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Cell transfection with FAK mutated at Tyr-97 suppressed the strain-induced phosphorylation of ERK2 at Tyr-187. ROS 17/2.8 cells were transfected with the mutant FAK-Y397F or pcDNA3.1/lacZ control vector (pcDNA3 vector). Lysates were immunoprecipitated with an anti-HA mouse monoclonal antibody. Immunoprecipitates (IP) were analyzed by SDS-PAGE followed by the transfer of proteins and immunoblotting with an anti-FAK-Tyr(P)-397 rabbit phosphospecific antibody (Anti-FAK 397 IB) (A), or the lysates were immunoblotted with a rabbit polyclonal anti-ERK1/2 MAPK Tyr(P)-185/187 phosphospecific antibody (ERKY 185/187 IB) (B and C). Equal protein loading was routinely verified by stripping the blot and reblotting with an anti-ERK2 mouse monoclonal antibody (ERK (p42) IB).
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The mechanisms involved in the mechanical loading-induced increase in bone formation remain unclear. In this study, we showed that cyclic strain (CS) (10 min, 1% stretch at 0.25 Hz) stimulated the proliferation of overnight serum-starved ROS 17/2.8 osteoblast-like cells plated on type I collagen-coated silicone membranes. This increase was blocked...
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... Integrins are associated with FAK and PYK2, which are non-receptor tyrosine kinases at focal adhesions 8,19) . Notably, the activation of PYK2 induced by mechanical strain is regulated by intracellular Ca 2+ levels in osteoblasts 20) . ...
... Buckbinder et al. demonstrate that mouse bone marrow cultures isolated from PYK2 deficiency increase osteoblast differentiation, and PF431396 increases bone formation in ovariectomized rats 24) . However, in previous studies, mechanical strain demonstrates to an actuation of ERK2 through PYK2/FAK in osteoblast-like cells 20) . It induces chondrocyte proliferation and matrix synthesis through the integrin β1-calmodulin-dependent kinase II-Pyk2-ERK1/2 pathway 25) . ...
Low-intensity pulsed ultrasonic (LIPUS) is a noninvasive force promoting bone regeneration as mechanical stimulation. Integrins mediate numerous intracellular signaling pathways, including mitogen-activated protein kinase (MAPK) and calcium influx through focal adhesion. A non-receptor tyrosine kinase of proline-rich tyrosine kinase 2 (PYK2) associates with integrins at focal adhesions. A previous study reported that intracellular Ca²⁺ levels regulate the activation of PYK2 induced by mechanical strain in osteoblasts. However, the roles of PYK2 on the expression of extracellular matrix (ECM) proteins involved in osteogenesis and osteoblast differentiation induced by LIPUS remain unclear. We aimed to investigate the roles of PYK2 on the expression of transcription factors runt-related transcription factor 2 (Runx2) and osterix, which regulate osteoblast differentiation, ECM proteins, and the downstream intracellular signaling pathway, including PYK2 and focal adhesion kinase (FAK) in MC3T3-E1 cells when treated with LIPUS. LIPUS enhanced the phosphorylation of PYK2 and extracellular signal-regulated kinase2 (ERK2) and induced the expression of Runx2, osterix, type I collagen, osteocalcin, and calcium content in ECM. However, siPYK2 and PYK2 inhibitor PF431396 suppressed these stimulatory effects of LIPUS. These results suggest that LIPUS induces osteoblast differentiation mediated by the PYK2-ERK2 signaling in MC3T3-E1 cells.
... 24 FAK functions cooperatively with SFKs during mechanical signaling, and mechanical stimuli may increase the Src-FAK association. 25 The signaling of these tyrosine kinases might not be transduced by the same pathway under mechanical stimuli, but its underlying mechanisms require further investigation. Our study confirmed that MGF and hydraulic pressure may upregulate Fyn and FAK phosphorylation respectively in PDLSCs. ...
Clinical evidence shows that the mechanical stimulation obtained from occlusion could enhance periodontal ligament (PDL) remodeling. Mechano‐growth factor (MGF) is a growth factor produced specifically following mechanical stimulus Here, we aim to investigate the mechanical enhancement potential and mechanism of the MGF in PDL regeneration. In vivo study found that MGF produced from the PDL under occlusion force could strongly enhance PDL remodeling. In vitro experiments and mathematical modeling further confirmed the mechanical enhancement effect of MGF for PDLSC differentiation toward fibroblasts. A mechanochemical coupling effect of MGF mediated the enhancement of mechanical effect, which was modulated by Fyn‐FAK kinases signaling and subsequent MAPK pathway. Finally, enhanced PDL regeneration under the mechanochemical coupling of MGF and occlusal force was verified in vivo. There exists an additive mechanical effect of MGF mediated by Fyn‐FAK crosstalk and subsequent ERK1/2 and p38 phosphorylation, which could be developed as an MGF‐centered adjuvant treatment to optimize PDL remodeling, especially for patients with weakened bite force or destroyed periodontium.
... It is known that the interaction of integrins with the extracellular matrix is important for cell function and survival [27]. In addition, integrins also act on osteoblasts to promote osteoclast differentiation [28]. In the present study, two members of the integrin family were identified, ITGA1, ITGA8, among significantly DEGs in the NBW and IUGR groups. ...
Intrauterine growth restriction (IUGR) is a major problem associated with piglet growth performance. The incidence of IUGR is widespread in Rongchang pigs. The pituitary gland is important for regulating growth and metabolism, and research has identified genes associated with growth and development. The pituitary gland of newborn piglets with normal birth weight (NBW group, n = 3) and (IUGR group, n = 3) was collected for transcriptome analysis. A total of 323 differentially expression genes (DEGs) were identified (|log2(fold-change)| > 1 and q value < 0.05), of which 223 were upregulated and 100 were downregulated. Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that the DEGs were mainly related to the extracellular matrix, regulation of the multicellular organismal process, tissue development and angiogenesis, which participate in the growth and immune response in IUGR piglets. Moreover, 7 DEGs including IGF2, THBS1, ITGA1, ITGA8, EPSTI1, FOSB, and UCP2 were associated with growth and immune response. Furthermore, based on the interaction network analysis of the DEGs, two genes, IGF2 and THBS1, participated in cell proliferation, embryonic development and angiogenesis. IGF2 and THBS1 were also the main genes participating in the IUGR. This study identified the core genes involved in IUGR in piglets and provided a reference for exploring the effect of the pituitary gland on piglet growth.
... On experiencing fluid flow, osteocyte signaling generally involves release of nitric oxide (NO), Adenosine triphosphate (ATP), Ca 2þ , and activation of ERK1/2, which, in turn, regulates numerous bone remodeling pathways including receptor activator of nuclear factor kappa-B ligand (RANKL) expression, cell proliferation, matrix metalloproteinase-13 (MMP13) expression, and osteogenic differentiation of MSCs. [45][46][47][48] Key differences in osteocyte responses to oscillating and unidirectional flow have been reported. 18,[49][50][51][52][53][54] For example, unidirectional flow increases intracellular calcium through the release of intracellular stores and membrane channel activation, while no such activation of membrane channels has been reported in cells subjected to oscillatory flows. ...
Multifaceted changes in the mechanobiological environment of skeletal joints, at multiple length scales, are central to the development of diseases-like osteoarthritis (OA). Recent evidence demonstrates related mechanical alterations in both bone and cartilage tissues, with crosstalk between the tissues being an important factor in acute and chronic degenerative processes. However, recapitulating multicellular tissue systems in the laboratory to study the entire osteochondral unit remains challenging. Thus, the development of accurate and reproducible OA model systems and the selection of the most suitable model for individual experimental approaches are critical. This review first discusses recent progress in understanding mechanosensory processes in healthy and osteoarthritic joints. Subsequently, we review advancements in the development of in vitro and ex vivo model systems ranging from 2D monocultures through to joint organ-on-a-chip models. Use of these systems allows for the study of multiple cell types in controlled, reproducible, and dynamic environments, which can incorporate precisely controlled mechanical and biochemical stimuli, and biophysical cues. The way in which these models have, and will continue to, improve our ability to recapitulate complex mechanical/paracrine signaling pathways in osteochondral tissues is then discussed. As the accuracy of model systems advances, they will have a significant impact on both our understanding of the pathobiology of OA and in identifying and screening therapeutic targets to improve treatment of this complex disease.
... These forces are potentially affected by cyclic stretch stimulation. It has been reported that the exposure to cyclic stretch results in an increase in cell elongation and realignment of intracellular actin cytoskeletons nearly perpendicular to the direction of uniaxial cyclic stretch (Boutahar et al. 2004;Kaunas et al. 2005;Na et al. 2008). Actin stress fibers in the cyclically stretched cells reorient in the direction of zero normal strain to minimize the effect of the applied stress or strain (Takemasa et al. 1998;Nagayama et al. 2012). ...
Ultraviolet (UV) radiation exerts adverse effects on genome stability, alters the normal state of life, and causes several diseases by inducing DNA damage. Although mechanical stimulation such as stretching has significant effects on the prevention and treatment of diseases, its influence on nuclear morphology and/or intranuclear functions involving resistance to DNA damage remains unknown. Here, we investigated the effects of mechanical stimulation by cyclic stretching on nuclear morphology and resistance of DNA to UV damage in NIH3T3 fibroblasts. Adherent cells on silicone elastic membranes were subjected to ~ 10% cyclic uniaxial stretch at a frequency of 0.5 Hz for 12 h. As a result, the intracellular actin cytoskeleton and nucleus were found to be elongated and aligned in the direction of zero normal strain (~ 62° with respect to the stretch direction) in an actomyosin tension-dependent manner. The nuclei of the stretched cells were dramatically compressed by the reorganized actin stress fibers located on their apical and both sides, and a significant increase in the intranuclear DNA density was observed. Intercellular tension, as assessed with live cell atomic force microscopy imaging, also increased following exposure to cyclic stretch. The UV radiation-induced DNA damage, estimated from the fluorescence intensity of the phospho-histone γ-H2AX, significantly decreased in these stretched cells. These results indicate that the cyclic stretch-induced morphological changes in the nucleus may improve the UV radiation resistance of cells, probably owing to the intracellular force-induced condensation of chromatin. To our knowledge, this is the first study to demonstrate the inhibition of the UV radiation-induced DNA damage by mechanical stimulation.
... Actin stress bers re-orient in the direction of zero normal strain so as to minimize the stress or strain applied to them. These reorientation responses have been investigated in detail by many research groups using various types of cells including endothelial cells [5,6], smooth muscle cells [7], broblasts [8], and osteoblasts [9,10]. Thus, it is possible that directional cyclic stretch stimulation of cells may facilitate the wound healing process and establish ordered tissue formation. ...
Cells sense the mechanical properties of their surrounding environment and activate intracellular signaling pathways that play important roles in cell survival, proliferation, differentiation, and migration. Migration of cells into an injury site is crucial for repair after injury and requires cytoskeletal reorganization and remodeling of focal adhesions that connect the cytoskeleton to the extracellular matrix. Thus, it is possible that a directional cyclic stretch stimulation of cells may facilitate the wound healing process and establish ordered tissue formation. Here, we investigated the effects of directional cyclic uniaxial stretch on wound repair processes of monolayer epithelial-like cells that was scratch wounded. We controlled the direction of scratched wound in cell tissue to be i) perpendicular to the stretch direction (perpendicular stretch), ii) parallel to the direction of the zero normal strain in the substrate θ0 (～60º) (oblique stretch), and iii) parallel to stretch direction (parallel stretch). We found that cyclic stretching perpendicular to the scratched wound direction did not improve cell migration, whereas oblique stretching, by which cells were induced to align in the zero normal strain direction θ0, significantly facilitated cell migration for wound closure even though the migration direction was varied. We further found that cell migration for wound closure was improved most efficiently by cyclic stretching parallel to the wound direction, which facilitated polymerization of actin cytoskeleton aligning in the migration direction and vinculin–actin interactions. These results indicate that cell migration for wound healing is significantly influenced not only by the normal strain applied to cells but also by shear strain under cyclic strain fields, and cells for wound healing preferentially migrate to the direction in which both the normal and shear strains applied to them become smaller.
... Further light on the pathway of proliferation was gleaned in osteoblast-like cells where CS activated FAK, c-Src, and proline-rich tyrosine kinase 2 (PYK2). Although c-Src was not found to be necessary, FAK associated with PYK2 and led to ERK2 phosphorylation mediated by the Ras/Raf/MEK pathway (Boutahar et al., 2004). It is logical that the same pathway would function in pulmonary endothelia since 10% CS at 1 Hz activates ERK1/2 phosphorylation in bovine ECs, though the p21ras/PI3K pathway may also be involved (Ikeda et al., 1999). ...
Patients with critical illness such as acute lung injury often undergo mechanical ventilation in the intensive care unit. Though lifesaving in many instances, mechanical ventilation often results in ventilator induced lung injury (VILI), characterized by overdistension of lung tissue leading to release of edemagenic agents, which further damage the lung and contribute to the mortality and progression of pulmonary inflammation. The endothelium is particularly sensitive, as VILI associated mechanical stress results in endothelial cytoskeletal rearrangement, stress fiber formation, and integrity loss. At the heart of these changes are integrin tethered focal adhesions (FAs) which participate in mechanosensing, structure, and signaling. Here, we present the known roles of FA proteins including c-Src, talin, FAK, paxillin, vinculin, and integrins in the sensing and response to cyclic stretch and VILI associated stress. Attention is given to how stretch is propagated from the extracellular matrix through integrins to talin and other FA proteins, as well as signaling cascades that include FA proteins, leading to stress fiber formation and other cellular responses. This unifying picture of FAs aids our understanding in an effort to prevent and treat VILI.
... The proline-rich tyrosine kinase, Pyk2, belongs to the family of focal adhesion kinases, and regulates the proliferation, adhesion and migration of mesenchymal cells [12][13][14][15][16][17][18]. We and others have shown that Pyk2 genetic deletion leads to increased bone bass, through both increased bone formation by osteoblasts and decreased bone degradation by osteoclasts [16,19]. ...
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.
... Bone mass maintenance is regulated by various stimuli which can be grouped into biochemical (growth factors and hormones) and mechanical. Regardless of the type of mechanical stimulation (low-power ultrasound, fluid flow, centrifugation, static load, vibration, or electromagnetic field), it is recognized by the bone cells after a process called mechanotransduction that is responsible for producing biochemical reactions from a mechanical (physical) phenomenon, determining a cellular response, which may be bone growth or bone resorption (Duncan and Turner, 1995, Boutahar et al., 2004, Nomura and Takano-Yamamoto, 2000. Mechanotransduction is the process by which physical forces are converted into biochemical signals that are then integrated into cellular responses. ...
... Referring to substrate stretch stress, which was simulated by Flexcell, MAPK signaling pathways were found to be involved. Boutahar et al. [27] and Hatton et al. [28] found that mechanical stress could phosphorylate extracellular signal-regulated kinase (ERK 1/2) in a dose-dependent manner in osteoblasts without affecting the other two MAPK pathways-namely P38 and JNK. However, the study of Matsuda et al. [29] showed that ERK 1/2 and JNK were both phosphorylated when periodontal ligament cells were treated with mechanical stress. ...
Purpose:
To evaluate the effects of leptin/leptin receptor (LepR) combined with mechanical stress on the development of ossification of the posterior longitudinal ligament (OPLL), which is a disease characterized by ectopic bone formation of the posterior longitudinal ligament (PLL) and can lead to radiculopathy and myelopathy.
Methods:
Six human samples of the PLL were analyzed for the expression of leptin and LepR by RT-PCR and western blotting. PLL cells were stimulated with leptin and mechanical stress delivered via a Flexcell tension system, and osteogenic differentiation was evaluated by RT-PCR and western blotting analysis of osteogenic marker expression as well as by alkaline phosphatase (ALP) staining and alizarin red S staining. Activation of mitogen-activated protein kinase (MAPK), Janus kinase (JAK) 2-signal transducer, activator of transcription (STAT) 3 and phosphatidylinositol 3-kinase (PI3K)-Akt was evaluated by western blotting.
Results:
Samples from the OPLL group had higher LepR mRNA and protein levels and lower leptin levels than those from healthy controls. Exposure to leptin and Flexcell increased the number of ALP-positive cells and calcium nodules in a dose-dependent manner; this effect was accompanied by upregulation of the osteogenic markers osteocalcin, runt-related transcription factor 2 (RUNX2) and osteopontin. Extracellular signal-regulated kinase, P38 MAPK, JAK2, STAT3, PI3K and Akt signaling, was also activated by the combined effects of leptin and mechanical stress.
Conclusions:
Leptin and LepR are differentially expressed in OPLL tissues, and the combined use of leptin/LepR and mechanical stress promotes osteogenic differentiation of PLL cells via MAPK, JAK2-STAT3 and PI3K/Akt signaling. These slides can be retrieved under Electronic Supplementary Material.
