Interaction of IGF-I and 1 alpha, 25(OH)2D3 on receptor expression and growth stimulation in rat growth plate chondrocytes.
Growth plate cartilage cell express receptors for, and are affected by both IGF-I and 1 alpha, 25(OH)2D3. The studies were undertaken to investigate interaction between these two hormone systems, that is, (i) to study effects of 1 alpha, 25(OH)2D3 on IGF-type 1 receptors (IGFIR), on IGF-I stimulated cell replication, colony formation, and on alkaline phosphatase activity (AP), and conversely, (ii) to study the effect of IGF-I on vitamin D receptor (VDR) expression on 1 alpha, 25(OH)2D3 stimulated growth parameters and on AP activity. Freshly isolated rat tibial chondrocytes were grown in monolayer cultures, (serum-free) or in agarose stabilized suspension cultures (0.1% FCS). Vitamin D receptor and IGFIR were visualized by immunostaining with the monoclonal antibody (mAb) 9A7 gamma and mAb alpha IR3, respectively, and quantitated by RT-PCR for mRNA and by Scatchard analysis using [3H]-1,25(OH)2D3 and [125I]-alpha IR3. Cell proliferation was measured by [3H]-thymidine incorporation, growth curves in monolayer cultures, and by colony formation in agarose-stabilized suspension cultures. IGF-I dose-dependently increased [3H]-thymidine incorporation. 1 alpha, 25(OH)2D3, but not 1 beta, 25(OH)2D3 was stimulatory at low ((10-12 M) and slightly inhibitory at high (10-8 M) concentrations. The effect of IGF-I was additive to that of 1 alpha, 25 (OH)2D3 [IGF-I 60 ng/ml, 181 +/- 12.7; 1 alpha, 25(OH)2D3 10(-12) M, 181 +/- 9.8%, IGF-I + 1 alpha, 25(OH)2D3, 247 +/- 16.7%, P < 0.05 by ANOVA] and specifically obliterated by polyclonal IGF-I antibody (AB-1). Interaction could also be confirmed in suspension cultures. IGFIR mRNA and [125I]-alphaIR3 binding was increased by low (10(-12) m) but not by high (10(-8) M) concentrations of 1 alpha, 25(OH)2D3. Homologous up-regulation by IGF-I (60 ng/ml) was specifically inhibited by AB-1 and markedly amplified by coincubation with 1 alpha, 25(OH)2D3 (10(-12)m). Immunostaining with alpha IR3 showed specific IGFIR expression in rat growth cartilage, but not liver tissue. Stimulation of chondrocytes with 1 alpha, 25(OH)2D3 or IGF-I suggested some increase of receptor expression in single cells, but the predominant effect was increased recruitment of receptor positive cells, Vitamin D receptor expression was markedly stimulated (fourfold) by IGF-I (60 ng/ml), but not IGF-II and inhibited by actinomycin D. This study shows that IGF-I and 1 alpha, 25(OH)2D3 mutually up-regulate their respective receptors in growth plate chondrocytes. In parallel, they have additive effects on cell proliferation and colony formation suggesting independent effector pathways.
Klaus
G
G
Department of Pediatrics, University of Marburg, Germany. klaus@post.med.uni-marburg.de
Weber
L
L
Rodríguez
J
J
Fernández
P
P
Klein
T
T
Grulich-Henn
J
J
Hügel
U
U
Ritz
E
E
Mehls
P
P
D000818:Animals; D001483:Base Sequence; D002117:Calcitriol / Q000008:administration & dosage*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects; D004357:Drug Synergism; D006132:Growth Plate / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000008:administration & dosage*; D007700:Kinetics; D016133:Polymerase Chain Reaction; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D018167:Receptors, Calcitriol / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
D017931:DNA Primers; D018167:Receptors, Calcitriol; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D002117:Calcitriol