Differential effects of insulin-like growth factor (IGF)-I and IGF-II on the expression of IGF binding proteins (IGFBPs) in a rat neuroblastoma cell line: isolation and characterization of two forms of IGFBP-4.
The isolation and hormonal regulation of two low molecular weight insulin-like growth factor binding proteins (IGFBPs) present in the conditioned medium (CM) of the rat neuroblastoma cell line B104 cells has been performed. IGFBPs were purified by ZnSO4 precipitation, insulin-like growth factor-I 1IGF-I) affinity chromatography, and reverse phase HPLC. Final isolation and N-terminal analysis was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotting to polyvinylidene difluoride membranes, and sequencing of the bound proteins. Two IGFBPs, with apparent Mr of 28K and 24K were coisolated and sequenced. Both proteins had identical N-terminal sequences and appear to be two forms of IGFBP-4. Treatment of the IGFBPs with endoglycosidase-F caused a shift in the apparent Mr of the 28K IGFBP to 24K. However, there was no change in the apparent Mr of the 24K IGFBP. The data from this study suggest that the IGFBP-4 exists as both a glycosylated and nonglycosylated protein. Treatment of B104 cells with IGF-I increased the expression of both the 24K and 28K IGFBPs and also resulted in the appearance of IGFBP-3 and an unknown IGFBP at 29K. When added to subconfluent cells, IGF-I was also mitogenic in B104 cells. Similar to IGF-I, IGF-II treatment increased cell number and resulted in the appearance of IGFBP-3 and the 29K IGFBP. However, IGF-II treatment resulted in a significant decrease (approximately 50%) in the 24K IGFBP and also decreased the 28K IGFBP. This decrease in the expression of the 24K and 28K IGFBPs was dose-dependent and was blocked by addition of IGF-I to the cells. When an IGF-II receptor antibody was added to the cells it mimicked the effects of IGF-II on B104 cells, suggesting that the inhibitory effects of IGF-II are mediated through the type II IGF receptor. Although both IGF-I and IGF-II affected the amount of the 24K IGFBP in the CM, neither peptide affected the expression of the messenger RNA for the 24K IGFBP. In conclusion, we have isolated two IGFBPs from the CM of B104 cells. Both the 24K and 28K IGFBPs appear to be isoforms of the same protein, and sequence data suggest these proteins are two forms of IGFBP-4. IGF-I increases the expression of both of these IGFBPs, whereas IGF-II decreases their expression.(ABSTRACT TRUNCATED AT 400 WORDS)
Ceda
G P
GP
Department of Geriatrics, University of Parma, Italy.
Fielder
P J
PJ
Henzel
W J
WJ
Louie
A
A
Donovan
S M
SM
Hoffman
A R
AR
Rosenfeld
R G
RG
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003470:Culture Media; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology*; D008024:Ligands; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D013002:Somatomedins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D003470:Culture Media; D018969:Insulin-Like Growth Factor Binding Proteins; D008024:Ligands; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II