High concentrations of transforming growth factor b (TGF-) are found in the bone matrix, reflecting a pivotal role of this growth factor in the coupling of bone resorption and formation. TGF-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-. Little is known about how bone-forming cells respond to different concentrations of TGF-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-receptors (T R) I, II and III (betaglycan) on human osteoblast-like cells from adult donors, and examined the T R presentation on these cells after a preceding exposure to TGF-1. Affinity crosslinking studies with disuccinimidylsuberate showed the presence of all three receptor types. Preincubation with TGF-1 markedly reduced 125 I-TGF-1 binding in a time-dependent and dose-dependent manner and revealed a 95% reduction after an 18-h preincubation with 200 pM TGF-1. In parallel, Scatchard analysis showed that the binding affinity did not change as a consequence of TGF-1 preincubation. Immunoblotting analyses revealed an almost complete disappearance of immunoreactive T R-II and T R-III proteins after a 24-h preincubation with TGF-1. Using semi-quantitative reverse transcription PCR, no effect of TGF-1 on the expression of T R-II mRNA was observed. These studies demonstrate a ligand-induced downregulation of T Rs-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-1 decrease the responsiveness of osteoblasts towards this growth factor.

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder usually characterized by either a reduction in the production of normal collagen I or the synthesis of abnormal collagen. The variability in the clinical phenotype is not in each case sufficiently explained by the underlying mutation in the collagen I genes. Also, biochemical differences between mutant collagen from different tissues suggest additional regulatory mechanisms possibly involved in matrix deposition and maturation, two processes in which transforming growth factor-β (TGF-β) plays an important role. We, therefore, studied the cell surface expression and functional properties of TGF-β receptors I, II and III on osteoblasts from a group of OI patients compared to healthy controls. Receptor number and affinity were determined by Scatchard analysis of binding data and TGF-β receptor II gene expression was assessed by RT-PCR. Ligand-induced downregulation of TGF-β receptors was analyzed to demonstrate the dynamic response to exogenous stimuli. All experiments were performed in parallel in human osteoblastic cells from OI patients and from age-matched controls. TGF-β receptors I, II and III (betaglycan) were present on osteoblasts from both healthy donors and OI patients. The receptor numbers were significantly higher (29,000 per cell) on OI osteoblasts than on age-matched control osteoblasts (12,000 per cell) in spite of similar steady state levels for TGF-β receptor II mRNA in OI and control cells. Furthermore, receptor affinity was not significantly different in OI osteoblasts (181 vs. 177 nM^–1), and the receptor number did not depend on the culture substrate. With respect to dynamic adaption, ligand-induced downregulation of TGF-β receptors was reduced in OI osteoblasts. In conclusion, the human osteoblastic cells from patients with OI investigated all have an elevated number of cell surface receptors for TGF-β, without any evidence for a transcriptional regulation of TGF-β receptor II. On the functional level, there is some evidence for an impaired adaptive behavior of receptor presentation, whereas receptor affinity is unchanged.

The cell surface expression of receptors for TGF-beta was studied in human osteoblasts derived from femoral trabecular bone of a total of 19 patients aged 2-83 years. All cell populations investigated showed a similar profile of expression of TGF-beta receptors (TbetaR) I, II and III (betaglycan). There were no significant differences in cell differentiation or proliferative behaviour between the age groups. The TGF-beta receptor number per cell significantly increased with age, while the receptor affinity tended to decrease. IGF-I did not influence TbetaR expression in vitro. The results indicate an age-dependent and IGF-I independent increase of osteoblastic TGF-beta receptors in human osteoblast-like cells in vitro.