Regulation of insulin-like growth factor-binding protein expression by thyroid hormone in rat GH₃ pituitary tumor cells

Rat pituitary GH₃ tumor cells express GH and insulin-like growth factor-I (IGF-I) mRNAs and possess specific IGF-I and IGF-II receptors which mediate the inhibitory effect of IGF on GH secretion. T₃ increases the rate of cell replication and GH gene transcription, and causes an increase in IGF-I binding to cell membranes. Since the IGFs circulate in association with specific binding proteins (IGFBPs) that appear to modulate the access of IGFs to their receptors, we have investigated the effect of T₃ treatment on the expression of IGFBPs in GH₃ cells. Cells were grown in serum-free defined medium, and IGFBP secretion was determined by Western ligand blotting of conditioned medium after the addition of T₃ and/or various peptides for 48-72 h. The conditioned medium of GH₃ cells revealed a complex of bands migrating at 40-45 Mr, a pattern typical of rat (r) IGFBP-3. T₃ treatment resulted in an increase in rIGFBP-3. IGF-I, IGF-II, and insulin did not alter rIGFBP-3 levels. After concentrating (10-fold) conditioned medium samples, two additional bands at 24K and 28K mol wt were also seen. These bands corresponded in size to rIGFBP-4 (24K) and its glycosylated form (28K). The mRNAs for both rIGFBP-3 and rIGFBP-4 were present in GH₃ cells; T₃ treatment increased steady state levels of rIGFBP-3 mRNA, but did not affect BP-4 mRNA levels. To learn whether the increased expression of IGFBPs could be responsible for the increased IGF-I binding seen after T₃ treatment, [¹²⁵I]IGF-I was cross-linked to GH₃ membranes, and the proteins were separated on a 5-15% gradient sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. T₃ treatment induced a large increase in the intensity of bands migrating at 135K and 280K, representing the α-subunit of the IGF-I receptor and an incompletely reduced α-α-dimer, respectively. No membrane-associated IGFBPs were detected. In conclusion, GH₃ cells express two IGFBPs, rIGFBP-3 and rIGFBP-4, which are differentially regulated by T₃. The increased binding of IGF-I to GH₃ cell membranes after T₃ treatment indicates that thyroid hormone induces an up-regulation of IGF-I receptors and that the increased IGF-I binding to GH₃ membranes is not due to increased expression of membrane-associated IGFBPs.