Regulation of insulin-like growth factor I messenger ribonucleic acid levels by serum in cultured rat fibroblasts.
Fibroblasts represent one of the in vivo sites of extrahepatic insulin-like growth factor I (IGF-I) production. In this study, cultured fibroblasts prepared from the skin of neonatal rats were used as a model to assess the role of serum in regulating IGF-I messenger RNA (mRNA) levels. IGF-I mRNA, as demonstrated by Northern blot analysis, was present in the cultured fibroblasts, and serum free media which was conditioned by fibroblasts for 20 h contained 108 pg/ml of immunoreactive IGF-I. Fetal calf serum (FCS) decreased steady state IGF-I mRNA levels, as measured by solution hybridization/RNase protection assay, in fibroblasts in a time- and dose-dependent fashion. Incubation of fibroblasts for 18 h in the presence of 0.3%, 0.6%, or 1% FCS decreased IGF-I mRNA levels to 76%, 56%, and 46% of the levels present in control cells which were maintained in serum free media with 0.25% BSA. Maximal inhibition to approximately 20% of control levels was seen with 4-10% FCS. In contrast, basic fibroblast growth factor and beta-actin mRNA levels increased 2- and 4-fold, respectively, with increasing concentrations of FCS. Treatment of the cells with 10 micrograms/ml cycloheximide resulted in partial abrogation of the inhibitory effect of FCS while protein synthesis in the cells was decreased to 6% of control levels. The addition of 2 micrograms/ml of insulin or 15-100 ng/ml of IGF-I to the fibroblasts did not reproduce the inhibitory effect of FCS. Finally, the inhibitory factor(s) present in the FCS was partially removed/inactivated by charcoal stripping or heat inactivating the serum, but delipidation of the FCS by chloroform extraction had no effect on the inhibitory effect of FCS. In summary, FCS contains a factor(s) that decreases IGF-I mRNA levels in cultured fibroblasts in a time- and dose-dependent fashion. The partial abrogation of the inhibitory effect of FCS with cycloheximide treatment suggests that this effect is at least partially dependent upon new protein synthesis. Furthermore, the studies using delipidated, heat-inactivated, and charcoal-stripped serum suggest that the inhibitory factor(s) is a peptide.
Lowe
W L
WL
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City.
Kummer
M
M
Karpen
C W
CW
Wu
X D
XD
D000199:Actins / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D001769:Blood; D015152:Blotting, Northern; D002478:Cells, Cultured; D003470:Culture Media; D003513:Cycloheximide / Q000494:pharmacology; D015342:DNA Probes; D016222:Fibroblast Growth Factor 2 / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis / Q000235:genetics*; D007930:Leucine / Q000378:metabolism; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D012867:Skin / Q000378:metabolism
D000199:Actins; D003470:Culture Media; D015342:DNA Probes; D012333:RNA, Messenger; D016222:Fibroblast Growth Factor 2; D012313:RNA; D007334:Insulin-Like Growth Factor I; D003513:Cycloheximide; D007930:Leucine