Ligand-induced downregulation of receptors for TGF-β in human osteoblast-like cells from adult donors

High concentrations of transforming growth factor b (TGF-β) are found in the bone matrix, reflecting a pivotal role of this growth factor in the coupling of bone resorption and formation. TGF-β strongly stimulates the synthesis of extracellular matrix proteins, but in vitro studies show an inhibitory effect on the final mineralization process, which in vivo occurs despite high concentrations of TGF-β. Little is known about how bone- forming cells respond to different concentrations of TGF-β and if they can transiently adapt receptor numbers in order to modulate cellular activity. Against this background, we studied the cell-surface expression of TGF-β receptors (TβR) I, II and III (betaglycan) on human osteoblast-like cells from adult donors, and examined the TβR presentation on these cells after a preceding exposure to TGF-β1. Affinity crosslinking studies with disuccinimidylsuberate showed the presence of all three receptor types. Preincubation with TGF-β1 markedly reduced ¹²⁵I-TGF-β1 binding in a time-dependent and dose-dependent manner and revealed a 95% reduction after an 18-h preincubation with 200 pM TGF-β1. In parallel, Scatchard analysis showed that the binding affinity did not change as a consequence of TGF-β1 preincubation. Immunoblotting analyses revealed an almost complete disappearance of immunoreactive TβR-II and TβR-III proteins after a 24-h preincubation with TGF-β1. Using semi-quantitative reverse transcription PCR, no effect of TGF-β1 on the expression of TβR-II mRNA was observed. These studies demonstrate a ligand-induced downregulation of TβRs-II and - III on human osteoblast-like cells, without any evidence for recovery within the first 24 h, both in the presence and after the removal of the ligand. The underlying mechanism appears to be based on post-transcriptional events. The results suggest that high concentrations of active TGF-β1 decrease the responsiveness of osteoblasts towards this growth factor.