Degradation of IGF-binding protein-3 by proteases in cultured FRTL-5 rat thyroid cells

Abstract

Abstract In this study, we have found that IGF-binding protein-3 (IGFBP-3) in calf serum added to tissue culture medium is degraded by cultured FRTL-5 cells and a major 31 kDa fragment of IGFBP-3 is produced. When FRTL-5 rat thyroid cells were cultured in 6H medium (modified F-12M medium containing TSH, insulin, hydrocortisone, somatostatin, transferrin, and glycyl-histidyl-lysine) containing 5% calf serum, both 44–46 and 31 kDa IGFBPs were found in conditioned medium by ligand blot analysis using 125I-labelled IGF-II. However, predominantly the 44–46 kDa IGFBP was detected in unconditioned 6H medium containing 5% calf serum. When calf serum in the media was replaced by human serum similar results were obtained, and the 44–46 kDa and 31 kDa IGFBPs were recognized using a human IGFBP-3 antibody following Western blot analysis. FRTL-5 cells secreted only small amounts of an endogenous 29 kDa IGFBP, thought to be IGFBP-5. To separate the 31 kDa fragment of IGFBP-3 from the endogenous IGFBP-5, culture media were fractionated by concanavalin-A–Sepharose chromatography and aliquots of both flow-through and eluate from the column were analyzed by ligand blotting. A 31 kDa IGFBP was found in the eluate fractions from concanavalin-A–Sepharose chromatography following the separation of conditioned 6H medium supplemented with calf serum, suggesting that this species was an N-linked glycoprotein and could be derived from the degradation of serum IGFBP-3 by FRTL-5 cells. Using a modified zymographic assay, we examined whether the degradation of IGFBP-3 could depend on the cell membrane. Confluent FRTL-5 cells were washed with PBS and overlaid with liquid agarose solution. After the agarose had solidified, unconditioned 6H medium containing 5% calf serum was incubated with the cells at 37 °C for 16 h. Both 44–46 and 31 kDa IGFBP species were found in the overlying, conditioned medium by ligand blot. However, the 31 kDa IGFBP was not found in medium in the absence of FRTL-5 cells, and no IGFBP could be found in serum-free conditioned medium from agarose-covered FRTL-5 cells. This suggests that the 44–46 kDa IGFBP-3 in serum was degraded to yield a 31 kDa fragment, while any endogenous IGFBP-5 could not pass out of the agarose. The degradation of 44–46 kDa IGFBP-3 in the modified zymographic assay was inhibited by phenylmethylsulfonyl fluoride, EDTA, and aprotinin, but not by leupeptin. In summary, these results indicated that IGFBP-3 in calf serum added to culture medium could be degraded by FRTL-5 cells and that this may involve calcium-dependent serine proteases. Journal of Endocrinology (1997) 152, 265–274