Macrophages synthesize and secrete a 25-kilodalton protein that binds insulin-like growth factor-I.
Primary (thymus) and secondary (spleen) murine lymphoid tissues express a 25-kDa protein that binds IGF-I. To determine the cellular source of this insulin-like growth factor binding protein (IGFBP), 11 murine or human cell lines representing T, B, and myeloid cells at various stages of differentiation were characterized by IGF-I affinity cross-linkage and Western ligand blotting. Mature myeloid cells, but not T or B cells, secrete a 25-kDa protein that is capable of binding IGF-I. CSF-1-derived bone marrow macrophages also synthesize this 25-kDa IGFBP. Thymic macrophages, which were estimated to secrete 2 ng of binding protein/10(6) cells-h, were used in conjunction with [125I] IGF-I affinity cross-linking to develop a protein binding immunomobility-shift assay to identify which IGFBP is produced by these cells. An anti-IGFBP-4Ab, but not an anti-IGFBP-2 Ab or normal rabbit serum, shifted the [125I] IGF-IGFBP complex to a higher m.w. position, indicating that the single 25-kDa IGFBP is IGFBP-4. Northern blotting confirmed that transcripts for IGFBP-4 as well as IGF-I are expressed in thymic macrophages. A putative 278-bp IGFBP-4 cDNA fragment (residues 341-618) of rat) that contains two unique cysteine residues found only in IGFBP-4 was cloned and sequenced from thymic macrophages. These clones differed from the rat sequence at only six residues (97% homology), and the deduced amino acid sequence from the murine cDNA was identical with that of the rat sequence. Subsequent studies revealed that IGF-I stimulates DNA synthesis in thymic macrophages. However, two different IGF-I analogues differing in the amino-terminus that bind equally well to the IGF-I receptor but poorly to IGFBPs are as effective as IGF-I at 100-fold lower concentrations. These data demonstrate that murine macrophages are a source of a single 25-kDa secreted protein that binds IGF-, that the molecular identity of this protein is IGFBP-4, and that this binding protein may antagonize the extracellular effects of IGF-I.
Li
Y M
YM
Department of Animal Sciences, University of Illinois, Urbana-Champaign 61801, USA.
Arkins
S
S
McCusker
R H
RH
Donovan
S M
SM
Liu
Q
Q
Jayaraman
S
S
Dantzer
R
R
Kelley
K W
KW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D002448:Cell Adhesion; D002454:Cell Differentiation; D002460:Cell Line; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000096:biosynthesis* / Q000378:metabolism*; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000235:genetics; D018969:Insulin-Like Growth Factor Binding Proteins / Q000032:analysis; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000494:pharmacology; D007962:Leukocytes / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000494:pharmacology; D013154:Spleen / Q000737:chemistry; D013950:Thymus Gland / Q000737:chemistry; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D018970:Insulin-Like Growth Factor Binding Protein 1; D018971:Insulin-Like Growth Factor Binding Protein 2; D018974:Insulin-Like Growth Factor Binding Protein 4; D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I