Gene expression and secretion of insulin-like growth factor-II and insulin-like growth factor binding protein-2 from cultured sheep choroid plexus epithelial cells.
The gene expression of insulin-like growth factor II (IGF-II) and insulin-like growth factor binding protein-2 (IGFBP-2) has previously been demonstrated in rat and human choroid plexus by in situ hybridization analysis. In the present study we have characterized IGF-II and IGFBP-2 transcripts and proteins in primary cultures of epithelial cells from lateral choroid plexus of sheep brain. Northern blot analysis of total RNA showed one major IGF-II mRNA of 4.8 kb and four minor IGF-II transcripts of 1.5, 2.0, 3.0 and 6.0 kb as well as one IGFBP-2 transcript of 1.7 kb. Radioreceptor assay of conditioned medium from the cultured choroid plexus epithelial cells showed inhibition of [125I]IGF-I and [125I]IGF-II binding to mouse NIH 3T3 fibroblasts, the displacement curves being identical to that of unlabelled IGF-II. The conditioned medium was fractionated by gel filtration on a Bio-Gel P-60 column, and analysis by IGF-II radioreceptor assay showed two peaks of IGF-II-binding inhibitory activity of M(r) 7.5-10 and 25 kDa, suggesting the presence of both IGF-II, and an IGFBP. Western immunoblot analysis of conditioned medium with antibodies toward IGF-II and IGFBP-2 demonstrated proteins with M(r) 6 kDa and 32 kDa, respectively. Protein binding assays of the conditioned medium with [125I]IGF-I or [125]IGF-II demonstrated that the IGFBP present in the conditioned medium preferentially binds IGF-II. In conclusion, cultured sheep choroid plexus epithelial cells synthesize and secrete IGF-II and IGFBP-2, suggesting that the choroid plexus epithelium is the main source of these polypeptides in the cerebrospinal fluid.
Holm
N R
NR
Department of Clinical Chemistry, Bispebjerg Hospital, Copenhagen, Denmark.
Hansen
L B
LB
Nilsson
C
C
Gammeltoft
S
S
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000378:metabolism; D002478:Cells, Cultured; D002831:Choroid Plexus / Q000378:metabolism*; D017077:Culture Media, Conditioned; D004848:Epithelium / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D012756:Sheep; D014158:Transcription, Genetic
D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D018971:Insulin-Like Growth Factor Binding Protein 2; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II