CHARACTERIZATION OF THE INSULIN-LIKE GROWTH-FACTORS (IGF) AXIS IN A CULTURED MOUSE LEYDIG-CELL LINE (TM-3)

Characterization of the insulin-like growth factor (IGF) axis in a cul tured mouse Leydig cell line (TM-3) was performed. Radioimmunoassayabl e IGF-I and IGF-II concentrations in TM-3 conditioned media (CM) were below the assay detection levels. Affinity cross-linking of IGF-I and IGF-II to crude membranes prepared from TM-3 cells revealed both type 1 and type 2 receptors and a 31 kDa IGF binding protein (IGFBP). Immun oprecipitation of solubilized crude membranes indicated that the 31 kD a protein was membrane associated IGFBP-4. Western ligand blots of CM from TM-3 cells demonstrated the presence of 24 and 28 kDa bands as ma jor IGFBPs, and 25, 40, and 44 kDa as minor ones. The 28 kDa band coul d be invisible after Endoglycosidase-F treatment, suggesting that the 28 kDa IGFBP was a glycosylated form of IGFBP. The 24 kDa and 28 kDa b ands were immunoprecipitated with an antibody to IGFBP-A. Treatment of TM-3 cells with IGF-I increased the levels of the 24 kDa IGFBP-4, as well as 25, 28, 40, and 44 kDa IGFBPs, IGF-I treatment also resulted i n the appearance of a 29 kDa IGFBP. Neither the 25 nor 29 kDa bands we re deglycosylated with Endoglycosidase-F nor immunoprecipitated by ant ibodies to rIGFBP-1 and -2. On the other hand, IGF-II treatment result ed in a significant decrease in the 24 kDa IGFBP-4. When [Leu(27)]IGF- II, which binds to the type 2 receptor and to IGFBPs, but not to the t ype 1 receptor, was added to the cells, it mimicked the effect of IGF- II on the 24 kDa IGFBP-4. Although both IGF-I and -II affected the lev el of 24 kDa IGFBP-4 in CM, neither peptide affected the expression of messenger RNA for IGFBP-4. Northern blot analysis of total RNA prepar ed from TM-3 cells treated with IGF-I shared the expression of mRNA fo r IGFBP-5, which was not seen in untreated cells, suggesting that the 29 kDa IGFBP seen in the CM of TM-3 cells treated with IGF-I, might be IGFBP-5. The presence of mRNA for IGFBP-6 was noted under basal condi tions. In conclusion, neither IGF-I nor IGF-II was detectable in TM-3 CM. TM-3 cells have type 1 and type 2 IGF receptors and membrane assoc iated IGFBP-4. TM-3 cells can produce IGFBP-4 and glycosylated IGFBP-4 as major IGFBPs, and 40 and 44 kDa IGFBPs and a 25 kDa IGFBP (presuma bly IGPBP-6) under basal conditions. Treatment with IGF-I increased le vels of a 29 kDa IGFBP (presumably IGFBP-5). The divergent effects of IGF-I and -II on the concentrations of IGFBP-4 in CM are post-transcri ptional.