Marc D. McKee

Endocrine Regulation of Energy Metabolism by the Skeleton

The regulation of bone remodeling by an adipocyte-derived hormone implies that bone may exert a feedback control of energy homeostasis. To test this hypothesis we looked for genes expressed in osteoblasts, encoding signaling molecules and affecting energy metabolism. We show here that mice lacking the protein tyrosine phosphatase OST-PTP are hypoglycemic and are protected from obesity and glucose intolerance because of an increase in beta-cell proliferation, insulin secretion, and insulin sensitivity. In contrast, mice lacking the osteoblast-secreted molecule osteocalcin display decreased beta-cell proliferation, glucose intolerance, and insulin resistance. Removing one Osteocalcin allele from OST-PTP-deficient mice corrects their metabolic phenotype. Ex vivo, osteocalcin can stimulate CyclinD1 and Insulin expression in beta-cells and Adiponectin, an insulin-sensitizing adipokine, in adipocytes; in vivo osteocalcin can improve glucose tolerance. By revealing that the skeleton exerts an endocrine regulation of sugar homeostasis this study expands the biological importance of this organ and our understanding of energy metabolism.

Bisphosphonate-Associated Osteonecrosis of the Jaw: Report of a Task Force of the American Society for Bone and Mineral Research

ONJ has been increasingly suspected to be a potential complication of bisphosphonate therapy in recent years. Thus, the ASBMR leadership appointed a multidisciplinary task force to address key questions related to case definition, epidemiology, risk factors, diagnostic imaging, clinical management, and future areas for research related to the disorder. This report summarizes the findings and recommendations of the task force.

Calcification of Vascular Smooth Muscle Cell Cultures: Inhibition by Osteopontin

Calcification of vascular tissue is a common complication in aging, atherosclerosis, diabetes, renal failure, aortic stenosis, and prosthetic valve replacement. Osteopontin is a noncollagenous adhesive protein routinely found at sites of dystrophic calcification and synthesized at high levels by macrophages in calcified aortic valves and atherosclerotic plaques. In the present study, we have characterized the calcification of bovine aortic smooth muscle cell (BASMC) cultures in vitro and have studied the effects of exogenous osteopontin on mineral deposition. Induction of calcification in BASMC cultures was alkaline phosphatase-dependent and was characterized by a multilayer cell morphology. Mineral deposition occurred in the basal matrix of multilayered areas as indicated by von Kossa staining, and transmission electron microscopy and electron diffraction identified the mineral as apatite. Ultrastructural analysis of the cultures showed the presence of extracellular matrix vesicles, calcifying collagen fibrils, and nodular-type calcifications similar to those found in calcified heart valves and atherosclerotic plaques. Purified osteopontin (0.05 to 5 microgram/mL) dose dependently inhibited calcification of BASMC cultures, whereas vitronectin and fibronectin had no effect. In contrast to the inhibitory mechanism of levamisole on mineral deposition, osteopontin did not inhibit alkaline phosphatase activity or reduce phosphorus levels in the culture medium. Addition of calcium to the cultures overcame the inhibitory effect of osteopontin on BASMC culture calcification and resulted in decreased levels of calcium in the culture medium and increased levels in the cell layer. Moreover, using high-resolution, colloidal-gold immunocytochemistry, osteopontin was found intimately associated with growing apatite crystals. These data indicate that the effect of osteopontin, although calcium-dependent, was not mediated by simple calcium chelation but most likely by direct interaction of osteopontin with crystal surfaces. These studies suggest that BASMCs can be used to model vascular calcification in vitro and that soluble osteopontin released near sites of vascular calcification may represent an adaptive mechanism aimed at preventing vascular calcification.

Chemical modification of titanium surfaces for covalent attachment of biological molecules

The surface of implantable biomaterials is in direct contact with the host tissue and plays a critical role in determining biocompatibility. In order to improve the integration of implants, it is desirable to control interfacial reactions such that nonspecific adsorption of proteins is minimized and tissue-healing phenomena can be controlled. In this regard, our goal has been do develop a method to functionalize oxidized titanium surfaces by the covalent immobilization of bioactive organic molecules. Titanium first was chemically treated with a mixture of sulfuric acid and hydrogen peroxide to eliminate surface contaminants and to produce a consistent and reproducible titanium oxide surface layer. An intermediary aminoalkylsilane spacer molecule was then covalently linked to the oxide layer, followed by the covalent binding of either alkaline phosphatase or albumin to the free terminal NH2 groups using glutaraldehyde as a coupling agent. Surface analyses following coating procedures consisted of X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Enzymatic activity of coupled alkaline phosphatase was assayed colorimetrically, and surface coverage by bound albumin was evaluated by SEM visualization of colloidal gold immunolabeling. Our results indicate that the linkage of the aminoalkylsilane to the oxidized surface is stable and that bound proteins such alkaline phosphatase and albumin retain their enzymatic activity and antigenicity, respectively. The density of immunolabeling for albumin suggests that the binding and surface coverage obtained is in excess of what would be expected for inducing biological activity. In conclusion, this method offers the possibility of covalently linking selected molecules with known biological activity to oxidized titanium surfaces in order to guide and promote the tissue healing that occurs during implant integration in bone and soft tissues.

Extracellular matrix mineralization is regulated locally; different roles of two gla-containing proteins

Extracellular matrix mineralization (ECMM) is a physiologic process in the skeleton and in teeth and a pathologic one in other organs. The molecular mechanisms controlling ECMM are poorly understood. Inactivation of Matrix gla protein (Mgp) revealed that MGP is an inhibitor of ECMM. The fact that MGP is present in the general circulation raises the question of whether ECMM is regulated locally and/or systemically. Here, we show that restoration of Mgp expression in arteries rescues the arterial mineralization phenotype of Mgp−/− mice, whereas its expression in osteoblasts prevents bone mineralization. In contrast, raising the serum level of MGP does not affect mineralization of any ECM. In vivo mutagenesis experiments show that the anti-ECMM function of MGP requires four amino acids which are γ-carboxylated (gla residues). Surprisingly, another gla protein specific to bone and teeth (osteocalcin) does not display the anti-ECMM function of MGP. These results indicate that ECMM is regulated locally in animals and uncover a striking disparity of function between proteins sharing identical structural motifs.

Mice lacking osteopontin show normal development and bone structure but display altered osteoclast formation in vitro.

We have used homologous recombination in embryonic stem cells to generate mice with a targeted disruption of the osteopontin (Opn, or Spp1, for secreted phosphoprotein 1) gene. Mice homozygous for this disruption fail to express osteopontin (OPN) as assessed at both the mRNA and protein level, although an N‐terminal fragment of OPN is detectable at extremely low levels in the bones of −/− animals. The Opn−/− mice are fertile, their litter size is normal, and they develop normally. The bones and teeth of animals not expressing OPN are morphologically normal at the level of light and electron microscopy, and the skeletal structure of young animals is normal as assessed by radiography. Ultrastructurally, proteinaceous structures normally rich in OPN, such as cement lines, persist in the bones of the Opn−/− animals. Osteoclastogenesis was assessed in vitro in cocultures with a feeder layer of calvarial osteoblast cells from wild‐type mice. Spleen cells from Opn−/− mice cells formed osteoclasts 3‐ to 13‐fold more frequently than did control Opn+/+ cells, while the extent of osteoclast development from Opn−/− bone marrow cells was about 2‐ to 4‐fold more than from the corresponding wild‐type cells. Osteoclast development occurred when Opn−/− spleen cells were differentiated in the presence of Opn−/− osteoblasts, indicating that endogenous OPN is not required for this process. These results suggest that OPN is not essential for normal mouse development and osteogenesis, but can modulate osteoclast differentiation.

Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification.

Ectopic calcification, the abnormal calcification of soft tissues, can have severe clinical consequences especially when localized to vital organs such as heart valves, arteries, and kidneys. Recent observations suggest that ectopic calcification, like bone biomineralization, is an actively regulated process. These observations have led a search for molecular determinants of ectopic calcification. A candidate molecule is osteopontin (OPN), a secreted phosphoprotein invariantly associated with both normal and pathological mineral deposits. In the present study, OPN was found to be a natural inhibitor of ectopic calcification in vivo. Glutaraldehyde-fixed aortic valve leaflets showed accelerated and fourfold to fivefold greater calcification after subcutaneous implantation into OPN-null mice compared to wild-type mice. In vitro and in vivo studies suggest that OPN not only inhibits mineral deposition but also actively promotes its dissolution by physically blocking hydroxyapatite crystal growth and inducing expression of carbonic anhydrase II in monocytic cells and promoting acidification of the extracellular milieu. These findings suggest a novel mechanism of OPN action and potential therapeutic approach to the treatment of ectopic calcification.

Osteopontin at mineralized tissue interfaces in bone, teeth, and osseointegrated implants: Ultrastructural distribution and implications for mineralized tissue formation, turnover, and repair

Currently available data describing the gene expression and regulation, secretion, distribution, and protein chemistry of osteopontin (OPN) all are consistent with the notions of this protein functioning as an inhibitor of mineralization and/or as a mediator of cell‐matrix and matrix‐matrix/mineral adhesion (cohesion) during the formation, turnover, and repair of normal and pathological mineralized tissues. The properties and overall integrity of mineralized tissues are in part dictated by the nature of their interfaces—sites where organic and inorganic components of the extracellular matrix interact to provide biomechanical strength, regulate mineral ion homeostasis, and influence cellular events involved in mineralized tissue modeling, remodeling, and repair. High‐resolution, colloidal‐gold immunocytochemistry has been used to characterize the proteinaceous composition of these interfaces and to establish that the phosphorylated sialoprotein, OPN, is a major component found at these sites where it accumulates as a dense, planar “coating” of organic material termed either a cement line or a lamina limitans. Structural/functional features of OPN predict an ability of this protein to regulate calcification in the matrix proper of mineralized tissues and to participate, more specifically, in cell‐matrix and matrix‐matrix/mineral adhesion in laminae limitantes and cement lines, respectively. From the ultrastructural immunocytochemical data presented herein for OPN illustrating the cellular expression and extracellular matrix distribution of this protein, it is demonstrated that the production of OPN is one of the earliest, and latest, secretory activities of the osteoblast lineage and that this activity manifests itself morphologically as a cement line or a lamina limitans, respectively, at bone matrix interfaces. In laminae limitantes at bone surfaces, OPN appears to be involved in osteoclast adhesion and possibly haptotaxis. An OPN‐containing cement line is also present at hard tissue interfaces in rat tooth, against osseointegrated titanium and hydroxyapatite implants and at the margins of surgically created bone defects—and there may influence biological adhesion in a manner similar to that proposed for normal bone. It is suggested, therefore, that in addition to its potential for influencing cell adhesion/dynamics in bones and teeth, OPN in cement lines may act as an interfacial adhesion promotor between apposing substrates, therein maintaining the overall integrity of bone during the bone remodeling sequence and “bonding” dissimilar tissues (or biocompatible materials) together in biological composites such as teeth and osseointegrated implants.

Inactivation of the Osteopontin Gene Enhances Vascular Calcification of Matrix Gla Protein–deficient Mice: Evidence for Osteopontin as an Inducible Inhibitor of Vascular Calcification In Vivo

Osteopontin (OPN) is abundantly expressed in human calcified arteries. To examine the role of OPN in vascular calcification, OPN mutant mice were crossed with matrix Gla protein (MGP) mutant mice. Mice deficient in MGP alone (MGP−/− OPN+/+) showed calcification of their arteries as early as 2 weeks (wk) after birth (0.33 ± 0.01 mmol/g dry weight), and the expression of OPN in the calcified arteries was greatly up-regulated compared with MGP wild-types. OPN accumulated adjacent to the mineral and colocalized to surrounding cells in the calcified media. Cells synthesizing OPN lacked smooth muscle (SM) lineage markers, SM α-actin and SM22α. However, most of them were not macrophages. Importantly, mice deficient in both MGP and OPN had twice as much arterial calcification as MGP−/− OPN+/+ at 2 wk, and over 3 times as much at 4 wk, suggesting an inhibitory effect of OPN in vascular calcification. Moreover, these mice died significantly earlier (4.4 ± 0.2 wk) than MGP−/− OPN+/+ counterparts (6.6 ± 1.0 wk). The cause of death in these animals was found to be vascular rupture followed by hemorrhage, most likely due to enhanced calcification. These studies are the first to demonstrate a role for OPN as an inducible inhibitor of ectopic calcification in vivo.

Osteopontin Deficiency Increases Mineral Content and Mineral Crystallinity in Mouse Bone

Fourier transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) were used to characterize the mineral in bones of two different lines of Opn-deficient (Opn-/-) mice and their background-matched wild-type controls (Opn+/+). Sections of tibia and femur from 12-week-old and 16-week-old mice were evaluated with a spatial resolution between 10 microm (FTIRM) and 7 microm (FTIRI). FTIRI was used to examine 400 microm x 400 microm areas in cortical bone and trabecular bone and FTIRM examined selected 20 microm x 20 microm areas at sites within these anatomically defined areas. Despite the absence of an obvious phenotype in Opn-deficient mice, being undetectable by radiographic and histological methods, FTIRM analyses revealed that the relative amount of mineral in the more mature areas of the bone (central cortical bone) of Opn-knockout mice was significantly increased. Moreover, mineral maturity (mineral crystal size and perfection) throughout all anatomic regions of the Opn-deficient bone was significantly increased. The 2-dimensional, color-coded data (images) produced by FTIRI showed similar increases in mineral maturity in the Opn-/- bone, however, the crystallinity parameters were less sensitive, and significance was not achieved in all areas analyzed. Nonetheless, the findings of increased mineral content and increased crystal size/perfection in both lines of Opn-deficient mice at both ages are consistent with in vitro data indicating that Opn is a potent inhibitor of mineral formation and mineral crystal growth and proliferation, and also support a role for Opn in osteoclast recruitment and function.