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

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

Enhancement of osteoblast activity on nanostructured NiTi/hydroxyapatite coatings on additive manufactured NiTi metal implants by nanosecond pulsed laser sintering

Background: The osteoinductive behaviors of nitinol (NiTi)-based metal implants for bone regeneration are largely dependent on their surface composition and topology. Continuous-mode laser sintering often results in complete melting of the materials and aggregation of particles, which lack control of heat transfer, as well as microstructural changes during sintering of the nanocomposite materials. Methods: In the current study, in situ direct laser deposition was used to additively manufacture three-dimensional NiTi structures from Ni and Ti powders. The mechanical property of NiTi has been shown to be similar to bone. Nanosecond pulsed laser sintering process was then utilized to generate a nanoporous composite surface with NiTi alloy and hydroxyapatite (HA) by ultrafast laser heating and cooling of Ni, Ti, and HA nanoparticles mixtures precoated on the 3D NiTi substrates; HA was added in order to improve the biocompatibility of the alloy. We then studied the underlying mechanism in the formation of NiTi/HA nanocomposite, and the synergistic effect of the sintered HA component and the nanoporous topology of the composite coating. In addition, we examined the activity of bone-forming osteoblasts on the NiTi/HA surfaces. For this, osteoblast cell morphology and various biomarkers were examined to evaluate cellular activity and function. Results: We found that the nanoscale porosity delivered by nanosecond pulsed laser sintering and the HA component positively contributed to osteoblast differentiation, as indicated by an increase in the expression of collagen and alkaline phosphatase, both of which are necessary for osteoblast mineralization. In addition, we observed topological complexities which appeared to boost the activity of osteoblasts, including an increase in actin cytoskeletal structures and adhesion structures. Conclusion: These findings demonstrate that the pulsed laser sintering method is an effective tool to generate biocompatible coatings in complex alloy-composite material systems with desired composition and topology. Our findings also provide a better understanding of the osteoinductive behavior of the sintered nanocomposite coatings for use in orthopedic and bone regeneration applications

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

A Pyk2 Inhibitor Incorporated into a PEGDA-Gelatin Hydrogel Promotes Osteoblast Activity and Mineral Deposition

Pyk2 is a non-receptor tyrosine kinase that belongs to the family of focal adhesion kinases. Studies from our laboratory and others demonstrated that mice lacking the Pyk2 gene (Ptk2B) have high bone mass, which was due to increased osteoblast activity, as well as decreased osteoclast activity. It was previously reported that a chemical inhibitor that targets both Pyk2 and its homolog FAK, led to increased bone formation in ovariectomized rats. In the current study, we developed a hydrogel containing poly(ethylene glycol) diacrylate (PEGDA) and gelatin which was curable by visible-light and was suitable for the delivery of small molecules, including a Pyk2-targeted chemical inhibitor. We characterized several critical properties of the hydrogel, including viscosity, gelation time, swelling, degradation, and drug release behavior. We found that a hydrogel composed of PEGDA1000 plus 10% gelatin (P1000:G10) exhibited Bingham fluid behavior that can resist free flowing before in situ polymerization, making it suitable for use as an injectable carrier in open wound applications. The P1000:G10 hydrogel was cytocompatible and displayed a more delayed drug release behavior than other hydrogels we tested. Importantly, the Pyk2-inhibitor-hydrogel retained its inhibitory activity against the Pyk2 tyrosine kinase, and promoted osteoblast activity and mineral deposition in vitro. Overall, our findings suggest that a Pyk2-inhibitor based hydrogel may be suitable for the treatment of craniofacial and appendicular skeletal defects and targeted bone regeneration

The Effect of the Kalirin Gene on Osteoid Development

Lrp5 Gene Over-expression Leads to Decreased Tooth Movement. R. Holland*, C. Bain, A. Robling, A. Utreja. (Indiana University School of Dentistry) Lrp5 (Low-density lipoprotein receptor-related protein 5) is a co-receptor of the Wnt intracellular signaling system. The Wnt system is known to play an important role in bone biology. Increased activity of the Wnt system leads to an increase in bone mass. The aim of this study was to compare tooth movement in genetically modified Lrp5 knock-in mice to control mice. Thirty-six C57BL/6 wildtype mice were utilized. Eighteen possessed a A214V mutation and eighteen possessed a G171V mutation in the Lrp5 gene. Eighteen wild type C57BL/6 mice served as control. Experimental tooth movement was produced by attaching a 3mm closed-coil nickel-titanium spring from the first molar to the incisors. Tooth movement occurred for three weeks prior to sacrifice. For all but six mice, a microchromotagraphy scan was taken after sacrifice followed by a histologic analysis. The remaining six mice were Mice were injected with Calcin, Alizeren and Tetracycline at eight, four and two days pre-sacrifice respectively. These mice were processed in plastic and analyzed with fluorescent microscopy. Genetic knock-in mice showed higher percent bone volume (BV/TV), lower bone surface/volume ratio (BS/BV) and increased trabecular thickness (Tb.Th) compared to the control group. Cellular expression of the Sclerostin gene was higher per unit area in the genetic knock-in mice compared to the control mice. Fluorescent microscopy showed decrease in bone turnover in the genetic knock-in mice compared to the control. This study demonstrates that overexpression of the Lrp5 gene leads to an increased bone density, decreased bone turnover and a decreased rate of tooth movement in mice. Further study should be completed on the Wnt signaling pathway as this could lead to improvement of future orthodontic treatments

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

ROLE OF OSTEOCLASTS IN THE BIOCORROSION OF METAL IMPLANTS

poster abstractMini implants (MIs), typically composed of stainless steel (SS) or titanium alloy (Ti), have recently emerged as superior alternatives to traditional dental and orthopedic implants. When a metal implant is inserted into bone, a process called bone remodeling is triggered near the implant. Bone remodeling involves the activity of osteoblasts (OBs), which produce new bone tissue, and osteoclasts (OCs), which degrade and digest bone. OCs degrade bone by acidifying the extracellular environment and secreting hydrolytic enzymes that degrade the extracellular matrix. However, the acidification of the extracellular environment can potentially lead to the biological corrosion of metal implants after implantation. This may have important consequences such as cell toxicity, decreased osseointegration of the implant, and implant loosening. The objective of this study is to determine if implants made from Ti are more resistant to OC-mediated biocorrosion than stainless steel (SS) implants. We hypothesize that biocorrosive activity by OCs will be greater on SS than titanium. To assess the biocorrosive effects of OCs on SS and Ti, the top face of 150 µm thick sections of each metal were scanned using a Proscan 2000 Scantron to provide accurate three dimensional surface measurements of the metals before introduction of OCs. OC precursors were isolated from the bone marrow of C57/bl6 mice and differentiated with macrophage colony stimulating factor and receptor activator of NF-kappaB ligand for 7 days in the presence of either SS or Ti metals. The metals discs were then removed and rescanned with the Proscan Scantron and changes in the surface measurements before and after OC growth was calculated. OCs were fixed and stained for tartrate-resistant acid phosphatase, a marker of mature OCs, and counted. Our preliminary findings revealed that the surface roughness of SS was reduced to a greater extent than Ti metals. OC number was also reduced in cultures containing SS compared with Ti. These findings suggest SS may be more susceptible to OC-mediated biocorrosion than Ti-based metal implants. Although the physiological implications are unclear, we speculate that sustained corrosion of SS can negatively affect the long-term stability of implants in vivo

Morphological and Structural Evaluation of Hydration/Dehydration Stages of MgSO4 Filled Composite Silicone Foam for Thermal Energy Storage Applications

Salt hydrates, such as MgSO4·7H2O, are considered attractive materials for thermal energy storage, thanks to their high theoretical storage density. However, pure salt hydrates present some challenges in real application due to agglomeration, corrosion and swelling problems during hydration/dehydration cycles. In order to overcome these limitations, a composite material based on silicone vapor-permeable foam filled with the salt hydrate is here presented. For its characterization, a real-time in situ environmental scanning electron microscopy (ESEM) investigation was carried out in controlled temperature and humidity conditions. The specific set-up was proposed as an innovative method in order to evaluate the morphological evolution of the composite material during the hydrating and dehydrating stages of the salt. The results evidenced an effective micro-thermal stability of the material. Furthermore, dehydration thermogravimetric/differential scanning calorimetric (TG/DSC) analysis confirmed the improved reactivity of the realized composite foam compared to pure MgSO4·7H2O.This work was partially funded by the Ministerio de Ciencia, Innovación y Universidades de España (RTI2018-093849-B-C31). This work was partially supported by ICREA under the ICREA Academia program

Kalirin Decreases Bone Mass Through Effects in Both Osteoclasts and Osteoblasts

poster abstractBone homeostasis is maintained by the balance between osteoclasts which degrade bone and osteoblasts, which form new bone. When the activity of either of these cells is dysregulated, bone loss can ensue, leading to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. The activity of osteoclasts and osteoblasts is regulated by local and systemic factors, as well as by key signaling proteins expressed in these cells. Kalirin is a novel GTP-exchange factor protein that plays a role in signaling pathways leading to cytoskeletal remodeling and dendritic spine formation in neurons, but its function in other cells is unknown. Western blotting and real time PCR confirmed that Kalirin is expressed in osteoclasts and osteoblasts, suggesting it may play a role in regulating bone cell function and bone mass. We used micro-CT to examine the bone phenotype of 14 week old female mice lacking Kalirin in all tissues (Kal-KO). Kal-KO mice exhibited a 40% lower trabecular bone volume in the distal femur compared to wild-type (WT) mice (n=9/group, p<0.05). We next quantified osteoclasts in histological sections by counting multinucleated cells expressing tartrate-resistant acid phosphatase (TRAP), a marker of mature osteoclasts. We found 48% higher osteoclast surface/bone surface in trabecular bone of Kal-KO mice, compared to WT mice (n=6/group, p<0.05). Osteoclast differentiation is controlled by osteoblasts, which secrete receptor activator of NF-kB ligand (RANKL), macrophage colony stimulating factor (MCSF) and osteoprotegerin (OPG), a decoy receptor for RANKL. We examined if Kalirin could regulate osteoclast differentiation in vitro. Osteoclasts were generated from the bone marrow of WT or Kal-KO mice by incubation with RANKL and MCSF for 7 days, and TRAP+ multinucleated cells were counted. Consistent with our in vivo studies, osteoclast number was significantly higher in cultures from Kal-KO mice, compared to WT mice. We next examined if Kalirin altered the ratio of secreted RANKL and OPG secreted by osteoblasts. Osteoblasts were generated from the calvaria of 2 day old neonates and the level of secreted RANKL and OPG in conditioned media was quantified by ELISA. Consistent with increased osteoclast differentiation, we found a higher RANKL/OPG ratio in conditioned media from Kal-KO osteoblasts, compared to WT cells. These data confirm a role for Kalirin in the regulation of trabecular bone mass through effects in both osteoclasts and osteoblasts