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.