Mechanosensitive Ion Channel Piezo1 Research Articles

Mechanosensitive Ion Channel Piezo1 regulates tight junctions between endothelium and mediates the occurrence of aortic dissection

Abstract Aims Exploring whether changes in endothelial Piezo1 mechanically sensitive ion channels can lead to thoracic aortic dissection（TAD） and its specific mechanisms of action. Methods and results IF showed aberrant Piezo1 expressions in the CD31+ endothelial cells in thoracic aortas of patients with TAD. In a β-aminopropionitrile (BAPN)-induced TAD mouse model, knockout of Piezo1 in endothelial cells shows a protective effect on aortic dissection, while the crucial tight junction (TJ) components ZO-1 was significantly increased by En Face staining. For identified how the Piezo1 ion channel influences the ZO-1 between endothelium, we used mass spectrometry analysis and RNA sequencing to identify a key factor Trib1 mediating the ubiquitination degradation of ZO-1. Also, we found the low expression of Trib1 in endothelial cell of Piezo1 ECKO mouse with BAPN feeding. Finally, we rescued the expression of TRIB1 by targeting endothelial cells with AAV9-TIE2-TRIB1, demonstrating that Piezo1 indeed promotes the occurrence of aortic dissection through degrading the TRIB1-COP1-ZO-1 complex. Conclusion The activation of Piezo1 promotes the occurrence of （TAD） through Trib1 mediated degradation of endothelial tight junctions. Inhibiting endothelial Piezo has the potential to become a specific therapeutic and preventive target for（TAD）.

Piezo1 expression in mature osteocytes is dispensable for the skeletal response to mechanical loading

Mechanical loading is essential for bone growth and homeostasis, with osteocytes considered the primary mechanosensors. Deleting the mechanosensitive ion channel Piezo1 from Dmp1-Cre-targeted cells, which include both osteoblasts and osteocytes, resulted in reduced bone mass and impaired skeletal responses to mechanical stimuli. To specifically isolate Piezo1's role in osteocytes, we used Sost-Cre mice to generate mice with Piezo1 deletion exclusively in mature osteocytes. These mice exhibited lower bone mineral density, decreased cancellous bone mass, and reduced cortical thickness with decrease periosteal expansion. However, unlike mice lacking Piezo1 in both osteoblasts and osteocytes, those with Piezo1 deletion in mature osteocytes responded normally to mechanical loading. These findings suggest that Piezo1 expression in mature osteocytes contributes to bone maintenance under normal physiological condition, but is dispensable for the skeletal response to mechanical loading.

LOX-mediated ECM mechanical stress induces Piezo1 activation in hypoxic-ischemic brain damage and identification of novel inhibitor of LOX

Hypoxic-ischemic encephalopathy (HIE) poses a significant challenge in neonatal medicine, often resulting in profound and lasting neurological deficits. Current therapeutic strategies for hypoxia-ischemia brain damage (HIBD) remain limited. Ferroptosis has been reported to play a crucial role in HIE and serves as a potential therapeutic target. However, the mechanisms underlying ferroptosis in HIBD remain largely unclear. In this study, we found that elevated lysyl oxidase (LOX) expression correlates closely with the severity of HIE, suggesting LOX as a potential biomarker for HIE. LOX expression levels and enzymatic activity were significantly increased in HI-induced neuronal models both in vitro and in vivo. Notably, we discovered that HI-induced brain tissue injury results in increased stiffness and observed a selective upregulation of the mechanosensitive ion channel Piezo1 in both brain tissue of HIBD and primary cortex neurons. Mechanistically, LOX increases its catalytic substrates, the Collagen I/III components, promoting extracellular matrix (ECM) remodeling and possibly mediating ECM cross-linking, which leads to increased stiffness at the site of injury and subsequent activation of the Piezo1 channel. Piezo1 senses these stiffness stimuli and then induces neuronal ferroptosis in a GPX4-dependent manner. Pharmacological inhibition of LOX or Piezo1 ameliorated brain neuronal ferroptosis and improved learning and memory impairments. Furthermore, we identified traumatic acid (TA) as a novel LOX inhibitor that effectively suppresses LOX enzymatic activity, mitigating neuronal ferroptosis and promoting synaptic plasticity. In conclusion, our findings elucidate a critical role for LOX-mediated ECM mechanical stress-induced Piezo1 activation in regulating ferroptotic cell death in HIBD. This mechanistic insight provides a basis for developing targeted therapies aimed at ameliorating neurological outcomes in neonates affected by HIBD.

Piezo1 expression in neutrophils regulates shear-induced NETosis

Neutrophil infiltration and subsequent extracellular trap formation (NETosis) is a contributing factor in sterile inflammation. Furthermore, neutrophil extracellular traps (NETs) are prothrombotic, as they provide a scaffold for platelets and red blood cells to attach to. In circulation, neutrophils are constantly exposed to hemodynamic forces such as shear stress, which in turn regulates many of their biological functions such as crawling and NETosis. However, the mechanisms that mediate mechanotransduction in neutrophils are not fully understood. In this study, we demonstrate that shear stress induces NETosis, dependent on the shear stress level, and increases the sensitivity of neutrophils to NETosis-inducing agents such as adenosine triphosphate and lipopolysaccharides. Furthermore, shear stress increases intracellular calcium levels in neutrophils and this process is mediated by the mechanosensitive ion channel Piezo1. Activation of Piezo1 in response to shear stress mediates calpain activity and cytoskeleton remodeling, which consequently induces NETosis. Thus, activation of Piezo1 in response to shear stress leads to a stepwise sequence of cellular events that mediates NETosis and thereby places neutrophils at the centre of localized inflammation and prothrombotic effects.

Microscale geometrical modulation of PIEZO1 mediated mechanosensing through cytoskeletal redistribution

The microgeometry of the cellular microenvironment profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains unclear. Herein, we describe a fluorescent micropipette aspiration assay that allows for simultaneous visualization of intracellular calcium dynamics and cytoskeletal architecture in real-time, under varied micropipette geometries. By integrating elastic shell finite element analysis with fluorescent lifetime imaging microscopy and employing PIEZO1-specific transgenic red blood cells and HEK cell lines, we demonstrate a direct correlation between the microscale geometry of aspiration and PIEZO1-mediated calcium signaling. We reveal that increased micropipette tip angles and physical constrictions lead to a significant reorganization of F-actin, accumulation at the aspirated cell neck, and subsequently amplify the tension stress at the dome of the cell to induce more PIEZO1’s activity. Disruption of the F-actin network or inhibition of its mobility leads to a notable decline in PIEZO1 mediated calcium influx, underscoring its critical role in cellular mechanosensing amidst geometrical constraints.

PIEZO1 Promotes Odontoblast-Mediated Reactionary Dentinogenesis via SEMA3A

Located at the interface of the dentin–pulp complex, the odontoblasts are specialized cells responsible for dentin synthesis and nociceptive signal detection in response to external stimuli. Recent studies have shown that the mechanosensitive ion channel PIEZO1 is involved in bone formation and remodeling through the influx of calcium ions, and it is abundantly expressed in odontoblasts. However, the specific role of PIEZO1 in reactionary dentinogenesis and the underlying mechanisms remain elusive. In this study, we found intense PIEZO1 expression in the plasma membrane and cytoplasm of odontoblasts in healthy human third molars, mouse mandibular molars, and human odontoblast-like cells (hOBLCs). In hOBLCs, PIEZO1 positively regulated DSPP, DMP1, and COL1A1 expression through the Ca2+/PI3K-Akt/SEMA3A signaling pathway. In addition, exogenous SEMA3A supplementation effectively reversed reduced mineralization capacity in PIEZO1-knockdown hOBLCs. In vivo, Piezo1 expression peaked at day 7 and returned to baseline at day 21 in a wild-type mice dentin injury model, with Sema3a presenting a similar expression pattern. To investigate the specific role of PIEZO1 in odontoblast-mediated reactionary dentinogenesis, mice with a conditional knockout of Piezo1 in odontoblasts were generated, and no significant differences in teeth phenotypes were observed between the control and conditional knockout (cKO) mice. Nevertheless, cKO mice exhibited reduced reactionary dentin formation and decreased Sema3a and Dsp positive staining after dentin injury, indicating impaired dental pulp repair by odontoblasts. In summary, these findings suggest that PIEZO1 enhances the mineralization capacity of hOBLCs in vitro via the Ca2+/PI3K-Akt/SEMA3A signaling pathway and contributes to reactionary dentinogenesis in vivo.

The activation of Piezo1 channel promotes invasion and migration via the release of extracellular ATP in cervical cancer

BackgroundThe mechanosensitive ion channel Piezo1 has emerged as a potential prognostic and therapeutic target in different types of cancers. The aim of this study was to determine the expression levels and underlying mechanisms of Piezo1 in the invasion and migration processes in cervical cancer. MethodsInitially, we employed qRT-PCR, western blot, and immunohistochemical staining techniques to assess the disparity in Piezo1 expression in cervical cancer tissues and cells. Subsequently, we conducted wound healing, transwell assays and phalloidin staining to observe the effects of stable Piezo1 silencing and Piezo1 selective agonist Yoda1 on the invasion and migration capabilities. The release of extracellular ATP was assessed using the enhanced ATP assay kit. Furthermore, we conducted rescue experiments to investigate whether the activation of Piezo1 facilitates cervical cancer invasion and migration through extracellular ATP. Finally, we constructed xenograft tumor models to determine weather the Piezo1 selective agonist Yoda1 influenced the tumor growth in vivo. ResultsIn our study, we found that Piezo1 expression was elevated in both cervical cancer tissues and cells, with the highest levels observed in patients with lymph node metastasis. Knocking down Piezo1 resulted in a significant reduction in the invasion and migration capabilities of cervical cancer cells, whereas the use of the Piezo1 selective agonist Yoda1 enhanced these capabilities. Moreover, the activation of Piezo1 channels was found to regulate the release of extracellular ATP. Mechanistically, the activation of Piezo1 might facilitate cervical cancer invasion, migration, and pseudopodium formation through the release of extracellular ATP. And Piezo1 was an important molecule for the tumor growth of cervical cancer in vivo. ConclusionOur findings revealed that Piezo1 facilitated the invasion and migration of cervical cancer by releasing extracellular ATP, which might hold potential as a valuable target for prognostic and therapeutic interventions in cervical cancer.

Stiffness sensing via Piezo1 enhances macrophage efferocytosis and promotes the resolution of liver fibrosis.

Tissue stiffening is a predominant feature of fibrotic disorders, but the response of macrophages to changes in tissue stiffness and cellular context in fibrotic diseases remains unclear. Here, we found that the mechanosensitive ion channel Piezo1 was up-regulated in hepatic fibrosis. Macrophages lacking Piezo1 showed sustained inflammation and impaired spontaneous resolution of early liver fibrosis. Further analysis revealed an impairment of clearance of apoptotic cells by macrophages in the fibrotic liver. Macrophages showed enhanced efferocytosis when cultured on rigid substrates but not soft ones, suggesting stiffness-dependent efferocytosis of macrophages required Piezo1 activation. Besides, Piezo1 was involved in the efficient acidification of the engulfed cargo in the phagolysosomes and affected the subsequent expression of anti-inflammation genes after efferocytosis. Pharmacological activation of Piezo1 increased the efferocytosis capacity of macrophages and accelerated the resolution of inflammation and fibrosis. Our study supports the antifibrotic role of Piezo1-mediated mechanical sensation in liver fibrosis, suggesting that targeting PIEZO1 to enhance macrophage efferocytosis could induce fibrosis regression.

PIEZO1 as a new target for hyperglycemic stress-induced neuropathic injury: The potential therapeutic role of bezafibrate

Hyperglycemic stress can directly lead to neuronal damage. The mechanosensitive ion channel PIEZO1 can be activated in response to hyperglycemia, but its role in hyperglycemic neurotoxicity is unclear. The role of PIEZO1 in hyperglycemic neurotoxicity was explored by constructing a hyperglycemic mouse model and a high-glucose HT22 cell model. The results showed that PIEZO1 was significantly upregulated in response to high glucose stress. In vitro experiments have shown that high glucose stress induces changes in neuronal cell morphology and membrane tension, a key mechanism for PIEZO1 activation. In addition, high glucose stress upregulates serum/glucocorticoid-regulated kinase-1 (SGK1) and activates PIEZO1 through the Ca2+ pool and store-operated calcium entry (SOCE). PIEZO1-mediated Ca2+ influx further enhances SGK1 and SOCE, inducing intracellular Ca2+ peaks in neurons. PIEZO1 mediated intracellular Ca2+ elevation leads to calcium/calmodulin-dependent protein kinase 2α (CaMK2α) overactivation, which promotes oxidative stress and apoptosis signalling through p-CaMK2α/ERK/CREB and ox-CaMK2α/MAPK p38/NFκB p65 pathways, subsequently inducing synaptic damage and cognitive impairment in mice. The intron miR-107 of pantothenic kinase 1 (PANK1) is highly expressed in the brain and has been found to target PIEZO1 and SGK1. The PANK1 receptor is activated by peroxisome proliferator-activated receptor α (PPARα), an activator known to upregulate miR-107 levels in the brain. The clinically used lipid-lowering drug bezafibrate, a known PPARα activator, may upregulate miR-107 through the PPARɑ/PANK1 pathway, thereby inhibiting PIEZO1 and improving hyperglycemia-induced neuronal cell damage. This study provides a new idea for the pathogenesis and drug treatment of hyperglycemic neurotoxicity and diabetes-related cognitive dysfunction.

Extracellular matrix stiffness mediates insulin secretion in pancreatic islets via mechanosensitive Piezo1 channel regulated Ca2+ dynamics

The pancreatic islet is surrounded by ECM that provides both biochemical and mechanical cues to the islet β-cell to regulate cell survival and insulin secretion. Changes in ECM composition and mechanical properties drive β-cell dysfunction in many pancreatic diseases. While several studies have characterized changes in islet insulin secretion with changes in substrate stiffness, little is known about the mechanotransduction signaling driving altered islet function in response to mechanical cues. We hypothesized that increasing matrix stiffness will lead to insulin secretion dysfunction by opening the mechanosensitive ion channel Piezo1 and disrupting intracellular Ca2+ dynamics in mouse and human islets. To test our hypothesis, mouse and human cadaveric islets were encapsulated in a biomimetic reverse thermal gel (RTG) scaffold with tailorable stiffness that allows formation of islet focal adhesions with the scaffold and activation of Piezo1 in 3D. Our results indicate that increased scaffold stiffness causes insulin secretion dysfunction mediated by increases in Ca2+ influx and altered Ca2+ dynamics via opening of the mechanosensitive Piezo1 channel. Additionally, inhibition of Piezo1 rescued glucose-stimulated insulin secretion (GSIS) in islets in stiff scaffolds. Overall, our results emphasize the role mechanical properties of the islet microenvironment plays in regulating function. It also supports further investigation into the modulation of Piezo1 channel activity to restore islet function in diseases like type 2 diabetes (T2D) and pancreatic cancer where fibrosis of the peri-islet ECM leads to increased tissue stiffness and islet dysfunction.

Intestinal Piezo1 aggravates intestinal barrier dysfunction during sepsis by mediating Ca2+ influx

IntroductionIntestinal barrier dysfunction is a pivotal factor in sepsis progression. The mechanosensitive ion channel Piezo1 is associated with barrier function; however, its role in sepsis-induced intestinal barrier dysfunction remains poorly understood.MethodsThe application of cecal ligation and puncture (CLP) modeling was performed on both mice of the wild-type (WT) variety and those with Villin-Piezo1flox/flox genetic makeup to assess the barrier function using in vivo FITC-dextran permeability measurements and immunofluorescence microscopy analysis of tight junctions (TJs) and apoptosis levels. In vitro, Caco-2 monolayers were subjected to TNF-α incubation. Moreover, to modulate Piezo1 activation, GsMTx4 was applied to inhibit Piezo1 activation. The barrier function, intracellular calcium levels, and mitochondrial function were monitored using calcium imaging and immunofluorescence techniques.ResultsIn the intestinal tissues of CLP-induced septic mice, Piezo1 protein levels were notably elevated compared with those in normal mice. Piezo1 has been implicated in the sepsis-mediated disruption of TJs, apoptosis of intestinal epithelial cells, elevated intestinal mucosal permeability, and systemic inflammation in WT mice, whereas these effects were absent in Villin-Piezo1flox/flox CLP mice. In Caco-2 cells, TNF-α prompted calcium influx, an effect reversed by GsMTx4 treatment. Elevated calcium concentrations are correlated with increased accumulation of reactive oxygen species, diminished mitochondrial membrane potential, and TJ disruption.ConclusionsThus, Piezo1 is a potential contributor to sepsis-induced intestinal barrier dysfunction, influencing apoptosis and TJ modification through calcium influx-mediated mitochondrial dysfunction.

Roles of mechanosensitive ion channel PIEZO1 in the pathogenesis of brain injury after experimental intracerebral hemorrhage

Secondary brain injury after intracerebral hemorrhage (ICH) is the main cause of poor prognosis in ICH patients, but the underlying mechanisms remain less known. The involvement of Piezo1 in brain injury after ICH was studied in a mouse model of ICH. ICH was established by injecting autologous arterial blood into the basal ganglia in mice. After vehicle, Piezo1 blocker, GsMTx4, Piezo1 activator, Yoda-1, or together with mannitol (tail vein injection) was injected into the left lateral ventricle of mouse brain, Piezo1 level and the roles of Piezo1 in neuronal injury, brain edema, and neurological dysfunctions after ICH were determined by the various indicated methods. Piezo1 protein level in neurons was significantly upregulated 24 h after ICH in vivo (human and mice). Piezo1 protein level was also dramatically upregulated in HT22 cells (a murine neuron cell line) cultured in vitro 24 h after hemin treatment as an in vitro ICH model. GsMTx4 treatment or together with mannitol significantly downregulated Piezo1 and AQP4 levels, markedly increased Bcl2 level, maintained more neurons alive, considerably restored brain blood flow, remarkably relieved brain edema, substantially decreased serum IL-6 level, and almost fully reversed the neurological dysfunctions at ICH 24 h group mice. In contrast, Yoda-1 treatment achieved the opposite effects. In conclusion, Piezo1 plays a crucial role in the pathogenesis of brain injury after ICH and may be a target for clinical treatment of ICH.

Simulations of Functional Motions of Super Large Biomolecules with a Mixed-Resolution Model.

Many large protein machines function through an interplay between large-scale movements and intricate conformational changes. Understanding functional motions of these proteins through simulations becomes challenging for both all-atom and coarse-grained (CG) modeling techniques because neither approach alone can readily capture the full details of these motions. In this study, we develop a multiscale model by employing the popular MARTINI CG model to represent a heterogeneous environment and structurally stable proteins and using the united-atom (UA) model PACE to describe proteins undergoing subtle conformational changes. PACE was previously developed to be compatible with the MARTINI solvent and membrane. Here, we couple the protein descriptions of the two models by directly mixing UA and CG interaction parameters to greatly simplify parameter determination. Through extensive validations with diverse protein systems in solution or membrane, we demonstrate that only additional parameter rescaling is needed to enable the resulting model to recover the stability of native structures of proteins under mixed representation. Moreover, we identify the optimal scaling factors that can be applied to various protein systems, rendering the model potentially transferable. To further demonstrate its applicability for realistic systems, we apply the model to a mechanosensitive ion channel Piezo1 that has peripheral arms for sensing membrane tension and a central pore for ion conductance. The model can reproduce the coupling between Piezo1's large-scale arm movement and subtle pore opening in response to membrane stress while consuming much less computational costs than all-atom models. Therefore, our model shows promise for studying functional motions of large protein machines.

Mechanical stiffness promotes skin fibrosis via Piezo1-Wnt2/Wnt11-CCL24 positive feedback loop

Skin fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) caused by fibrotic disorders of the skin. In recent years, ECM stiffness has emerged as a prominent mechanical cue that precedes skin fibrosis and drives its progression by promoting fibroblasts activation. However, how stiffness influences fibroblasts activation for skin fibrosis progression remains unknown. Here, we report a positive feedback loop mediated by the mechanosensitive ion channel Piezo1 and aberrant tissue mechanics in driving skin fibrosis. Piezo1 is upregulated in fibrotic skin in both humans and mice. Piezo1 knockdown dermal fibroblasts lose their fibroproliferative phenotypes despite being grown on a stiffer substrate. We show that Piezo1 acts through the Wnt2/Wnt11 pathway to mechanically induce secretion of C-C motif chemokine ligand 24 (CCL24, also known as eotaxin-2), a potent cytokine associated with fibrotic disorders. Importantly, adeno-associated virus (AAV)-mediated Piezo1 knockdown ameliorated the progression of skin fibrosis and skin stiffness in mice. Overall, increased matrix stiffness promotes skin fibrosis through the inflammatory Piezo1-Wnt2/Wnt11-CCL24 pathway. In turn, a stiffer skin microenvironment increases Piezo1 expression to exacerbate skin fibrosis aggression. Therefore, targeting Piezo1 represents a strategy to break the positive feedback loop between fibroblasts mechanotransduction and aberrant tissue mechanics in skin fibrosis.

Roles of the mechanosensitive ion channel Piezo1 in the renal podocyte injury of experimental hypertensive nephropathy.

Glomerular podocyte injury plays an essential role in proteinuria pathogenesis, a hallmark of chronic kidney disease, including hypertensive nephropathy. Although podocytes are susceptible to mechanical stimuli, their mechanotransduction pathways remain elusive. Piezo proteins, including Piezo1 and 2, are mechanosensing ion channels that mediate various biological phenomena. Although renal Piezo2 expression and its alteration in rodent dehydration and hypertension models have been reported, the role of Piezo1 in hypertensive nephropathy and podocyte injury is unclear. In this study, we examined Piezo1 expression and localization in the kidneys of control mice and in those of mice with hypertensive nephrosclerosis. Uninephrectomized, aldosterone-infused, salt-loaded mice developed hypertension, albuminuria, podocyte injury, and glomerulosclerosis. RNAscope in situ hybridization revealed that Piezo1 expression was enhanced in the podocytes, mesangial cells, and distal tubular cells of these mice compared to those of the uninephrectomized, vehicle-infused control group. Piezo1 upregulation in the glomeruli was accompanied by the induction of podocyte injury-related markers, plasminogen activator inhibitor-1 and serum/glucocorticoid regulated kinase 1. These changes were reversed by antihypertensive drug. Exposure of Piezo1-expressing cultured podocytes to mechanical stretch activated Rac1 and upregulated the above-mentioned markers, which was antagonized by the Piezo1 blocker grammostola mechanotoxin #4 (GsMTx4). Administration of Piezo1-specific agonist Yoda1 mimicked the effects of mechanical stretch, which was minimized by the Yoda1-specific inhibitor Dooku1 and Rac inhibitor. Rac1 was also activated in the above-mentioned hypertensive mice, and Rac inhibitor downregulated gene expression of podocyte injury-related markers in vivo. Our results suggest that Piezo1 plays a role in mechanical stress-induced podocyte injury.

Endothelial Response to the Combined Biomechanics of Vessel Stiffness and Shear Stress Is Regulated via Piezo1.

How endothelial cells sense and respond to dynamic changes in their biophysical surroundings as we age is not fully understood. Vascular stiffness is clearly a contributing factor not only in several cardiovascular diseases but also in physiological processes such as aging and vascular dementia. To address this gap, we utilized a microfluidic model to explore how substrate stiffness in the presence of shear stress affects endothelial morphology, senescence, proliferation, and inflammation. We also studied the role of mechanosensitive ion channel Piezo1 in endothelial responses under the combined effect of shear stress and substrate stiffness. To do so, we cultured endothelial cells inside microfluidic channels covered with fibronectin-coated elastomer with elastic moduli of 40 and 200 kPa, respectively, mimicking the stiffness of the vessel walls in young and aged arteries. The endothelial cells were exposed to atheroprotective and atherogenic shear stress levels of 10 and 2 dyn/cm2, respectively. Our findings show that substrate stiffness affects senescence under atheroprotective flow conditions and cytoskeleton remodeling, senescence, and inflammation under atherogenic flow conditions. Additionally, we found that the expression of Piezo1 plays a crucial role in endothelial adaptation to flow and regulation of inflammation under both atheroprotective and atherogenic shear stress levels. However, Piezo1 contribution to endothelial senescence was limited to the soft substrate and atheroprotective shear stress level. Overall, our study characterizes the response of endothelial cells to the combined effect of shear stress and substrate stiffness and reveals a previously unidentified role of Piezo1 in endothelial response to vessel stiffening, which potentially can be therapeutically targeted to alleviate endothelial dysfunction in aging adults.

Piezo1 Is Required for Myoblast Migration and Involves Polarized Clustering in Association with Cholesterol and GM1 Ganglioside.

A specific plasma membrane distribution of the mechanosensitive ion channel Piezo1 is required for cell migration, but the mechanism remains elusive. Here, we addressed this question using WT and Piezo1-silenced C2C12 mouse myoblasts and WT and Piezo1-KO human kidney HEK293T cells. We showed that cell migration in a cell-free area and through a porous membrane decreased upon Piezo1 silencing or deletion, but increased upon Piezo1 activation by Yoda1, whereas migration towards a chemoattractant gradient was reduced by Yoda1. Piezo1 organized into clusters, which were preferentially enriched at the front. This polarization was stimulated by Yoda1, accompanied by Ca2+ polarization, and abrogated by partial cholesterol depletion. Piezo1 clusters partially colocalized with cholesterol- and GM1 ganglioside-enriched domains, the proportion of which was increased by Yoda1. Mechanistically, Piezo1 activation induced a differential mobile fraction of GM1 associated with domains and the bulk membrane. Conversely, cholesterol depletion abrogated the differential mobile fraction of Piezo1 associated with clusters and the bulk membrane. In conclusion, we revealed, for the first time, the differential implication of Piezo1 depending on the migration mode and the interplay between GM1/cholesterol-enriched domains at the front during migration in a cell-free area. These domains could provide the optimal biophysical properties for Piezo1 activity and/or spatial dissociation from the PMCA calcium efflux pump.

Higher-intensity ultrasound accelerates fracture healing via mechanosensitive ion channel Piezo1.

Osteoporosis-related fractures are a major public health problem. Mechanobiological stimulation utilizing low-intensity pulsed ultrasound (LIPUS) is the most widely accepted modality for accelerating fracture healing. However, recent evidence has demonstrated the ineffectiveness of LIPUS, and the biophysical mechanisms of ultrasound-induced bone formation also remain elusive. Here, we demonstrate that ultrasound at a higher intensity than LIPUS effectively accelerates fracture healing in a mouse osteoporotic fracture model. Higher-intensity ultrasound promoted chondrogenesis and hypertrophic differentiation of chondrocytes in the fracture callus. Higher-intensity ultrasound also increased osteoblasts and newly formed bone in the callus, resulting in accelerated endochondral ossification during fracture healing. In addition, we found that accelerated fracture healing by ultrasound exposure was attenuated when the mechanosensitive ion channel Piezo1 was inhibited by GsMTx4. Ultrasound-induced new bone formation in the callus was attenuated in fractured mice treated with GsMTx4. Similar results were also confirmed in a 3D osteocyte-osteoblast co-culture system, where osteocytic Piezo1 knockdown attenuated the expression of osteoblastic genes after ultrasound exposure. Together these results demonstrate that higher-intensity ultrasound than clinically used LIPUS can accelerate endochondral ossification after fractures. Furthermore, our results suggest that mechanotransduction via Piezo1 mediates ultrasound-stimulated fracture healing and bone formation.

Piezo1 Activation Prevents Spheroid Formation by Malignant Melanoma SK-MEL-2 Cells.

Melanoma is a highly aggressive type of skin cancer produced through the malignant transformation of melanocytes, and it is usually associated with a poor prognosis. Clinically, melanoma has several stages associated with migration and invasion of the cells through the skin's layers, the rapid spreading of cells and the formation of tumors in multiple organs. The main problem is the emergence of resistance in melanoma to the applied methods of treatment; thus, it is of primary importance to find more crucial signaling pathways that control the progression of this type of cancer and could be targeted to prevent melanoma spreading. Here, we uncover novel aspects of the role of the mechanosensitive ion channel Piezo1 in melanoma tumor formation. Using a combinative approach, we showed the functional expression of mechanosensitive Piezo1 channels in the aggressive human melanoma SK-MEL-2 cell line. We found that chemical activation of Piezo1 by its agonist, Yoda1, prevents melanoma spheroid formation; thus, Piezo1 could be a potential target for selective modulation aimed at the prevention of melanoma development.

Three-Dimensional Matrix Stiffness Activates the Piezo1-AMPK-Autophagy Axis to Regulate the Cellular Osteogenic Differentiation.

Extracellular matrix (ECM) stiffness is a key stimulus affecting cellular differentiation, and osteoblasts are also in a three-dimensional (3D) stiff environment during the formation of bone tissues. However, it remains unclear how cells perceive matrix mechanical stiffness stimuli and translate them into intracellular signals to affect differentiation. Here, for the first time, we constructed a 3D culture environment by GelMA hydrogels with different amino substitution degrees and found that Piezo1 expression was significantly stimulated by the stiff matrix with high substitution; meanwhile, the expressions of osteogenic markers OSX, RUNX2, and ALP were also observably improved. Moreover, knockdown of Piezo1 in the stiff matrix revealed significant reduction of the abovementioned osteogenic markers. In addition, in this 3D biomimetic ECM, we also observed that Piezo1 can be activated by the static mechanical conditions of the stiff matrix, leading to the increase of the intracellular calcium content and accompanied with a continuous change in cellular energy levels as ATP was consumed during cellular differentiation. More surprisingly, we found that in the 3D stiff matrix, intracellular calcium as a second messenger promoted the activation of the AMP-activated protein kinase (AMPK) and unc-51-like autophagy-activated kinase 1 (ULK1) axis and modestly modulated the level of autophagy, bringing it more similar to differentiated osteoblasts, with increased ATP energy metabolism consumption. Our study innovatively clarifies the regulatory role of the mechanosensitive ion channel Piezo1 in a static mechanical environment on cellular differentiation and verifies the activation of the AMPK-ULK1 axis in the cellular ATP energy metabolism and autophagy level. Collectively, our research develops the understanding of the interaction mechanisms of biomimetic extracellular matrix biomaterials and cells from a novel perspective and provides a theoretical basis for bone regeneration biomaterials design and application.