Osteopontin (OPN) is a highly phosphorylated sialoprotein that is a prominent component of the mineralized extracellular matrices of bones and teeth. OPN is characterized by the presence of a polyaspartic acid sequence and sites of Ser/Thr phosphorylation that mediate hydroxyapatite binding, and a highly conserved RGD motif that mediates cell attachment/signaling. Expression of OPN in a variety of tissues indicates a multiplicity of functions that involve one or more of these conserved motifs. While the lack of a clear phenotype in OPN "knockout" mice has not established a definitive role for OPN in any tissue, recent studies have provided some novel and intriguing insights into the versatility of this enigmatic protein in diverse biological events, including developmental processes, wound healing, immunological responses, tumorigenesis, bone resorption, and calcification. The ability of OPN to stimulate cell activity through multiple receptors linked to several interactive signaling pathways can account for much of the functional diversity. In this review, we discuss the structural features of OPN that relate to its function in the formation, remodeling, and maintenance of bones and teeth.

The search for a protein nucleator of hydroxyapatite crystal formation has been a focus for the isolation and characterization of the major non-collagenous proteins in bone. Of the proteins characterized to date, bone sialoprotein (BSP) has emerged as the only bona fide candidate for nucleation. BSP is a highly glycosylated and sulphated phosphoprotein that is found almost exclusively in mineralized connective tissues. Characteristically, polyglutamic acid and arginine-glycine-aspartate (RGD) motifs with the ability to bind hydroxyapatite and cell-surface integrins, respectively, have been conserved in the protein sequence. Expression of the BSP gene, which is induced in newly formed osteoblasts, is up-regulated by hormones and cytokines that promote bone formation and down-regulated by factors that suppress bone formation. Thus, BSP has the biophysical and chemical properties of a nucleator, and its temporo-spatial expression coincides with de novo mineralization in bone and cementum. Moreover, BSP has been associated with mineral crystal formation in several pathologies, including breast carcinomas. However, the ability of BSP to mediate cell attachment and to signal through the RGD motif points to alternate functions for BSP which need further investigation. In combination, the hydroxyapatite-binding polyglutamic acid sequences and the RGD provide bi-functional entities through which BSP may mediate the targeting and attachment of normal and metastasizing cells to the bone surface.

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.

A number of 1-(omega-aminoalkyl)-5-hydroxy-2-(4-hydroxyphenyl)indoles were synthesized and studied for their binding affinities for the calf uterine estrogen receptor and estrogen antagonistic activities. Highest binding affinities were found with derivatives bearing a methyl group in position 3 and a hexamethylene chain between the indole and amino nitrogen atoms. Values for relative binding affinity (RBA) are between 20 and 30 for derivatives 5b, 5c, 5f, and 5h (17 beta-estradiol = 100). In the mouse uterine weight test, no significant agonistic (estrogenic) activity was observed up to a daily dose of 125 micrograms/animal, except for derivatives 5c, 5j, and 5l. 2-Phenylindoles with amino (5b), pyrrolidino (5f), piperidino (5h), and morpholino (5k) groups as the amino function completely suppressed estrone-stimulated uterine growth as a dose of 125 micrograms/animal (100% antagonism). Therefore, these derivatives can be considered as first examples of nonsteroidal pure antiestrogens.

Bone sialoprotein (BSP), a phosphorylated and sulphated glycoprotein that is expressed by mineralized connective tissues is also produced in tumors that metastasize to bone. To facilitate studies of BSP expression in normal and pathological human tissues a monoclonal antibody (BSP 1.2 mab) was raised against human bone BSP. BSP 1.2 mab was shown by ELISA assays to recognize the epitope "DEYSY" (amino acids 279-283) that is conserved in mammalian BSP sequences. However, whereas the antibody recognized recombinant BSPs expressed in bacteria, it did not recognize native forms of rat or pig BSP in which the first tyrosine of the DEYSY peptide sequence appears to be modified. Immunostaining of embryonic human tibiae and calvariae with BSP 1.2 mab showed strong reaction in osteoblasts and osteocytes with relatively weak staining of the bone matrix, suggesting that the BSP 1.2 mab epitope is partially masked in the bone matrix. BSP 1.2 mab also stained osteosarcoma cells and normal trophoblastic cells in the placenta in areas of microcrystalline deposits. Cancer cells in primary breast tumors, lymph nodes, and secondary bone metastases from individual patients were stained strongly by BSP 1.2 mab. Although BSP 1.2 mab also stained breast cancer carcinoma cell lines and SaOS2 osteosarcoma cells, biosynthesis of radiolabelled BSP could not be demonstrated in breast cancer cells. Notably, the staining of BSP in the breast cancer cells was diffuse contrasting the punctate staining, typical of secreted proteins, in SaOS2 cells. These studies, therefore, have identified a unique epitope in human BSP recognized by a monoclonal antibody, BSP 1.2 mab, which can be used for the unequivocal identification of BSP in normal and pathological human tissues.