Identification and characterization of insulin-like growth factor (IGF)-binding protein expression and secretion by adult human adrenocortical cells:differential regulation by IGFs and adrenocorticotropin

In previous studies we have shown that IGF-II stimulates basal as well as A CTH-induced cortisol secretion from adult human adrenocortical cells more p otently than IGF-I, and that both IGFs predominantly stimulate androgen bio synthesis. The steroidogenic effect of IGF-I and IGF-II is mediated through interaction with the IGF-I receptor, and modified by locally produced IGF- binding proteins (IGFBPs). In the present study, we identified and characte rized IGFBP synthesis in normal adult human adrenocortical cells in primary culture, and investigated the effect of ACTH and recombinant human IGF-I a nd -II on the regulation of IGFBP expression and secretion. Using RT-PCR, we identified the mRNA of all six high-affinity IGFBPs, in bo th adrenocortical tissue and monolayer cell cultures of adrenocortical cell s. Using Western ligand and immunoblotting and two-dimensional Western Liga nd blotting we confirmed the secretion of IGFBP-1, -2, -3, -4 and -5 by adr enocortical cells in primary culture. The quantification of IGFBPs indicated that IGFBP-3 accounts for almost hal f the binding activity in conditioned the regulation of the differentiated medium of unstimulated cells (47%), followed by IGFBP-4 (20%), IGFBP-5 (15% ), IGFBP-2 (12%) and IGFBP-1 (6%). After treatment with ACTH, the abundance of IGFBP-1 was upregulated significantly 2.6-fold, while IGFBP-3 was induc ed only slightly (1.3-fold). IGFBP-2, -4 and -5 remained unchanged. In cont rast, IGF-I and -II (6.5 nM) predominantly induced the abundance of IGFBP-5 (2- and 1.6-fold respectively) and IGFBP-3 (2- and 1.7-fold respectively), while IGFBP-1, -2 and -4 were unaltered. The induction of IGFBP-1 and -5 b y ACTH and IGFs, respectively, was paralleled by an increase in the amount of IGFBP-1 and -5 mRNA in these cells. In conclusion, all six high-affinity IGFBPs are expressed in the adult huma n adrenal gland, and the presence of at least five high-affinity IGFBPs has been demonstrated in conditioned medium of adult human adrenocortical cell s. Furthermore, the expression and secretion of IGFBP-1 is upregulated by A CTH, whereas IGFBP-5 is induced by IGF-I and -II. Together with earlier fin dings, these results suggest that IGFBPs play an important modulatory role in adrenocortical function.