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 cultured mouse Leydig cell line (TM-3) was performed. Radioimmunoassayable 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). Immunoprecipitation of solubilized crude membranes indicated that the 31 kDa 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 major IGFBPs, and 25, 40, and 44 kDa as minor ones. The 28 kDa band could be invisible after Endoglycosidase-F treatment, suggesting that the 28 kDa IGFBP was a glycosylated form of IGFBP. The 24 kDa and 28 kDa bands were immunoprecipitated with an antibody to IGFBP-4. 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 in the appearance of a 29 kDa IGFBP. Neither the 25 nor 29 kDa bands were deglycosylated with Endoglycosidase-F nor immunoprecipitated by antibodies to rIGFBP-1 and -2. On the other hand, IGF-II treatment resulted in a significant decrease in the 24 kDa IGFBP-4. When [Leu²⁷]IGF-II, which binds to the type 2 receptor and to IGFBPs, but not to the type 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 level of 24 kDa IGFBP-4 in CM, neither peptide affected the expression of messenger RNA for IGFBP-4. Northern blot analysis of total RNA prepared from TM-3 cells treated with IGF-I shared the expression of mRNA for 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 conditions. 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 associated 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 (presumably IGFBP-6) under basal conditions. Treatment with IGF-I increased levels 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-transcriptional.