Monkey retinal pigment epithelial cells in vitro synthesize, secrete, and degrade insulin-like growth factor binding proteins.
Cultured monkey retinal pigment epithelial (RPE) cells rapidly secrete large amounts of insulin-like growth factor binding proteins (IGF-BPs). IGF-II tracer binding activity in conditioned media is two to three times greater than that of IGF-I. Under reducing SDS-PAGE conditions, 125I-IGF affinity-crosslinked binding protein (BP) is visualized as a broad band between 36 +/- 2.9 and 49 +/- 3.3 kDa. Because the electrophoretic mobility of the crosslinked BP is increased under non-reducing conditions (33-45 kDa), intramolecular sulfhydryl bonding may be present. Frequently, the radiographic band representing affinity-crosslinked binding protein exhibits a complex pattern of non-uniform densities that suggests structural or functional IGF-BP micro-heterogeneity. IGF-BPs synthesized by RPE also exhibit heterogeneity with respect to the absence or presence of oligosaccharide side chains. In particular, the larger, but not the mid-sized or smaller IGF-BPs exhibit side chains linked to the core protein with N-glycosidic linkage. None of the crosslinked IGF-BPs exhibit O-linked side chains. Long-term (12, 24, 48 hr) conditioning studies revealed that IGF-BP fails to accumulate in culture media beyond 12 hr, but that replacement of conditioned media with fresh media allows a second period of binding protein accumulation. Other short-term (12 hr) experiments indicate that, in fresh medium, the levels of IGF-BP increase during the first 6-8 hr and then remain stable. To examine the processes contributing to these steady state levels of IGF-BP, aliquots of 8-hr conditioned medium were removed from the cells and either frozen on dry ice or incubated at 37 degrees C for 16 hr. Importantly, it was found that incubation at 37 degrees C resulted in a near total loss of binding activity. This is the first report of IGF-BP degrading activity in a cell culture system. These findings indicate that 1) primate RPE cells rapidly secrete a complex mixture of N-glycosylated and non-glycosylated IGF-BPs, and 2) the steady state levels of secreted IGF-BP are tightly regulated at least in part through a concomitant IGF-BP inactivating activity. Cultured RPE cells may be of utility in examining the mechanisms of IGF-BP synthesis, secretion, and degradation at the cellular level.
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
R J
RJ
Waldbillig
T J
TJ
Schoen
G J
GJ
Chader
B A
BA
Pfeffer
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D000882:Haplorhini; D018969:Insulin-Like Growth Factor Binding Proteins; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D013002:Somatomedins / Q000378:metabolism*
D002352:Carrier Proteins; D003470:Culture Media; D018969:Insulin-Like Growth Factor Binding Proteins; D013002:Somatomedins