Bone Resorption by Osteoclasts: Molecular Mechanism of Pyk2 dephosphorylation by Dynamin

poster abstractOsteoporosis is a bone disease that affects hundreds of millions of people worldwide and is characterized by low bone mass and structural deterioration of bone tissue which increases the risk of bone fracture, frailty, morbidity and mortality. Excessive bone loss is caused by osteoclasts which degrade the organic and inorganic components of bone. The specific aim of this study is to identify and characterize the signaling proteins in osteoclasts that are responsible for the bone resorbing activity of these cells. The non-receptor tyrosine kinase, Pyk2, is highly expressed in osteoclasts. Mice lacking Pyk2 have an increase in bone mass due to impairment in osteoclast function. It has been demonstrated that phosphorylation of Pyk2 at Y402 is very important for osteoclast spreading and bone resorption. Our group also reported that the GTPase dynamin controls osteoclast bone resorption in part by leading to the dephosphorylation of Pyk2, thus decreasing Pyk2’s kinase activity. In the current study we examined the intracellular mechanism by which dynamin regulates Pyk2 dephosphorylation. Our findings demonstrated that Pyk2 dephosphorylation is predominately due to GTPase activity of dynamin since expression of dynamin mutants that have reduced affinity for GTP or exhibit defective GTPase activity resulted in an increase in Pyk2 Y402 phosphorylation. We also found that that Pyk2 phosphorylation was rescued in the presence of phenyl arsine oxide (PAO), a chemical inhibitor of tyrosine phosphatases and our preliminary results indicate that the tyrosine phosphatase PTP-PEST is involved in the dynamin-mediated dephosphorylation of Pyk2. Understanding the intracellular mechanism that regulates osteoclast function may lead to the identification of novel proteins that can be targeted by anti-resorptive therapies to treat bone related diseases. Over the past few decades, bisphosphonates have played a significant role in the treatment of osteoporosis. Unfortunately, osteonecrosis of the jaw has been recently described as a harmful side effect of bisphosphonate therapy, emphasizing the need to develop alternative approaches to treat osteoporosis. Novel therapeutic approaches may one day involve inhibitors to tyrosine kinases such as Pyk2 or involve combination therapies where inhibitors are paired with bisphosphonates as a way to boost the efficacy of anti-resorptive therapies with fewer side-effects

Focal Adhesion Kinases in Adhesion Structures and Disease

Cell adhesion to the extracellular matrix (ECM) is essential for cell migration, proliferation, and embryonic development. Cells can contact the ECM through a wide range of matrix contact structures such as focal adhesions, podosomes, and invadopodia. Although they are different in structural design and basic function, they share common remodeling proteins such as integrins, talin, paxillin, and the tyrosine kinases FAK, Pyk2, and Src. In this paper, we compare and contrast the basic organization and role of focal adhesions, podosomes, and invadopodia in different cells. In addition, we discuss the role of the tyrosine kinases, FAK, Pyk2, and Src, which are critical for the function of the different adhesion structures. Finally, we discuss the essential role of these tyrosine kinases from the perspective of human diseases

Dynamin and PTP-PEST cooperatively regulate Pyk2 dephosphorylation in osteoclasts

Bone loss is caused by the dysregulated activity of osteoclasts which degrade the extracellular bone matrix. The tyrosine kinase Pyk2 is highly expressed in osteoclasts, and mice lacking Pyk2 exhibit an increase in bone mass, in part due to impairment of osteoclast function. Pyk2 is activated by phosphorylation at Y402 following integrin activation, but the mechanisms leading to Pyk2 dephosphorylation are poorly understood. In the current study, we examined the mechanism of action of the dynamin GTPase on Pyk2 dephosphorylation. Our studies reveal a novel mechanism for the interaction of Pyk2 with dynamin, which involves the binding of Pyk2's FERM domain with dynamin's plextrin homology domain. In addition, we demonstrate that the dephosphorylation of Pyk2 requires dynamin's GTPase activity and is mediated by the tyrosine phosphatase PTP-PEST. The dephosphorylation of Pyk2 by dynamin and PTP-PEST may be critical for terminating outside-in integrin signaling, and for stabilizing cytoskeletal reorganization during osteoclast bone resorption

Osteoblast differentiation and migration are regulated by Dynamin GTPase activity

Bone formation is controlled by osteoblasts but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the dynamin GTPase, which is associated with actin remodeling and migration in other cells, plays a role in osteoblast differentiation and migration. Dynamin mRNA was expressed in primary osteoblasts throughout differentiation (0–21 days). However, alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was decreased in osteoblasts over-expressing dynamin. Conversely, ALP activity was increased following shRNA-mediated knockdown of dynamin and in osteoblasts treated with the dynamin inhibitor, dynasore. Dynasore also reduced c-fos and osterix expression, markers of early osteoblasts, suggesting a role for dynamin in pre-osteoblast to osteoblast differentiation. Since dynamin GTPase activity is regulated by tyrosine phosphorylation, we examined the mechanism of dynamin dephosphorylation in osteoblasts. Dynamin formed a protein complex with the tyrosine phosphatase PTP-PEST and inhibition of phosphatase activity increased the level of phosphorylated dynamin. Further, PTP-PEST blocked the Src-mediated increase in the phosphorylation and GTPase activity of wild-type dynamin but not the phosphorylation mutant dynY231F/Y597F. Although ALP activity was increased in osteoblasts expressing GTPase-defective dynK44A, and to a lesser extent dynY231F/Y597F, osteoblast migration was significantly inhibited by dynK44A and dynY231F/Y597F. These studies demonstrate a novel role for dynamin GTPase activity and phosphorylation in osteoblast differentiation and migration, which may be important for bone formation

The Rho-GEF Kalirin regulates bone mass and the function of osteoblasts and osteoclasts

Bone homeostasis is maintained by the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation in the activity of the bone cells can lead to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. Kalirin is a novel GTP-exchange factor protein that has been shown to play a role in cytoskeletal remodeling and dendritic spine formation in neurons. We examined Kalirin expression in skeletal tissue and found that it was expressed in osteoclasts and osteoblasts. Furthermore, micro-CT analyses of the distal femur of global Kalirin knockout (Kal-KO) mice revealed significantly reduced trabecular and cortical bone parameters in Kal-KO mice, compared to WT mice, with significantly reduced bone mass in 8, 14 and 36 week-old female Kal-KO mice. Male mice also exhibited a decrease in bone parameters but not to the level seen in female mice. Histomorphometric analyses also revealed decreased bone formation rate in 14 week-old female Kal-KO mice, as well as decreased osteoblast number/bone surface and increased osteoclast surface/bone surface. Consistent with our in vivo findings, the bone resorbing activity and differentiation of Kal-KO osteoclasts was increased in vitro. Although alkaline phosphatase activity by Kal-KO osteoblasts was increased in vitro, Kal-KO osteoblasts showed decreased mineralizing activity, as well as decreased secretion of OPG, which was inversely correlated with ERK activity. Taken together, our findings suggest that deletion of Kalirin directly affects osteoclast and osteoblast activity, leading to decreased OPG secretion by osteoblasts which is likely to alter the RANKL/OPG ratio and promote osteoclastogenesis. Therefore, Kalirin may play a role in paracrine and/or endocrine signaling events that control skeletal bone remodeling and the maintenance of bone mass

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

The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton

Pyk2 deficiency enhances bone mass during midpalatal suture expansion

OBJECTIVE: To determine if Pyk2 deficiency increases midpalatal suture bone mass and preserves sutural integrity after maxillary expansion. SETTING AND SAMPLE: Thirty-six male Pyk2 knockout (KO) and control (WT) mice at 6 weeks of age. MATERIALS AND METHODS: Mice received nickel-titanium spring expanders delivering 0 g (no intervention control), 10 or 20 g force for 14 days. High-resolution micro-CT was used to determine bone volume/tissue volume (BV/TV), sutural width and intermolar width. Effects on osteoclasts, chondrocytes and suture morphology were determined by histomorphometry. RESULTS: Pyk2-KO controls (0 g) had 7% higher BV/TV compared with WT controls. Expanded Pyk2-KO maxillae also exhibited 12% (10 g) and 18% (20 g) higher BV/TV than WT mice. Although bone loss following expansion occurred in both genotypes, BV/TV was decreased to a greater extent in WT maxillae (-10% at 10g; -22% at 20 g) compared with Pyk2-KO maxillae (-11% only at 20 g). Expanded WT maxillae also showed a greater increase in sutural width, intermolar width and fibrous connective tissue width compared with expanded Pyk2-KO maxillae. Moreover, osteoclast number was increased 77% (10 g) and 132% (20 g) in expanded WT maxillae, but remained unchanged in expanded Pyk2-KO, compared to their respective controls. Cartilage area and chondrocyte number were increased to the same extent in expanded WT and Pyk2-KO sutures. CONCLUSIONS: These findings suggest that midpalatal suture expansion increases osteoclast formation in WT but not Pyk2-KO mice, leading to higher BV/TV in expanded Pyk2-KO maxillae. These studies suggest Pyk2-targeted strategies may be beneficial to increase bone density and preserve sutural integrity during maxillary expansion

Pyk2 deficiency potentiates osteoblast differentiation and mineralizing activity in response to estrogen or raloxifene

Bone remodeling is controlled by the actions of bone-degrading osteoclasts and bone-forming osteoblasts (OBs). Aging and loss of estrogen after menopause affects bone mass and quality. Estrogen therapy, including selective estrogen receptor modulators (SERMs), can prevent bone loss and increase bone mineral density in post-menopausal women. Although investigations of the effects of estrogen on osteoclast activity are well advanced, the mechanism of action of estrogen on OBs is still unclear. The proline-rich tyrosine kinase 2 (Pyk2) is important for bone formation and female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone mass, increased bone formation rate and reduced osteoclast activity. Therefore, in the current study, we examined the role of estrogen signaling on the mechanism of action of Pyk2 in OBs. As expected, Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In addition we found that Pyk2-KO OBs cultured in the presence of either 17β-estradiol (E2) or raloxifene, a SERM used for the treatment of post-menopausal osteoporosis, showed a further robust increase in alkaline phosphatase (ALP) activity and mineralization. We examined the possible mechanism of action and found that Pyk2 deletion promotes the proteasome-mediated degradation of estrogen receptor α (ERα), but not estrogen receptor β (ERβ). As a consequence, E2 signaling via ERβ was enhanced in Pyk2-KO OBs. In addition, we found that Pyk2 deletion and E2 stimulation had an additive effect on ERK phosphorylation, which is known to stimulate cell differentiation and survival. Our findings suggest that in the absence of Pyk2, estrogen exerts an osteogenic effect on OBs through altered ERα and ERβ signaling. Thus, targeting Pyk2, in combination with estrogen or raloxifene, may be a novel strategy for the prevention and/or treatment of bone loss diseases

Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms

Osteoblast differentiation and migration are necessary for bone formation during bone remodeling. Mice lacking the proline-rich tyrosine kinase Pyk2 (Pyk2-KO) have increased bone mass, in part due to increased osteoblast proliferation. Megakaryocytes (MKs), the platelet-producing cells, also promote osteoblast proliferation in vitro and bone-formation in vivo via a pathway that involves Pyk2. In the current study, we examined the mechanism of action of Pyk2, and the role of MKs, on osteoblast differentiation and migration. We found that Pyk2-KO osteoblasts express elevated alkaline phosphatase (ALP), type I collagen and osteocalcin mRNA levels as well as increased ALP activity, and mineralization, confirming that Pyk2 negatively regulates osteoblast function. Since Pyk2 Y402 phosphorylation is important for its catalytic activity and for its protein-scaffolding functions, we expressed the phosphorylation-mutant (Pyk2(Y402F) ) and kinase-mutant (Pyk2(K457A) ) in Pyk2-KO osteoblasts. Both Pyk2(Y402F) and Pyk2(K457A) reduced ALP activity, whereas only kinase-inactive Pyk2(K457A) inhibited Pyk2-KO osteoblast migration. Consistent with a role for Pyk2 on ALP activity, co-culture of MKs with osteoblasts led to a decrease in the level of phosphorylated Pyk2 (pY402) as well as a decrease in ALP activity. Although, Pyk2-KO osteoblasts exhibited increased migration compared to wild-type osteoblasts, Pyk2 expression was not required necessary for the ability of MKs to stimulate osteoblast migration. Together, these data suggest that osteoblast differentiation and migration are inversely regulated by MKs via distinct Pyk2-dependent and independent signaling pathways. Novel drugs that distinguish between the kinase-dependent or protein-scaffolding functions of Pyk2 may provide therapeutic specificity for the control of bone-related diseases

Lnk Deficiency Leads to TPO-Mediated Osteoclastogenesis and Increased Bone Mass Phenotype

The Lnk adapter protein negatively regulates the signaling of thrombopoietin (TPO), the main megakaryocyte (MK) growth factor. Lnk-deficient (-/-) mice have increased TPO signaling and increased MK number. Interestingly, several mouse models exist in which increased MK number leads to a high bone mass phenotype. Here we report the bone phenotype of these mice. MicroCT and static histomorphometric analyses at 20 weeks showed the distal femur of Lnk-/- mice to have significantly higher bone volume fraction and trabecular number compared to wild-type (WT) mice. Notably, despite a significant increase in the number of osteoclasts (OC), and decreased bone formation rate in Lnk-/- mice compared to WT mice, Lnk-/- mice demonstrated a 2.5-fold greater BV/TV suggesting impaired OC function in vivo. Additionally, Lnk-/- mouse femurs exhibited non-significant increases in mid-shaft cross-sectional area, yet increased periosteal BFR compared to WT femurs was observed. Lnk-/- femurs also had non-significant increases in polar moment of inertia and decreased cortical bone area and thickness, resulting in reduced bone stiffness, modulus, and strength compared to WT femurs. Of note, Lnk is expressed by OC lineage cells and when Lnk-/- OC progenitors are cultured in the presence of TPO, significantly more OC are observed than in WT cultures. Lnk is also expressed in osteoblast (OB) cells and in vitro reduced alkaline phosphatase activity was observed in Lnk-/- cultures. These data suggest that both direct effects on OB and OC as well as indirect effects of MK in regulating OB contributes to the observed high bone mass. J. Cell. Biochem. 118: 2231-2240, 2017