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The ideal implant surface plays a substantial role in maintaining bone homeostasis by simultaneously promoting osteoblast differentiation and limiting overactive osteoclast activity to a certain extent, which leads to satisfactory dynamic osseointegration. However, the rational search for implant materials with an ideal surface structure is challenging and a hot research topic in the field of tissue engineering. In this study, we constructed titanium dioxide titanium nanotubes (TNTs) by anodic oxidation and found that this structure significantly promoted osteoblast differentiation and inhibited osteoclast formation and function while simultaneously inhibiting the total protein levels of proline-rich tyrosine kinase 2 (PYK2) and focal adhesion kinase (FAK). Knockdown of the PYK2 gene by siRNA significantly suppressed the number and osteoclastic differentiation activity of mouse bone marrow mononuclear cells (BMMs), while overexpression of PYK2 inhibited osteogenesis and increased osteoclastic activity. Surprisingly, we found for the first time that neither knockdown nor overexpression of the FAK gene alone caused changes in osteogenesis or osteoclastic function. More importantly, compared with deletion or overexpression of PYK2/FAK alone, coexpression or cosilencing of the two kinases accelerated the effects of TNTs on osteoclastic and osteogenic differentiation on the surface of cells. Furthermore, in vivo experiments revealed a significant increase in positiveexpression-PYK2 cells on the surface of TNTs, but no significant change in positiveexpression -FAK cells was observed. In summary, PYK2 is a key effector molecule by which osteoblasts sense nanotopological mechanical signals and maintain bone homeostasis around implants. These results provide a referable molecular mechanism for the future development and design of homeostasis-based regulatory implant biomaterials.

Neutrophils are first responders in infection and inflammation. They are able to roll, adhere and transmigrate through the endothelium to reach the site of infection, where they fight pathogens through secretion of granule contents, production of reactive oxygen species, extrusion of neutrophil extracellular traps, and phagocytosis. In this study we explored the role of the non-receptor focal adhesion kinase Pyk2 in neutrophil adhesion and activation. Using a specific Pyk2 pharmacological inhibitor, PF-4594755, as well as Pyk2-deficient murine neutrophils, we found that Pyk2 is activated upon integrin αMβ2-mediated neutrophil adhesion to fibrinogen. This process is triggered by Src family kinases-mediated phosphorylation and supported by Pyk2 autophosphorylation on Y402. In neutrophil adherent to fibrinogen, Pyk2 activates PI3K-dependent pathways promoting the phosphorylation of Akt and of its downstream effector GSK3. Pyk2 also dynamically regulates MAP kinases in fibrinogen-adherent neutrophils, as it stimulates p38MAPK but negatively regulates ERK1/2. Pharmacological inhibition of Pyk2 significantly prevented adhesion of human neutrophils to fibrinogen, and neutrophils from Pyk2-knockout mice showed a reduced ability to adhere compared to wildtype cells. Accordingly, neutrophil adhesion to fibrinogen was reduced upon inhibition of p38MAPK but potentiated by ERK1/2 inhibition. Neutrophil adherent to fibrinogen, but not to polylysine, were able to produce ROS upon lipopolysaccharide challenge and ROS production was completely suppressed upon inhibition of Pyk2. By contrast PMA-induced ROS production by neutrophil adherent to either fibrinogen or polylysine was independent from Pyk2. Altogether these results demonstrate that Pyk2 is an important effector in the coordinated puzzle regulating neutrophil adhesion and activation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Rho/Rho-kinase downstream of G 12/13 plays an important role in the regulation of calcium-independent platelet shape change. We have previously shown that proline-rich tyrosine kinase 2 (Pyk2) is activated downstream of G 12/13 pathways. In this study, we evaluated the role of Pyk2 in G 12/13 -induced platelet shape change. We used low concentrations of YFLLRNP, a heptapeptide binding to protease-activated receptor 1 (PAR1), or PAR4-activating peptide AYPGKF in the presence of Gα q inhibitor YM254890 to selectively stimulate G 12/13 pathways. We found that G 12/13 -induced platelet shape change was completely inhibited in the presence of Pyk2 inhibitors AG17 and TAT-Pyk2-CT, suggesting an important role of Pyk2 in platelet shape change. In addition, AYPGKF-induced shape change in G q  -/- platelets was completely inhibited in the presence of AG17 or RhoA/p160 ROCK inhibitor Y27632, confirming the role of Pyk2 in RhoA-dependent shape change. Furthermore, AYPGKF-induced platelet aggregation and dense granule secretion were inhibited by blocking Pyk2 or RhoA. Finally, G 12/13 -induced myosin phosphatase target subunit 1 (MYPT1) phosphorylation was inhibited by AG17, confirming that Pyk2 regulates RhoA/p160 ROCK activation in platelets. These results demonstrate that Pyk2 downstream of G 12/13 pathways regulates platelet shape change as well as platelet aggregation and dense granule secretion through the regulation of RhoA/p160 ROCK ., Competing Interests: Declaration of competing interest Authors have declared no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mitochondrial dysfunction caused by Ca 2+ overload plays an important role in ischemia-induced brain damage. Mitochondrial calcium uniporter (MCU), located on the mitochondrial inner membrane, is the major channel responsible for mitochondrial Ca 2+ uptake. Activated proline-rich tyrosine kinase 2 (Pyk2) can directly phosphorylate MCU, which enhances mitochondrial Ca 2+ uptake in cardiomyocytes. It has been suggested that the Pyk2/MCU pathway may be a novel therapeutic target in stress-induced cellular apoptosis. In this study, we explored the role of the Pyk2/MCU pathway in the ischemic brain following a stroke injury. We found that the Pyk2/MCU pathway is activated in a rat cerebral ischemia model, and is responsible for mitochondrial dysfunction and neuronal apoptosis. Inhibiting the Pyk2/MCU pathway with a Pyk2 inhibitor (PF-431396) prevented mitochondrial Ca 2+ overload, mitochondrial injury, proapoptotic protein release, and cell death. Interestingly, human urinary kallidinogenase (HUK) alleviated neuronal ischemic injury by inhibiting the Pyk2/MCU pathway, suggesting that the Pyk2/MCU pathway may be a protective target for ischemic stroke treatment., (Copyright © 2017. Published by Elsevier B.V.)

Mechanical stimuli such as fluid shear and cyclic tension force induced extracellular adenosine triphosphate (ATP) release in osteoblasts. In particular, cyclic tension force-induced ATP enhances bone formation through P2X7 activation. Proline-rich tyrosine kinase 2 (PYK2) mediate osteoblasts differentiation is induced by mechanical stimuli. Furthermore, activation of PYK2 also was a response to integrin by mechanical stimuli. Extracellular matrix protein (ECMP)s, which are important factors for bone formation are expressed by osteoblasts. However, the effect of the interaction of 2'(3)-Ο-(4-Benzoylbenzoyl) adenosine-5'-triphosphate (BzATP), which is the agonist of the mechanosensitive receptor P2X7, with PYK2 on ECMP production is poorly understood. Thus, our purpose was to investigate the effects of PYK2 on BzATP-induced ECMP production in osteoblasts. BzATP increased phospho-PYK2 protein expression on days 3 and 7 of culture. Furthermore, the PYK2 inhibitor PF431394 inhibited the stimulatory effect of BzATP on the expression of type I collagen, bone sialoprotein and osteocalcin expression. PF431396 did not inhibit the stimulatory effect of BzATP on osteopontin (OPN) mRNA expression. These results suggest that mechanical stimuli activate P2X7 might induce ECMPs expression through PYK2 except in the case of OPN expression. Altogether, mechanical stimuli-induced ECMPs production might be implicated by extracellular ATP secretion or integrin via PYK2 activation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Wnt/β-catenin and Wnt/Ca 2+ pathways are involved in cellular processes during embryonic development and the interaction between them in the same cell decides the outcome of cellular functions. In this study, we showed that Wnt3a triggers the Wnt/Ca 2+ signaling pathway, indicated by an increase of cytosolic free calcium ([Ca 2+ ] i ) and activation of calmodulin dependent kinase II (CaMKII) during the differentiation of human neuronal progenitor cells (hNPCs). Wnt3a via the increase of [Ca 2+ ] i activates proline-rich tyrosine kinase 2 (Pyk2), which subsequently regulates phosphorylation of glycogen synthase kinase 3β (GSK3β) and β-catenin stabilization. Our findings suggest that Pyk2 plays an important role in the coordination of stabilization of β-catenin in the crosstalk between Wnt/β-catenin and Wnt/Ca 2+ signaling pathways upon Wnt3a stimulation in differentiating hNPCs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Fertilization is a multi-step process that begins with plasma membrane interactions that enable sperm - oocyte binding followed by fusion of the sperm and oocyte plasma membranes. Once membrane fusion has occurred, sperm incorporation involves actin remodeling events within the oocyte cortex that allow the sperm head to penetrate the cortical actin layer and gain access to the ooplasm. Despite the significance for reproduction, the control mechanisms involved in gamete binding, fusion, and sperm incorporation are poorly understood. While it is known that proline - rich tyrosine kinase 2 (PYK2 or PTK2b) kinase activity plays an important role in fertilization, its specific function has not been addressed. The present study made use of a zona-free mouse oocyte fertilization assay to investigate the relationship between PYK2 activity and sperm - oocyte binding and fusion, as well as localized changes in actin polymerization and sperm incorporation. In this assay, the majority of bound sperm had no apparent effect on the oocyte and only a few became incorporated into the ooplasm. However, a subset of bound sperm were associated with a localized response in which PYK2 was recruited to the oocyte cortex where it frequently co-localized with a ring or disk of f-actin. The frequency of sperm-oocyte binding sites that exhibited this actin response was reduced in pyk2 -/- oocytes and the pyk2 -/- oocytes proved less efficient at incorporating sperm, indicating that this protein kinase may have an important role in sperm incorporation. The response of PYK2 to sperm-oocyte interaction appeared unrelated to gamete fusion since PYK2 was recruited to sperm - binding sites under conditions where sperm - oocyte fusion was prevented and since PYK2 suppression or ablation did not prevent sperm - oocyte fusion. While a direct correlation between the PYK2 response in the oocyte and the successful incorporation of individual bound sperm remains to be established, these findings suggest a model in which the oocyte is not a passive participant in fertilization, but instead responds to sperm contact by localized PYK2 signaling that promotes actin remodeling events required to physically incorporate the sperm head into the ooplasm., (Copyright © 2017 Elsevier Inc. All rights reserved.)

STAP-2 is an adaptor molecule regulating several signaling pathways, including TLRs and cytokine/chemokine receptors in immune cells. We previously reported that STAP-2 enhances SDF-1α-induced Vav1/Rac1-mediated T-cell chemotaxis. However, the detailed mechanisms of STAP-2 involvement in enhancing T-cell chemotaxis remain unknown. In the present study, we demonstrate that STAP-2 directly interacts with Pyk2, which is a key molecule in the regulation of SDF-1α/CXCR4-mediated T-cell chemotaxis, and increases phosphorylation of Pyk2. Pyk2 itself can induce STAP-2 Y250 phosphorylation, and this phosphorylation is critical for maximal interactions between STAP-2 and Pyk2. Finally, SDF-1α-induced T-cell chemotaxis is inhibited by treatment with Pyk2 siRNA or AG17, an inhibitor of Pyk2, in Jurkat cells overexpressing STAP-2. Taken together, the Pyk2/STAP-2 interaction is a novel mechanism to regulate SDF-1α-dependent T-cell chemotaxis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tetraspanin family proteins regulate morphology, motility, fusion, and signaling in various cell types. We investigated the role of the tetraspanin 7 (Tspan7) isoform in the differentiation and function of osteoclasts. Tspan7 was up-regulated during osteoclastogenesis. When Tspan7 expression was reduced in primary precursor cells by siRNA-mediated gene knock-down, the generation of multinuclear osteoclasts was not affected. However, a striking cytoskeletal abnormality was observed: the formation of the podosome belt structure was inhibited and the microtubular network were disrupted by Tspan7 knock-down. Decreases in acetylated microtubules and levels of phosphorylated Src and Pyk2 in Tspan7 knock-down cells supported the involvement of Tspan7 in cytoskeletal rearrangement signaling in osteoclasts. This cytoskeletal defect interfered with sealing zone formation and subsequently the bone-resorbing activity of mature osteoclasts on dentin surfaces. Our results suggest that Tspan7 plays an important role in cytoskeletal organization required for the bone-resorbing function of osteoclasts by regulating signaling to Src, Pyk2, and microtubules., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Historically, great efforts have been made to elucidate the biochemical pathways that direct the complex process of wound healing; however only recently has there been recognition of the importance that mechanical signals play in the process of tissue repair and scar formation. The body's physiologic response to injury involves a dynamic interplay between mechanical forces and biochemical cues which directs a cascade of signals leading ultimately to the formation of fibrotic scar. Fibroblasts are a highly mechanosensitive cell type and are also largely responsible for the generation of the fibrotic matrix during scar formation and are thus a critical player in the process of mechanotransduction during tissue repair. Mechanotransduction is initiated at the interface between the cell membrane and the extracellular matrix where mechanical signals are first translated into a biochemical response. Focal adhesions are dynamic multi-protein complexes through which the extracellular matrix links to the intracellular cytoskeleton. These focal adhesion complexes play an integral role in the propagation of this initial mechanical cue into an extensive network of biochemical signals leading to widespread downstream effects including the influx of inflammatory cells, stimulation of angiogenesis, keratinocyte migration, fibroblast proliferation and collagen synthesis. Increasing evidence has demonstrated the importance of the biomechanical milieu in healing wounds and suggests that an integrated approach to the discovery of targets to decrease scar formation may prove more clinically efficacious than previous purely biochemical strategies., (Copyright © 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)