1,25-Dihydroxyvitamin D3 differentially regulates the production of insulin-like growth factor I (IGF-I) and IGF-binding protein-4 in mouse osteoblasts.
1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces differentiation and inhibits proliferation in many cell types including bone cells. These effects may be mediated by the modulation of the insulin-like growth factor (IGF) regulatory system. Therefore we investigated the effects of 1,25-(OH)2D3 on transcript and protein levels of both IGF-I and IGF binding proteins (IGFBPs) in clonal mouse osteoblasts. Subconfluent cultures were treated in serum-free medium with 1,25-(OH)2D3. Secreted IGF-I was measured using a RIA under conditions eliminating the interference of IGFBPs. 1,25-(OH)2D3 (10(-11)-10(-8) M) inhibited IGF-I release in a dose dependent manner at 24 h (maximally to 30 +/- 5% of control, mean +/- SEM of seven independent experiments). In a time course study IGF-I increased in the media of control cultures over a 48-h period, while IGF-I secretion was completely prevented from 6 h onward in 1,25-(OH)2D3 treated cultures. Northern blot analysis revealed four IGF-I transcripts of 0.9, 1.8, 4.4, and 7.5 kilobases (kb). 1,25-(OH)2D3 decreased levels of the 7.5 kb IGF-I transcript from 4-48 h, with maximal inhibition occurring at 24 h (25% of control). Western ligand blots of the culture medium demonstrated secretion of a 25-kilodalton IGFBP, which comprised greater than or equal to 90% of the secreted IGFBPs. The 25-kilodalton IGFBP had previously been shown to have sequence similarity with IGFBP-4, a binding protein which inhibits the action of IGFs on bone cells. 1,25-(OH)2D3 treatment increased secretion of IGFBP-4 up to 14-fold over 24 h. 1,25-(OH)2D3 also increased IGFBP-4 (2.2 kb) transcript levels within 30 min, with the maximal stimulation of 8-fold occurring after 8 h. [3H]Thymidine incorporation into cells was inhibited by 1,25-(OH)2D3 both under basal and serum-stimulated conditions. Our results are consistent with the hypothesis that the effects of 1,25-(OH)2D3 on osteoblast proliferation may be mediated in part by decreased levels of IGF-I and increased concentrations of inhibitory IGFBP-4. It is proposed that this alteration in the IGF system may be an important functional autocrine or paracrine switch in the transition of osteoblasts from states of proliferation to differentiation.
Scharla
S H
SH
Mineral Metabolism Laboratory, J. L. Pettis Memorial Veterans' Hospital, Loma Linda, California 92373.
Strong
D D
DD
Mohan
S
S
Baylink
D J
DJ
Linkhart
T A
TA
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002117:Calcitriol / Q000494:pharmacology*; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D018974:Insulin-Like Growth Factor Binding Protein 4; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D002352:Carrier Proteins; D018974:Insulin-Like Growth Factor Binding Protein 4; D012333:RNA, Messenger; D012339:RNA, Ribosomal, 28S; D007334:Insulin-Like Growth Factor I; D002117:Calcitriol