INTERACTION OF IGF-I AND 1-ALPHA,25(OH)(2)D-3 ON RECEPTOR EXPRESSION AND GROWTH-STIMULATION IN RAT GROWTH-PLATE CHONDROCYTES

Growth plate cartilage cells express receptors for, and are affected b y both IGF-I and 1<alpha,25(OH)(2)D-3. The studies were undertaken to investigate interaction between these two hormone systems, that is, (i ) to study effects of 1 alpha,25(OH)(2)D-3 on IGF-type 1 receptors (IG FIR), on IGF-I stimulated cell replication, colony formation, and on a lkaline phosphatase activity (AP), and conversely, (ii) to study the e ffect of IGF-I on vitamin D receptor (VDR) expression on 1 alpha 25(OH )(2)D-3 stimulated growth parameters and on AP activity. Freshly isola ted rat tibial chondrocytes were grown in monolayer cultures. (serum-f ree) or in agarose stabilized suspension cultures (0.1% FCS). Vitamin D receptor and IGFIR were visualized by immunostaining with the monocl onal antibody (mAb) 9A7 gamma and mAb alpha IR3, respectively and quan titated by RT-PCR far mRNA and by Scatchard analysis using [H-3]-1.25( OH)(2)D-3 and [I-125]-alpha JR3. Cell proliferation was measured by [H -3]-thymidine incorporation, growth curves in monolayer cultures, and by colony formation in agarose-stabilized suspension cultures. IGF-I d ose-dependently increased [H-3]-thymidine incorporation. 1 alpha.25(OH )(2)D-3, but not 1 beta,25(OH)(2)D-3 was stimulatory at low (10(-12) M ) and slightly inhibitory at high (10(-8) M) concentrations. The effec t of IGF-I was additive to that of 1 alpha 25(OH)(2)D-3 [IGF-I 60 ng/m l, 181 +/- 12.7; 1 alpha,25(OH)(2)D-3 10(-12) M, 181 +/- 9.8%, IGF-I - 1 alpha,25(OH)(2)D-3, 247 +/- 16.7%; P < 0.05 by ANOVA] and specifica lly obliterated by polyclonal IGF-I antibody (AB-1). Interaction could also be confirmed in suspension cultures. IGFIR mRNA and [I-125]-alph a IR3 binding was increased by low (10(-12) M) but not by high (10(-8) M) concentrations of 1 alpha.25(OH)(2)D-3. Homologous up-upregulation by IGF-I (60 ng/ml) was specifically inhibited by AB-1 and markedly a mplified by coincubation with 1 alpha,25(OH)(2)D-3 (10(-12) M). Immuno staining with alpha IR3 showed specific IGFIR expression in rat growth cartilage, but not liver tissue. Stimulation of chondrocytes with 1 a lpha,25(OH)(2)D-3 or IGF-I suggested some increase of receptor express ion in single cells, but the predominant effect was increased recruitm ent of receptor positive cells, Vitamin D receptor expression was mark edly stimulated (fourfold) by IGF-I (60 ng/ml), but not IGF-II and inh ibited by actinomycin D. This study shows that IGF-I and 1 alpha,25(OH )(2)D-3 mutually up-regulate their respective receptors in growth plat e chondrocytes. In parallel: they have additive effects on cell prolif eration and colony formation suggesting independent effector pathways.