INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-2 - THE EFFECT OF HUMAN CHORIONIC-GONADOTROPIN ON ITS GENE-REGULATION AND PROTEIN SECRETION AND ITS BIOLOGICAL EFFECTS IN RAT LEYDIG-CELLS

Human CG (hCG) and insulin-like growth factor-I (IGF-I) have synergistic effects on Leydig cell function. Leydig cells express high affinity IGF-I receptors. The number of IGF-I receptors and IGF-I receptor mRNA levels can be up-regulated by hCG. The most abundant mRNA species of the IGF binding proteins (IGFBPs) in rat Leydig cells is IGFBP-2. In the present study, we investigated the effect of hCG on IGFBP-2 transcription, mRNA accumulation, and protein production/secretion. Biological effects of IGFBP-2 on Leydig cells were also examined. Rat Leydig cells were purified from testes using centrifugal elutriation followed by Percoll gradient centrifugation. Cells were cultured for 24 h and then treated with or without hCG (10 ng/ml) for 6 h. The expression of IGFBP-2 mRNA was decreased by hCG in a dose-dependent manner, and at a concentration of 10 ng/ml the expression of IGFBP-2 mRNA was reduced by 50%. As early as 2 h after the addition of hCG, there was a significant decrease in IGFBP-2 mRNA accumulation. To evaluate the mechanism(s) responsible for decreased IGFBP-2 gene expression by hCG, the effect of hCG on the rate of transcription and stability of the mRNA was determined. Human CG (10 ng/ml) reduced the IGFBP-2 transcription rate by 32%/h in comparison with the control, while the half-life (t(1/2)) of mRNA remained unaltered (hCG-treated cells, 0.58 h; control cells, 0.51 h). IGFBP-2 with a molecular size of 33 kilodaltons was detected as a major band in the Western ligand blot. Human CG (10 ng/ml) markedly decreased the IGFBP-2 protein level in conditioned media by 35%. This suggests that reduction of IGFBP-2 protein levels by hCG in the Leydig cells was mainly due to decreased transcription rate. Finally, the effects of recombinant hlGFBP-2 (rhlGFBP-2) on Leydig cell function were evaluated. Recombinant hlGFBP-2 in a concentration of 5 pmol/ml completely blocked the stimulating effects of IGF-I (1.2 pmol/ml) on Leydig cell-testosterone formation. In conclusion, IGFBP-2 protein production in rat Leydig cells is negatively regulated by hCG in vitro, mainly due to its reduced transcription rate. Furthermore, rhlGFBP-2 blocks the effects of IGF-I on Leydig cell steroidogenesis. These results suggest that IGFBP-2 plays an important modulating role in Leydig cell function. Synergistic effects of hCG and IGF-I on Leydig cell function are partly mediated by decreased IGFBP-2 production and increased IGF-I receptor expression.