Independent Changes in Type I and Type II Receptors for Transforming Growth Factor β Induced by Bone Morphogenetic Protein 2 Parallel Expression of the Osteoblast Phenotype

Abstract

Transforming growth factor beta (TGF-beta), a potent regulator of bone formation, has bifunctional effects on osteoblast replication and biochemical activity that appear differentiation dependent. We now show that cell surface binding sites for TGF-beta vary markedly among fibroblasts, bone-derived cells, and highly differentiated osteosarcoma cultures from fetal rats. Expression of betaglycan and type II receptors decline relative to type I receptor expression in parallel with an increase in osteoblast-like activity, predicting that the ratio among various TGF-beta binding sites could influence how its signals are perceived. Bone morphogenetic protein 2 (BMP-2), which induces osteoblast function, does not alter TGF-beta binding or biochemical activity in fibroblasts and has only small effects in less differentiated bone cells. In contrast, BMP-2 rapidly reduces TGF-beta binding to betaglycan and type II receptors in osteoblast-enriched primary cell cultures and increases its relative binding to type I receptors in these cells and in ROS 17/2.8 cultures. Pretreatment with BMP-2 diminishes TGF-beta-induced DNA synthesis in osteoblast-enriched cultures but synergistically enhances its stimulatory effects on either collagen synthesis or alkaline phosphatase activity, depending on the present state of bone cell differentiation. Therefore, BMP-2 shifts the TGF-beta binding profile on bone cells in ways that are consistent with progressive expression of osteoblast phenotype, and these changes distinguish the biochemical effects mediated by each receptor. Our observations indicate specific stepwise actions by TGF-beta family members during osteoblast differentiation, developing in part from changes imprinted by BMP-2 on TGF-beta receptor stoichiometry.

Multiple and Essential Sp1 Binding Sites in the Promoter for Transforming Growth Factor-β Type I Receptor

“…Mineralized nodules were only observed in population 3-5, were evident 3-4 days after adding ␤-glycerol phosphate, and accumulated throughout 2 weeks of incubation (8,9). Clonal rat osteosarcoma-derived osteoblast-like ROS 17/2.8 cultures (obtained from Dr. Gideon Rodan; Merck Sharp and Dohme Research Laboratories, West Point, PA) and first passage skin fibroblasts from rat fetuses used to isolate bone cells were cultured and treated by similar procedures (4).…”

“…Partitioning of Sp1 and Sp3 in these ways has been noted previously (14 -17) and may relate in part to multiple Sp3 isoforms arising from differential initiation codon utilization. 4 Binding to Sp1-like sites occurred with oligonucleotide probes XN1 (from Ϫ108 to Ϫ71) and XN2 (from Ϫ70 to Ϫ46) of region XN and nuclear extract from osteoblast-enriched cultures (Fig. 6A).…”

“…Functional Sp1 Binding Sites in the TGF-␤RI PromoterPrimary osteoblast-enriched cell cultures are derived from normal tissue and appear to be controlled in appropriate physiological ways. Because they express high levels of TGF-␤RI mRNA, protein, and promoter activity (4,5,7,9), and protein that associates with Sp1 binding sites, many subsequent studies were performed with extracts from this culture model. Probes SA1, AX2, and AX5 all formed slowly migrating radiolabeled complexes identical to those with 32 P-SP1, containing a consensus Sp1 binding site, and were effectively reduced by unlabeled consensus oligonucleotide SP1 (Fig.…”

“…Imposed on this are our previous observations that the proportions of TGF-␤RI mRNA and protein may vary with the osteoblast phenotype and that its levels are rapidly controlled by certain stimulatory and inhibitory bone growth regulators (4, 7). 2 Initial TGF-␤RI promoter activity studies substantiate that osteoblast-related variations in steady state mRNA levels are controlled at least in part at the level of gene transcription (4,5). 3 Therefore, the widespread expression of TGF-␤RI, driven by a constitutively active promoter, may in some instances be regulated by other cis-acting regulatory elements.…”

“…In some situations its activity is controlled by complex interactions with other cell surface components (1)(2)(3). However, in contrast to TGF-␤RII and the cell surface proteoglycan also termed TGF-␤RIII or betaglycan, expression of TGF-␤RI is maintained on differentiated bone cells (4). For these reasons, and because little is known about the molecular control of TGF-␤RI expression, we cloned the rat TGF-␤RI promoter and characterized several of its functional aspects in cultures of primary and continuous skeletal and nonskeletal cells derived from fetal rats.…”