The serum glycoprotein fetuin is expressed during embryogenesis in multiple tissues including limb buds and has been shown to promote bone remodeling and stimulate cell proliferation in vitro. In this report, we demonstrate that fetuin antagonizes the antiproliferative action of transforming growth factor-␤1 (TGF-␤1) in cell cultures. Surface plasmon resonance measurements show that fetuin binds directly to TGF-␤1 and TGF-␤2 and with greater affinity to the TGF-␤-related bone morphogenetic proteins (BMP-2, BMP-4, and BMP-6). In a competitive enzyme-linked immunosorbent assay, fetuin blocked binding of TGF-␤1 to the extracellular domain of TGF-␤ receptor type II (T␤RII), one of the primary TGF-␤-binding receptors. A comparison of fetuin and T␤RII shows homology in an 18 -19-amino acid sequence, which we have designated TGF-␤ receptor II homology 1 domain (TRH1). Since the TRH1 sequence is known to form a disulfide loop in fetuin, cyclized TRH1 peptides from both fetuin and T␤RII were chemically synthesized and tested for cytokine binding activity. Cyclized TRH1 peptide from T␤RII bound to TGF-␤1 with greater affinity than to BMP-2, while the cyclized TRH1 peptide from fetuin bound preferentially to BMP-2. Finally, fetuin or neutralizing anti-TGF-␤ antibodies blocked osteogenesis and deposition of calciumcontaining matrix in cultures of dexamethasone-treated rat bone marrow cells. In summary, these experiments define the TRH1 peptide loop as a cytokine-binding domain in both T␤RII and fetuin and suggest that fetuin is a natural antagonist of TGF-␤ and BMP activities.

Soluble transforming growth factor-␤ (TGF-␤)/bone morphogenetic protein (BMP)-binding proteins are widely distributed in mammalian tissues and control cytokine access to membrane signaling receptors. The serum and bone-resident glycoprotein ␣2-HS-glycoprotein/fetuin (ASHG) binds to TGF-␤/BMP cytokines and blocks TGF-␤1 binding to cell surface receptors. Therefore, we examined bone growth and remodeling phenotypes in ASHG-deficient mice. The skeletal structure of Ahsg ؊/؊ mice appeared normal at birth, but abnormalities were observed in adult Ahsg ؊/؊ mice. Maturation of growth plate chondrocytes was impaired, and femurs lengthened more slowly between 3 and 18 months of age in Ahsg ؊/؊ mice. However, bone formation was increased in Ahsg ؊/؊ mice as indicated by greater cortical thickness, accelerated trabecular bone remodeling, and increased osteoblast numbers on bone surfaces. The normal age-related increase in cortical thickness and bone mineral density was accelerated in Ahsg ؊/؊ mice and was associated with increased energy required to fracture. Bone formation in response to implanted BMP cytokine extended further from the implant in Ahsg ؊/؊ compared with Ahsg ؉/؉ mice, confirming the interaction between ASHG and TGF-␤/BMP cytokines in vivo. Our results demonstrate that ASHG blocks TGF-␤-dependent signaling in osteoblastic cells, and mice lacking ASHG display growth plate defects, increased bone formation with age, and enhanced cytokine-dependent osteogenesis.

BackgroundSkeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO3 −)n) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization.Principal Findings/MethodologyThe enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO4 3−) concentration while permitting the accumulation of a high total PO4 3− concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO4 3− and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4′,6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation.Conclusions/SignificanceWe hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biologica...

Aryl hydrocarbon receptor (AhR) ligands are environmental contaminants found in cigarette smoke and other sources of air pollution. The prototypical compound is TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), also known as dioxin. There is an increasing body of knowledge linking cigarette smoking to osteoporosis and periodontal disease, but the direct effects of smokeassociated aryl hydrocarbons on bone are not well understood. Through the use of resveratrol (3,5,4 -trihydroxystilbene), a plant antifungal compound that we have recently demonstrated to be a pure AhR antagonist, we have investigated the effects of TCDD on osteogenesis. It was postulated that TCDD would inhibit osteogenesis in bone-forming cultures and that this inhibition would be antagonized by resveratrol. We employed the chicken periosteal osteogenesis (CPO) model, which has been shown to form bone in vitro in a pattern morphologically and biochemically similar to that seen in vivo, as well as a rat stromal cell bone nodule formation model. In the CPO model, alkaline phosphatase (AP) activity was reduced by up to 50% (P<0·01 vs control) in the presence of 10 9 M TCDD and these effects were reversed by 10 6 M resveratrol (P<0·05 vs TCDD alone). TCDD-mediated inhibition of osteogenesis was restricted primarily to the osteoblastic differentiation phase (days 0-2) as later addition did not appear to have any effects. Message levels for important bone-associated proteins (in the CPO model) such as collagen type I, osteopontin, bone sialoprotein and AP were inhibited by TCDD, an effect that was antagonized by resveratrol. Similar findings were obtained using the rat stromal bone cell line. TCDD (at concentrations as low as 10 10 M) caused an approximately 33% reduction in AP activity, which was abrogated by 3·5 10 7 M resveratrol. TCDD also induced a marked reduction in mineralization (75%) which was completely antagonized by resveratrol. These data suggest that AhR ligands inhibit osteogenesis probably through inhibition of osteodifferentiation and that this effect can be antagonized by resveratrol. Since high levels of AhR ligands are found in cigarette smoke, and further since smoking is an important risk factor in both osteoporosis and periodontal disease, it may be postulated that AhR ligands are the component of cigarette smoke linking smoking to osteoporosis and periodontal disease. If so, resveratrol could prove to be a promising preventive or therapeutic agent for smoking-related bone loss.