Vitamin D and growth hormone regulate growth hormone/insulin-like growth factor (GH–IGF) axis gene expression in human fetal epiphyseal chondrocytes

ObjectiveCell proliferation and gene expression regulation were studied in human fetal epiphyseal chondrocytes to ascertain the involvement of GH–IGF axis components in human fetal growth regulation by 1,25-dihydroxyvitamin D3 (VitD) and growth hormone (GH).DesignChondrocytes from primary cultures were plated in serum-free medium for 48 h and incubated for a further 48 h with VitD (10−11 to 10−6 M) and/or IGF-I (100 ng/ml) and/or GH (500 ng/ml). We analyzed 3H-thymidine incorporation into DNA and IGF-I, IGFBP-3, GHR, SOX9, COL2A1, aggrecan and COMP gene expression by real-time quantitative PCR.ResultsVitD dose-dependently and significantly inhibited 3H-thymidine incorporation whereas GH had no effect on proliferation and, when combined with VitD, the same inhibition was observed as with VitD alone. IGF-I (100 ng/ml) significantly stimulated proliferation and opposed inhibition by VitD. VitD dose-dependently stimulated IGF-I (11.1 ± 19.8 at VitD 10−6 M), IGFBP-3 (2.6 ± 0.9), GHR (3.8 ± 2.8) and COMP (1.5 ± 0.6) expression whereas it inhibited SOX9 (0.7 ± 0.2), COL2A1 (0.6 ± 0.3) and aggrecan (0.6 ± 0.2) expression and had no significant effect on IGF-II. IGF-I stimulated IGF-I, IGFBP-3, SOX9, COL2A1 and aggrecan expression and opposed COL2A1 and aggrecan gene expression inhibition by VitD. GH alone had no effect on gene expression whereas, in the presence of VitD, significantly-increased IGF-I expression stimulation was observed above values obtained with VitD alone (17.5 ± 7.4).ConclusionsOur results suggest that VitD regulation of fetal growth cartilage could have consisted of parallel enhancing of cell differentiation and conditioning to a phenotype more sensitive to regulation by other hormones such as GH as shown by increased GHR and IGF-I expression, but not by IGF-II expression which was not regulated.