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 synergist ic effects on Leydig cell function. Leydig cells express high affinity IGF-I receptors. The number of IGF-I receptors and IGF-I receptor mRN A 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 transc ription, mRNA accumulation, and protein production/secretion. Biologic al effects of IGFBP-2 on Leydig cells were also examined. Rat Leydig c ells were purified from testes using centrifugal elutriation followed by Percoll gradient centrifugation. Cells were cultured for 24 h and t hen 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 signifi cant decrease in IGFBP-2 mRNA accumulation. To evaluate the mechanism( s) responsible for decreased IGFBP-2 gene expression by hCG, the effec t of hCG on the rate of transcription and stability of the mRNA was de termined. Human CG (10 ng/ml) reduced the IGFBP-2 transcription rate b y 32%/h in comparison with the control, while the half-life (t(1/2)) o f 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 a s a major band in the Western ligand blot. Human CG (10 ng/ml) markedl y decreased the IGFBP-2 protein level in conditioned media by 35%. Thi s suggests that reduction of IGFBP-2 protein levels by hCG in the Leyd ig cells was mainly due to decreased transcription rate. Finally, the effects of recombinant hlGFBP-2 (rhlGFBP-2) on Leydig cell function we re evaluated. Recombinant hlGFBP-2 in a concentration of 5 pmol/ml com pletely blocked the stimulating effects of IGF-I (1.2 pmol/ml) on Leyd ig cell-testosterone formation. In conclusion, IGFBP-2 protein product ion in rat Leydig cells is negatively regulated by hCG in vitro, mainl y due to its reduced transcription rate. Furthermore, rhlGFBP-2 blocks the effects of IGF-I on Leydig cell steroidogenesis. These results su ggest that IGFBP-2 plays an important modulating role in Leydig cell f unction. 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.