Breaking new ground to build bone

Osteoporosis affects 10 million Americans over the age of 50 (1). Bone loss is caused by an imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Fifty percent of women and 20% of men over 50 will have a fragility fracture in their lifetime, contributing to 1.5 million osteoporotic fractures each year (1, 2). Prevention and treatment for osteoporosis aim to slow bone remodeling and restore the balance between bone formation and destruction. There are two therapeutic approaches: antiresorptive agents, such as bisphosphonates, which inhibit osteoclastic bone resorption, and anabolic agents, such as teriparatide, recombinant human parathyroid hormone(1–34), which increase new bone formation by stimulating osteoblast activity (Fig. 1). In a recent issue of PNAS, Buckbinder et al. (3) define a role for proline-rich tyrosine kinase 2 (PYK2) as a negative regulator of osteoblast differentiation and novel target for osteoporosis therapy. An inhibitor of PYK2 kinase activity, PF-431396, stimulated new bone formation in a rat model of postmenopausal osteoporosis and is a promising new anabolic osteoporosis therapy.

PYK2 is a nonreceptor tyrosine kinase related to focal adhesion kinase (FAK). Unlike FAK, PYK2 is expressed abundantly only in the central nervous system and the hematopoietic lineage (4). Previous studies reviewed by Duong et al. (5) demonstrated a role for PYK2 in osteoclast activation. Ligand binding to αvβ3 integrin on osteoclasts activates c-Src, which in turn phosphorylates and activates PYK2 (Fig. 1) (6). PYK2 localizes to the sealing zone in osteoclasts and, by interacting with factors such as paxillin, Grb2, and p130^cas, promotes cytoskeletal reorganization, osteoclast migration, and resorptive pit formation (7). Calcitonin, an inhibitor of bone resorption, causes PYK2 dephosphorylation and relocalization away from the sealing zone (8). Similarly, PYK2 is important for normal morphology and function of macrophages, which share a common progenitor with osteoclasts. Macrophages isolated from PYK2−/− mice had impaired polarization, membrane ruffling, and migration in response to chemokine stimulation (9). PYK2 also has effects on osteoblasts, where treatment with bone-stimulatory fluoroaluminate increases PYK2 autophosphorylation, c-Src binding, and osteoblast spreading and attachment (4).

Buckbinder et al. (3) characterized the bones of PYK2−/− mice. Microcomputed tomography and bone histomorphometric analysis showed a high bone mass phenotype, suggesting that PYK2 negatively regulates bone mass. However, the high bone mass was caused by increased bone formation, rather than reduced bone resorption in PYK2−/− mice, where osteoclast function appeared normal. Analyses in vivo and in vitro showed no differences in osteoclast number, activity, or degree of differentiation between PYK2−/− and wild-type mice, in direct contrast to previous work supporting a role for PYK2 in osteoclast activation. Osteoblast differentiation and mineralization were increased in bone marrow cultures from PYK2−/− mice compared with control. Knockdown of PYK2 with shRNA increased osteoblast differentiation of human mesenchymal stem cells, supporting a role for PYK2 as a negative regulator of the osteoblastic lineage.

A recent study by Kim et al. (10) provides insight into the role of PYK2 in osteoblasts. In a bone injury repair model of transgenic mice deleted for FAK expression in osteoblasts, osteoprogenitor cells migrated to sites of skeletal injury but displayed delayed osteoblast maturation and bone repair. Osteoclast activity also was delayed. The delays were temporary, and new bone deposition was equal to control by 21 days postinjury. PYK2 relocalized from the perinuclear region of FAK null osteoblasts to focal adhesions and may compensate for the absence of FAK. The results of these two studies (3, 10) suggest a model in which relocalization and increased activity of PYK2 at focal adhesions prevents osteoblast maturation and delays bone healing (Fig. 1).

These studies raise numerous mechanistic questions. How does PYK2 affect osteoprogenitor maturation? Where does PYK2 act in the osteoblast lineage? Does PYK2 suppress known regulators of osteoblast differentiation and function, such as Runx2 or osterix, or stimulate the Wnt inhibitor Dkk1? How can the finding that deletion of PYK2 had no effect on osteoclast function be reconciled with previous data that PYK2 stimulates osteoclast activity? The role of c-Src and FAK in this process is unclear and will be important to reconcile because c-Src inhibitors are under development as antiresorptive agents. Crossing PYK2 null mice with targeted FAK null mice would test how these related kinases contribute to the bone phenotype of the mice. FAK null transgenic mice could be treated with a PYK2 inhibitor to determine whether PYK2 activity delays osteoblast maturation at sites of bone injury.

Lastly, Buckbinder et al. (3) tested PYK2 as a target for osteoporosis therapy in an ovariectomized rat model of postmenopausal osteoporosis. Animals were treated with vehicle, estrogen replacement, or a PF-431396, potent small molecule inhibitor of PYK2 kinase activity, for 28 days. The drug significantly increased bone formation and mineralization and rescued ovariectomy-induced bone loss. Estrogen replacement suppressed bone turnover and prevented bone loss compared with vehicle but, unlike PF-431396, did not increase bone formation.

There is a great need for osteoblast-stimulatory, bone-building osteoporosis therapy. Bisphosphonates are currently the first-line therapy for osteoporosis, but the long half-life in bone, chronic suppression of bone turnover, and association with osteonecrosis of the jaw (2) raise concerns about the long-term use of these agents. Alternative Food and Drug Administration (FDA)-approved antiresorptive therapy, selective estrogen receptor modulators, do not share these issues but are less commonly prescribed. Promising new antiresorptive agents, such as RANK ligand-neutralizing antibody, are under development.

Teriparatide is the only FDA-approved anabolic agent. It reduces vertebral fractures by >65% and stimulates significant increases in lumbar spine bone mineral density (11). It effectively restores bone microarchitecture and stimulates new bone formation more than antiresorptive therapies (12–14). Teriparatide is especially useful for patients who fail or cannot tolerate bisphosphonate therapy. However, its use is restricted to 2 years because of a black box warning applied when rat studies indicated an increased risk of osteosarcoma. Other animal studies indicate that parathyroid hormone may increase tumor growth in bone (15, 16). Patients with cancers in bone or who have received radiation to the skeleton are not candidates for teriparatide therapy (13, 16). Thus, therapeutic alternatives, such as PF-431396, could be useful for these patients.

Novel anabolic approaches for treating osteoporosis are welcome but also viewed with caution.

Another target for anabolic therapy is sclerostin, whose gene is mutated in sclerosteosis, a rare genetic disorder characterized by a generalized increase in bone mass (17). Patients homozygous for sclerostin mutation have very thick bones, particularly the skull, which cause nerve entrapment and increased intracranial pressure. Sclerostin is expressed by osteocytes, terminally differentiated osteoblastic cells embedded within bone that may secrete the factor to regulate bone homeostasis. Mechanical stress leads to osteocyte apoptosis and decreased levels of sclerostin, which permits new bone formation (18). Sclerostin acts downstream of bone morphogenetic protein signaling (17) and is a negative regulator of the Wnt pathway (18). The factor is expressed only in bone, and antibodies to sclerostin increase bone formation rate and density in mice, suggesting that such antibodies could provide a future anabolic therapy (18). Antibodies to another Wnt inhibitor, Dkk1, are also under development.

Statins represent a third anabolic osteoporosis therapy. They are lipid-lowering agents that inhibit 5-hydroxy-3-methylglutaryl-CoA reductase. In animal models, statins increase bone mass by a bone morphogenetic protein-2-mediated effect on osteoblasts (19). A limited number of studies have been reported in which statin therapy increased bone mass in patients. Effect of statins on bone mass have been difficult to demonstrate because of high drug uptake by the liver and limited systemic bioavailability (20). Randomized, controlled clinical trials in which statins are delivered efficiently to bone would test their value as an anabolic osteoporosis therapy. Strontium is in common use outside of the United States to treat osteoporosis. The mechanisms by which strontium alters bone resorption and formation are unclear, and the compound itself may confound measurement of bone mineral density.

Agents that stimulate bone formation, such as PF-431396, modulators of the Wnt pathway (sclerostin and Dkk1 antibodies), and statins could be useful additions to the limited armamentarium to treat osteoporosis. Novel anabolic approaches for treating osteoporosis are welcome but also viewed with caution. Future studies will determine whether the new anabolic agents increase risk for osteosarcoma, as seen with teriparatide in rats, and will likely be required before FDA approval is granted. Also crucial to approval will be clinical data on whether these anabolic agents improve bone quality, reduce fracture risk, and improve fracture healing, because bone mineral density represents only one parameter of bone quality. These new agents could have a significant impact on the future of osteoporosis treatment.