang
Charles H
CH
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019382:Human Growth Hormone / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis* / Q000235:genetics*; D053616:Janus Kinase 3; D051379:Mice; D008894:Milk Proteins; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011799:Quinazolines / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects*; D015534:Trans-Activators / Q000096:biosynthesis* / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D008894:Milk Proteins; D011799:Quinazolines; D012333:RNA, Messenger; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050799:STAT5 Transcription Factor; C494087:Stat3 protein, mouse; D015534:Trans-Activators; C113888:WHI P154; D019382:Human Growth Hormone; D007334:Insulin-Like Growth Factor I; D011505:Protein-Tyrosine Kinases; C507933:JAK3 protein, human; C507935:Jak3 protein, mouse; D053616:Janus Kinase 3