Proline-rich tyrosine kinase 2 regulates osteoprogenitor cells and bone formation, and offers an anabolic treatment approach for osteoporosis

Abstract

Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2؊/؊ mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2؊/؊ mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.human mesenchymal stem cell ͉ osteoclast ͉ osteoblast P roline-rich tyrosine kinase 2 (PYK2) and focal adhesion kinase (FAK) are nonreceptor tyrosine kinases, and together they constitute the focal adhesion kinase subfamily (1). Unlike FAK, PYK2 expression is relatively restricted, with highest levels in the brain and the hematopoietic system. PYK2Ϫ/Ϫ mice have been described previously and appear normally developed (2, 3). Characterization of the immune system of PYK2Ϫ/Ϫ animals revealed the absence of marginal zone B cells along and abnormal T cell-independent type II responses (2), as well as altered macrophage morphology, adhesion, and migration (3).Although PYK2 is expressed in both bone-forming osteoblasts and bone-resorbing osteoclasts, the skeletal phenotype of PYK2Ϫ/Ϫ mice has not been described. In vitro studies pointed to a positive role for PYK2 in osteoclast maturation and bone resorption. PYK2 localizes to the podosomes of osteoclasts (4), and, upon integrin binding, cell attachment, and actin ring formation, PYK2 associates with a variety of proteins including p130 CAS (5), Src (4), Cbl (6), integrins (4), gelsolin (7), and paxillin (8). Antisense depletion of PYK2 (9), but not the expression of a kinase inactive dominant negative mutant (10), blocked osteoclast spreading and bone resorption, indicating that PYK2 catalytic activity may be dispensable. The in vitro effects of bone anabolic stimuli suggested that PYK2 might have a positive role in osteoblasts as well. Treatment of osteoblast cells with fluoroaluminate led to increased PYK2 autophosphorylation, Src association, and kinase activity (11) and was associated with increased cell attachment and spreading (12). Likewise, in an anabolic model of mechanical loading, PYK2 autophosphorylation and kinase activity were stimulated in o...

Tyrosine 397 phosphorylation is critical for FAK-promoted Rac1 activation and invasive properties in oral squamous cell carcinoma cells

“…We further examined the impact of inhibiting pY397 on the Rac1 activity by treating OECM-1 cells with PF-431396, a highly selective pyrimidine-based inhibitor of FAK. 21 As shown in Figure 3d (right panel), treatment of 1 μM PF-431396 caused decreases in the levels of pY397 and Rac1-GTP as compared with the vehicle control. Coordinating with the changes in Rac1 activity, expression of Y397F mutant or treatment of PF-431396 led to inhibited cell migration and invasion (Figures 3e and f).…”

Osteoblasts in osteoporosis: past, emerging, and future anabolic targets

“…Another example is inhibition of the proline-rich tyrosine kinase 2 (PYK2 or PTK2B), a non-receptor tyrosine kinase expressed in bone cells. PYK2-deficient mice show high bone mass and increased MSC differentiation and bone formation, and inhibition of PYK2 was shown to prevent bone loss in OVX rats (125). It can also be considered to inhibit midkine, a heparin-binding growth factor, because midkine deficiency results in increased trabecular bone formation in mice (126).…”

Megakaryocytes Regulate Expression of Pyk2 Isoforms and Caspase-mediated Cleavage of Actin in Osteoblasts

“…The focal adhesion tyrosine kinases Pyk2 and FAK are linked to the proliferation, migration, and activity of a variety of mesenchymal, epithelial, and hematopoietic cell types (7)(8)(9)(10). Our group and others have reported that deletion of the Pyk2 gene in mice leads to an increase in bone mass, which is due to defects in the activity of OBs and osteoclasts (7,11).…”