Interaction of IGF-I and 1alpha,25(OH)2D3 on receptor expression and growth stimulation in rat growth plate chondrocytes (original) (raw)

1998, Kidney International

Interaction of IGF-I and 1␣,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 by both IGF-I and 1␣,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␣,25(OH) 2 D 3 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␣,25(OH) 2 D 3 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␥ and mAb ␣IR3, respectively, and quantitated by RT-PCR for mRNA and by Scatchard analysis using [ 3 H]-1,25(OH) 2 D 3 and [ 125 I]-␣IR3. Cell proliferation was measured by [ 3 H]-thymidine incorporation, growth curves in monolayer cultures, and by colony formation in agarose-stabilized suspension cultures. IGF-I dose-dependently increased [ 3 H]-thymidine incorporation. 1␣,25(OH) 2 D 3 , but not 1␤,25(OH) 2 D 3 was stimulatory at low (10 Ϫ12 M) and slightly inhibitory at high (10 Ϫ8 M) concentrations. The effect of IGF-I was additive to that of 1␣,25(OH) 2 D 3 [IGF-I 60 ng/ml, 181 Ϯ 12.7; 1␣,25(OH) 2 D 3 10 Ϫ12 M, 181 Ϯ 9.8%, IGF-I ϩ 1␣,25(OH) 2 D 3 , 247 Ϯ 16.7%; P Ͻ 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 [ 125 I]-␣IR3 binding was increased by low (10 Ϫ12 M) but not by high (10 Ϫ8 M) concentrations of 1␣,25(OH) 2 D 3. Homologous up-upregulation by IGF-I (60 ng/ml) was specifically inhibited by AB-1 and markedly amplified by coincubation with 1␣,25(OH) 2 D 3 (10 Ϫ12 M). Immunostaining with ␣IR3 showed specific IGFIR expression in rat growth cartilage, but not liver tissue. Stimulation of chondrocytes with 1␣,25(OH) 2 D 3 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␣,25(OH) 2 D 3 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. METHODS Materials 1␣,25(OH) 2-[26,27-methyl-3 H]cholecalciferol (158 Ci/mmol) was obtained from Amersham Buchler (Braunschweig, Germany); unlabeled 1␣,25(OH) 2 D 3 and 1␤,25(OH) 2 D 3 , were gifts from