A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction.
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ABSTRACT:
SUBMITTER: Sato T
PROVIDER: S-EPMC7329900 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundMechanotransduction (MTD) is an important physiopathological signalling pathway associated with cardiovascular disease such as hypertension. Phosphorylation of focal adhesion kinase (FAK) is a MTD-sensing protein. This study tested the hypothesis that mTOR-FAK MTD signaling axis was crucial for focal adhesion (FA) maturation and cell proliferation.MethodsShock-wave was adopted as a tool for MTD and mTOR-FAK signaling.ResultsAfter demonstrating a failure in FAK phosphorylation after microfilament depolymerization, we attempted to identify the upstream regulator out of three kinases known to be activated in pressure-stimulated MTD [i.e., GSK-3β, Akt, and mTORC1 (mammalian target of rapamycin complex 1)]. Of the three specific inhibitors, only rapamycin, an inhibitor of mTORC1, was found to inhibit FAK phosphorylation, suggesting that mTORC1 is the upstream regulator in shock-wave-elicited FAK phosphorylation. Moreover, mTOR and its readout protein S6K were found to be activated by shock-wave stimulation. On the other hand, microscopic examination revealed not only MTD-induced increase in the number of actin stress fibers, but also alternative subcellular localization of mTORC1 as vesicle-like inclusions on microfilaments. Besides, rapamycin was found to destruct the granular pattern of mTORC1, while dissociation between F-actin and mTORC1 was noted after cytochalasin D administration. Since mTORC1 and FAK are essential for cell proliferation, we performed proliferation assay for mesenchymal stem cell (MSC) with and without shock-wave administration/rapamycin treatment/FAK depletion. The results demonstrated significant enhancement of cell proliferation after shock-wave stimulation but remarkable suppression after rapamycin and siFAK treatment.ConclusionOur findings suggest not only a co-ordinated regulation of FAK phosphorylation by mTORC1 and microfilaments, but also the participation of mTORC1-FAK signalling in MSC proliferation.
| S-EPMC5411911 | biostudies-literature
Project description:A paradox in bone tissue is that tissue-level strains due to animal and human locomotion are too small to initiate intracellular chemical responses directly. A model recently was proposed to resolve this paradox, which predicts that the fluid flow through the pericellular matrix in the lacunar-canalicular porosity due to mechanical loading can induce strains in the actin filament bundles of the cytoskeleton that are more than an order of magnitude larger than tissue level strains. In this study, we greatly refine this model by using the latest ultrastructural data for the cell process cytoskeleton, the tethering elements that attach the process to the canalicular wall and their finite flexural rigidity EI. We construct a much more realistic 3D model for the osteocyte process and then use large-deformation "elastica" theory for finite EI to predict the deformed shape of the tethering elements and the hoop strain on the central actin bundle. Our model predicts a cell process that is 3 times stiffer than in a previous study but hoop strain of >0.5% for tissue-level strains of >1,000 microstrain at 1 Hz and >250 microstrain at frequencies >10 Hz. We propose that this strain-amplification model provides a more likely hypothesis for the excitation of osteocytes than the previously proposed fluid-shear hypothesis.
| S-EPMC534548 | biostudies-literature
Project description:Osteocytes are accepted as the primary mechanosensing cell in bone, but how they translate mechanical signals into biochemical signals remains unclear. Adenylyl cyclases (AC) are enzymes that catalyze the production of second messenger cyclic adenosine monophosphate (cAMP). Osteocytes display a biphasic, cAMP response to fluid shear with an initial decrease in cAMP concentrations and then an increased concentration after sustained mechanical stimulation. To date, AC6, a calcium-inhibited AC, is the primary isoform studied in bone. Since osteocytes are calcium-responsive mechanosensors, we asked if a calcium-stimulated isoform contributes to mechanotransduction. Using a transcriptomic dataset of MLO-Y4 osteocyte-like cells from the NIH Gene Expression Omnibus, we identified AC3 as the only calcium-stimulated isoform expressed. We show that inhibiting AC3 in MLO-Y4 cells results in decreased cAMP-signaling with fluid shear and increased osteogenic response to fluid flow (measured as Ptgs2 expression) of longer durations, but not shorter. AC3 likely contributes to osteocyte mechanotransduction through a signaling axis involving the primary cilium and GSK3β. We demonstrate that AC3 localizes to the primary cilium, as well as throughout the cytosol and that fluid-flow regulation of primary cilia length is altered with an AC3 knockdown. Regulation of GSK3β is downstream of the primary cilium and cAMP signaling, and with western blots we found that GSK3β inhibition by phosphorylation is increased after fluid shear in AC3 knockdown groups. Our data show that AC3 contributes to osteocyte mechanotransduction and warrants further investigation to pave the way to identifying new therapeutic targets to treat bone disease like osteoporosis.
| S-EPMC8406666 | biostudies-literature
Project description:Focal adhesion kinase (FAK) and Src family kinases (SFK) are known to play critical roles in mechanotransduction and other crucial cell functions. Recent reports indicate that they reside in different microdomains of the plasma membrane. However, little is known about their subcellular domain-dependent roles and responses to extracellular stimuli. Here, we employed fluorescence resonance energy transfer (FRET)-based biosensors in conjunction with collagen-coupled agarose gels to detect subcellular activities of SFK and FAK in three-dimensional (3D) settings. We observed that SFK and FAK in the lipid rafts and nonrafts are differently regulated by fluid flow and pro-inflammatory cytokines. Inhibition of FAK in the lipid rafts blocked SFK response to fluid flow, while inhibition of SFK in the non-rafts blocked FAK activation by the cytokines. Ex-vivo FRET imaging of mouse cartilage explants showed that intermediate level of interstitial fluid flow selectively decreased cytokine-induced SFK/FAK activation. These findings suggest that SFK and FAK exert distinctive molecular hierarchy depending on their subcellular location and extracellular stimuli.
| S-EPMC5567257 | biostudies-literature
Project description:The mechanism by which extracellular cues influence intracellular biochemical cascades that guide axons is important, yet poorly understood. Because of the mechanical nature of axon extension, we explored whether the physical interactions of growth cones with their guidance cues might be involved. In the context of mouse spinal commissural neuron axon attraction to netrin-1, we found that mechanical attachment of netrin-1 to the substrate was required for axon outgrowth, growth cone expansion, axon attraction and phosphorylation of focal adhesion kinase (FAK) and Crk-associated substrate (CAS). Myosin II activity was necessary for traction forces >30 pN on netrin-1. Interestingly, while these myosin II-dependent forces on netrin-1 substrates or beads were needed to increase the kinase activity and phosphorylation of FAK, they were not necessary for netrin-1 to increase CAS phosphorylation. When FAK kinase activity was inhibited, the growth cone's ability to recruit additional adhesions and to generate forces >60 pN on netrin-1 was disrupted. Together, these findings demonstrate an important role for mechanotransduction during chemoattraction to netrin-1 and that mechanical activation of FAK reinforces interactions with netrin-1 allowing greater forces to be exerted.
| S-EPMC3461192 | biostudies-literature
Project description:Osteocytes secrete paracrine factors that regulate the balance between bone formation and destruction. Among these molecules, sclerostin (encoded by the gene SOST) inhibits osteoblastic bone formation and is an osteoporosis drug target. The molecular mechanisms underlying SOST expression remain largely unexplored. Here, we report that histone deacetylase 5 (HDAC5) negatively regulates sclerostin levels in osteocytes in vitro and in vivo. HDAC5 shRNA increases, whereas HDAC5 overexpression decreases SOST expression in the novel murine Ocy454 osteocytic cell line. HDAC5 knockout mice show increased levels of SOST mRNA, more sclerostin-positive osteocytes, decreased Wnt activity, low trabecular bone density, and reduced bone formation by osteoblasts. In osteocytes, HDAC5 binds and inhibits the function of MEF2C, a crucial transcription factor for SOST expression. Using chromatin immunoprecipitation, we have mapped endogenous MEF2C binding in the SOST gene to a distal intergenic enhancer 45 kB downstream from the transcription start site. HDAC5 deficiency increases SOST enhancer MEF2C chromatin association and H3K27 acetylation and decreases recruitment of corepressors NCoR and HDAC3. HDAC5 associates with and regulates the transcriptional activity of this enhancer, suggesting direct regulation of SOST gene expression by HDAC5 in osteocytes. Finally, increased sclerostin production achieved by HDAC5 shRNA is abrogated by simultaneous knockdown of MEF2C, indicating that MEF2C is a major target of HDAC5 in osteocytes.
| S-EPMC4342334 | biostudies-literature
Project description:Mechanical force is a fundamental regulator of bone development and homeostasis. Mechanosensitive osteocytes are the most abundant bone cells that form interconnected dendrites to respond to mechanical stimuli and interact with the bone-forming osteoblasts and the bone-remodeling osteoclasts. However, the molecular mechanisms underlying osteocyte maturation and dendrite formation remain unclear. By generating a Piezo1 "knock-out" osteocyte cell line, we identified a key role of Piezo1-mediated mechanotransduction in osteocyte differentiation. QRT-PCR analysis revealed delayed osteocyte differentiation in the Piezo1 KO cells relative to WT cells. By performing bulk RNA sequencing of WT and Piezo1 KO OCY454 cells at early (D1), intermediate (D14), and late (D28) stages of differentiation allowed for the identification of key signaling pathways in driving normal osteocyte dfiferentiation, as well as those regulated by Piezo1 mechanotransduction.
2024-09-20 | GSE277264 | GEO
Project description:BackgroundThe key focal adhesion protein β1 integrin plays an essential role in early skeletal development. However, roles of β1 integrin expression in osteocytes during the regulation of bone homeostasis and mechanotransduction are incompletely understood.Materials and methodsTo study the in vivo function of osteocyte β1 integrin in bone, we utilized the 10-kb Dmp1 (Dentin matrix acidic phosphoprotein 1)-Cre to generate mice with β1 integrin deletion in this cell type. Micro-computerized tomography, bone histomorphometry and immunohistochemistry were performed to determine the effects of osteocyte β1 integrin loss on bone mass accrual and biomechanical properties. In vivo tibial loading model was applied to study the possible involvement of osteocyte β1 integrin in bone mechanotransduction.ResultsLoss of β1 integrin expression in osteocytes resulted in a severe low bone mass and impaired biomechanical properties in load-bearing long bones and spines, but not in non-weight-bearing calvariae, in mice. The loss of β1 integrin led to enlarged size of lacunar-canalicular system, abnormal cell morphology, and disorientated nuclei in osteocytes. Furthermore, β1 integrin loss caused shortening and disorientated collagen I fibers in long bones. Osteocyte β1 integrin loss did not impact the osteoclast activities, but significantly reduced the osteoblast bone formation rate and, in the meantime, enhanced the adipogenic differentiation of the bone marrow stromal cells in the bone microenvironment. In addition, tibial loading failed to accelerate the anabolic bone formation and improve collagen I fiber integrity in mutant mice.ConclusionsOur studies demonstrate an essential role of osteocyte β1 integrin in regulating bone homeostasis and mechanotransduction. The transnational potential of this article : This study reveals the regulatory roles of osteocyte β1 integrin in vivo for the maintenance of bone mass accrual, biomechanical properties, extracellular matrix integrity as well as bone mechanobiology, which defines β1 integrin a potential therapeutic target for skeletal diseases, such as osteoporosis.
| S-EPMC9119859 | biostudies-literature
Project description:Adipose tissues dynamically remodel their cellular composition in response to external cues by stimulating beige adipocyte biogenesis; however, the developmental origin and pathways regulating this process remain insufficiently understood owing to adipose tissue heterogeneity. Here, we employed single-cell RNA-seq and identified a unique subset of adipocyte progenitor cells (APCs) that possessed the cell-intrinsic plasticity to give rise to beige fat. This beige APC population is proliferative and marked by cell-surface proteins, including PDGFRα, Sca1, and CD81. Notably, CD81 is not only a beige APC marker but also required for de novo beige fat biogenesis following cold exposure. CD81 forms a complex with αV/β1 and αV/β5 integrins and mediates the activation of integrin-FAK signaling in response to irisin. Importantly, CD81 loss causes diet-induced obesity, insulin resistance, and adipose tissue inflammation. These results suggest that CD81 functions as a key sensor of external inputs and controls beige APC proliferation and whole-body energy homeostasis.
| S-EPMC7415677 | biostudies-literature
Project description:IntroductionThe implantation of biomaterials into soft tissue leads to the development of foreign body response, a non-specific inflammatory condition that is characterized by the presence of fibrotic tissue. Epithelial-mesenchymal transition (EMT) is a key event in development, fibrosis, and oncogenesis. Emerging data support a role for both a mechanical signal and a biochemical signal in EMT. We hypothesized that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive channel, is a mediator of EMT.MethodsNormal human primary epidermal keratinocytes (NHEKs) were seeded on collagen-coated plastic plates or varied stiffness polyacrylamide gels in the presence or absence of TGFβ1, Immunofluorescence, immunoblot, and polymerase chain reaction analysis were performed to determine expression level of EMT markers and signaling proteins. Knock-down of TRPV4 function was achieved by siRNA transfection or by GSK2193874 treatment.ResultsWe found that knock-down of TRPV4 blocked both matrix stiffness- and TGFβ1-induced EMT in NHEKs. In a murine skin fibrosis model, TRPV4 deletion resulted in decreased expression of the mesenchymal marker, α-SMA, and increased expression of epithelial marker, E-cadherin. Mechanistically, our data showed that: i) TRPV4 was essential for the nuclear translocation of TAZ in response to matrix stiffness and TGFβ1; ii) Antagonism of TRPV4 inhibited both matrix stiffness-induced and TGFβ1-induced expression of TAZ proteins; and iii) TRPV4 antagonism suppressed both matrix stiffness-induced and TGFβ1-induced activation of Smad2/3, but not of AKT.ConclusionsThese data identify a novel role for TRPV4-TAZ mechanotransduction signaling axis in regulating EMT in NHEKs in response to both matrix stiffness and TGFβ1.
| S-EPMC6816769 | biostudies-literature