Mechanical gated ion channel Piezo1: Function,and role in macrophage inflammatory response

Abstract

Macrophages are present in many mechanically active tissues and are often subjected to varying degrees of mechanical stimulation. Macrophages play a crucial role in resisting pathogen invasion and maintaining tissue homeostasis. Piezo-type mechanosensitive channel component 1 (Piezo1) is the main cation channel involved in the rapid response to mechanical stimuli in mammals. This channel plays a crucial role in controlling blood pressure and motor performance and regulates urinary osmotic pressure and epithelial cell proliferation and division. In recent years, numerous studies have shown that in macrophages, Piezo1 not only plays a role in regulating the aforementioned physiological processes but also participates in multiple pathological processes such as inflammation and cancer. In this review, we summarize the research progress on Piezo1-mediated regulation of macrophage-mediated inflammatory responses through downstream signalling pathways and the aerobic glycolysis pathway.

Introduction

The body constantly receives mechanical stimuli from the external environment and responds to maintain a stable internal environment. ¹ Ion channels are the primary receptors that mediate signal transduction, and regulate ion concentrations on both sides of the cell membrane by promoting transmembrane ion transport . Ion channels are classified as voltage-gated ion channels, ligand-gated ion channels, or mechanically gated ion channels. Compared to the first two types of channels, the research progress on mechanically gated ion channels lags far behind. Research has shown that mechanoreceptors can induce channel opening by sensing changes in mechanical tension in the microenvironment, thereby converting mechanical stimulation into electrical and chemical signals and playing an important role in cell morphology and function.^2,3 Piezo-type mechanosensitive channel component 1 (Piezo1) was the first mechanically gated cation channel identified, is known as FAM38A, is found in mammals, and can mediate the passage of a variety of cations, especially Ca²⁺, which is critical for the functional regulation of histiocytes that are highly exposed to mechanical stimulation.^4,5

Macrophages are multifunctional cells of the innate immune system. The direct recognition of invading pathogens by macrophages is a key factor in the inflammatory response.The cell membrane can perceive the local microenvironment and respond to changes in temperature, pH, and pressure, leading to the opening of ion channels on the cell membrane . Although macrophages are exposed to force and pressure throughout the inflammatory cycle, little is known about how these mechanical processes regulate inflammatory responses. ⁶ Piezo1, which is a widely expressed mechanical ion channel in mammalian cells, plays an important role in the functional regulation of macrophages. In this paper, the role of Piezo1 in the regulation of macrophage involvement in the inflammatory response is reviewed.

Basic molecular structure and functions of Piezo1

Over time, organisms have evolved a highly conserved mechanically sensitive mechanism, known as mechanical-gated ion channels, which are capable of transforming mechanical stimuli into biological signals. These channels play crucial regulatory roles in hearing, pain, proprioception, embryogenesis, and many other bodily functions that rely on mechanical sensations.^7–9 The concept of a mechanically gated ion channel was first proposed by Corey and others after they recorded the current signals generated by mechanical stimulation of cochlear hair cells when studying the hearing of bullfrogs. ¹⁰ However, this assumption was not confirmed because signal transduction involves too many molecular processes. However, the main pathway by which organisms achieve rapid signal transduction involves specialized sensory cells that are rich in mechanical sensing (MS) transduction channels. ⁹ Cox and others discovered a variety of mechanically gated ion channels, including tandem pore domain potassium (K2P) channels and transient receptor potential (TRP) channels.^11,12 However, due to differences between organisms, research on mechanically gated ion channels in mammals has been lacking. In 2010, Coste and others discovered the first mechanically sensitive cationic current that produced excitability in mammalian cells in mouse neuroblastoma cell lines and identified the Piezo protein family. ⁴ The Piezo protein family consists of Piezo1 and Piezo2, which are responsible for the mechanical transmission of sensory information, including touch, proprioception, and pain. ¹³ The Piezo channel is a homologous trimer shaped like a propeller that has 38 transmembrane helices in three subunits that are arranged abnormally to form an iconic nano bowl configuration in the membrane plane. ¹⁴ Piezo channels are widely distributed in the lungs, bladder, bones, and dorsal root ganglion (DRG) neurons of vertebrates. ¹⁵

The central channel of Piezo1 includes an outer helix, an extracellular C-terminal domain, an inner helix, and an intracellular C-terminal domain. ¹³ The peripheral region is composed of 2200 amino acids, and is formed by the extracellular fan leaf region, peripheral transmembrane helices, and intracellular beam and anchor regions.^13,16 When Piezo1 is mechanically stimulated, the conformational change in the periphery causes the central pore to open, after which ions can be transferred through the membrane. ¹⁷ In addition, Piezo1 is expressed in many organs that often undergo mechanical stimulation, such as endothelial cells in blood or lymphatic vessels, and regulate vascular remodelling, valve morphology, and blood pressure; Overexpression of Piezo1 in osteoblasts regulates bone remodelling; Activation of Piezo1 in alveolar endothelial cells can protect the integrity of the alveolar capillary barrier structure; in urothelial cells, Piezo1 initiates the urination reflex and promotes urination after sensing stretch stimulation in the tube wall caused by urine accumulation.^18–22 In addition, Piezo1 gene mutations can lead to the occurrence of various human diseases, such as hereditary stem cell disease such as dehydrated hereditary stomatocytosis, congenital lymphodysplasia, and breast cancer.^23,24

According to previous reports, Yoda1(GlyT2-IN-1) is a specific activator of Piezo1 with high selectivity that acts on specific domains of Piezo1 and can activate Piezo1 in the absence of mechanical stimulation.^25,26 Syeda and colleagues confirmed through cell electrophysiology that Yoda1 could significantly affect the sensitivity of Piezo1 and could slow the dynamics of inactivation by stabilizing open channels; even in the absence of mechanical stimulation, Yoda1 could cause activation of Piezo1. ²⁷ In addition, the Piezo1 activators Jedi1 and Jedi2 are known to have high water solubility, fast starting current, and short decay duration. Jedi1 and Jedi2 can each bind to Piezo1 to create a synergistic effect and enhance the cells’ inward current.^1,28 There are many inhibitors of Piezo1, including ruthenium red, amyloid protein β, saturated fatty acids, polyunsaturated fatty acids, and GsMTx4(M-theraphotoxin-Gr1a, which is a peptide isolated from the venom of the spider Grammostola rosea); GsMTx4 is a current research hotspot. GsMTx4, which is a spider venom peptide, selectively inhibits Piezo and TRP cation channels by regulating membrane tension.^26,29–32 In addition, María and others have confirmed that GsMTx4 can inhibit the demyelination and neuronal damage induced by lysophosphatidylcholine (LPC) stimulation of Piezo1. ³³ Moreover, GsMTx4 can inhibit LPC-induced activation of astrocytes and microglia. ³² GsMTx4 is an important pharmacological tool for future research on the physiological function of Piezo1.

Macrophages and inflammatory reactions

Macrophages are the cells with the strongest phagocytic ability in vivo and have high plasticity. In addition to phagocytosis, macrophages also participate in antigen presentation, inflammatory reactions, angiogenesis and other pathophysiological processes during growth and development. Stimulated macrophages can polarize into classic macrophages (M1) or alternatively activated macrophages (M2).^34,35 M1 macrophages mainly play an antigen-presenting role and can participate in immune responses by secreting proinflammatory cytokines and chemokines.^36,37 In contrast, M2 macrophages mainly secrete the inhibitory cytokine interleukin-10 (IL-10) or transforming growth factor- β (TGF- β) to decrease the immune response. ³⁸ When inflammation occurs, the physical environment in which cells are located can undergo significant changes. Studies have shown that immune cells such as macrophages can perceive these changes through mechanically sensitive receptors, resulting in immune responses.^16,39

Piezo1 regulates macrophage involvement in the inflammatory response

Piezo1-CaMKII-Mst1/2-Rac1

Macrophages are immune cells that perform phagocytosis. During infection, Toll-like receptors (TLRs) on the surface of macrophages can recognize invading microorganisms and trigger innate immune responses. Studies have shown that Ca²⁺ influx caused by the binding of TLRs to Piezo1 can activate the Hippo kinases Mst1 and Mst2(Mst1/2,macrophage stimulating 1/2), thereby playing an important role in the macrophage-mediated inflammatory response. ⁴⁰ Jing and colleagues reported that the trend of changes in Piezo1 and TLR4 expression is very consistent when E. coli is used to infect bone marrow-derived macrophages (BMDMs). ⁴¹ Additionally,Piezo1- knockout mice had significantly higher levels of bacterial infection in the spleen, lung, liver, and kidney than Piezo1 wild-type mice. These results indicate that Piezo1 plays an important role in the regulation of Mst1/2 activation through TLR4 signalling, thereby controlling macrophage inflammation.

Yoda1 can accelerate the translocation of Ca²⁺ through Piezo1. ⁴² Ca²⁺/calmodulin-dependent protein kinase II (CaMK II) is a serine/threonine protein kinase that can phosphorylate many proteins within cells, thereby increasing the intracellular calcium ion concentration. ⁴³ After treating BMDMs with Yoda1 in calcium free medium, a decrease in the phosphorylation of Mob1 (a physiological substrate of Mst) was observed, indicating that eliminating extracellular Ca²⁺could inhibit the activation of Mst1/2 by Piezo1. These finding further confirmed that Ca²⁺ was an important second messenger through which Piezo1 activates Mst1/2. When macrophages were treated with an autocrine peptide-2 related inhibitory peptide (TFA, a potent CaMKII inhibitor), the results indicated that inhibiting CaMKII eliminated Yoda1 mediated activation of Piezo1. ⁴¹ These results indicate that the ion channel Piezo1 regulates the bactericidal activity of macrophages by influencing the cytoskeleton through the Ca²⁺- CaMKII -Mst1/2 axis.

Macrophage phagocytosis is mainly dependent on reorganization of the actin cytoskeleton. This process is regulated by the small GTP enzyme protein family. Ras-related C3 botulinum toxin substrate 1(Rac1) is a representative molecule of the small GTPase family. When Yoda1 was used to treat BMDMs, only Rac1 was activated, and the degree of activation was significantly different between Piezo1- deficient and wild-type mice. However, no increase in Rac1-GTP (the initial form of Rac1) was observed in wild-type BMDMs after Yoda1 treatment of Mst1/2 deficient BMDMs. ⁴¹ These results indicate that Piezo1 activates macrophage Mst1/2, thereby regulating Rac1-mediated signal transduction and enhancing macrophage phagocytosis of bacteria. Stimulation by LPS or E. coli can promote the binding of the receptor TLR4 and the mechanical transducer Piezo1 to induce calcium influx and activate the CaMKII Mst1/2-Rac1 axis to regulate the bactericidal activity of macrophages. Inhibiting CaMKII or knocking out Mst1/2 or Rac1 can decrease the bacterial clearance rate, which can have the same consequences as Piezo1 deficiency. ⁴¹

These studies indicate that during bacterial infection or LPS stimulation, the activation of Piezo1 in macrophages can be mediated by Ca²⁺ influx, which activates the intracellular CaMKII-Mst1/2-Rac1 pathway to regulate macrophage participation in inflammatory responses and protect the body from damage (Figure 1).

Figure 1.

Schematic diagram of Piezo1 in regulating macrophage-mediated inflammatory response through its downstream signalling pathways and aerobic glycolysis pathway. (α3：importins α3，α7：importins α7. Interaction between HIF1a and importins α3 and α7 is important for the import of HIF1a into the cell nucleus.).

Piezo1-CaMKII-ETS1-FGF2

In addition to participating in immune responses, macrophages can stimulate angiogenesis through the production of proangiogenic cytokines and growth factors, such as fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor-a (VEGF-a).^44–46 In addition, Piezo1 has been shown to play a key role in the development of the vascular system.^23,47 Angiogenesis is vital for postischemic recovery.^48,49 Xie and colleagues reported that the expression of Piezo1 in macrophages was significantly increased in ischaemic muscles and was positively correlated with time, in a mouse hindlimb ischaemia (HLI) model. ⁵ °Compared with wild-type mice, Piezo1- deficient mice exhibited increased capillary density and arteriole density, significantly improved blood flow recovery, and significantly reduced ischaemia and limb injury. ⁵⁰ Moreover, compared with wild-type mice, Piezo1- deficient mice exhibited significantly upregulated FGF2 expression after ischaemia and FGF2 expression in macrophages. ⁵⁰ These results suggest that a lack of Piezo1 in mouse macrophages can significantly improve the remodelling of blood vessels after ischaemia.

The activation of CaMKII depends on Ca²⁺. Research has shown that when Ca²⁺ is increased and binds to CaMKII, CaMKII is phosphorylated at the 286th amino acid, thereby activating CaMKII. ⁵¹ ETS1(ETS proto-oncogene 1, transcription factor gene) is a key factor in vascular remodelling, angiogenesis, and healing activation. ⁵² The activity of ETS1 has been reported to be regulated by phosphorylation at two different sites. Activated CaMKII phosphorylates ETS1 at a serine to inhibit ETS1 DNA binding. ⁵³ Xie and colleagues reported that as the stiffness of the BMDM medium increased, the phosphorylation of CaMKII and ETS1 gradually increased, similar to the effect of Yoda1 stimulation. ⁵⁰ However, in Piezo1-deficient BMDMs, there was an inhibitory effect on this pathway. When calcium- free culture medium was used, the CaMKII-ETS1 signalling pathway was blocked, and FGF2 expression increased. ⁵⁰ These results suggest that the CaMKII-ETS1 pathway in macrophages is regulated by Piezo1, which is associated with FGF2. Therefore, ischaemia can activate Piezo1 on the surface of macrophages, which inhibits the upregulation of FGF2 transcription mediated by CaMKII-ETS1 by mediating Ca²⁺ influx and participating in the inflammatory response after ischaemia, thereby promoting the progression of injury (Figure 1).

Piezo1-AKT-GSK3β-Ccnd1

Mechanical stimulation of M1 macrophages can play a role in regulating orthodontic tooth movement (OTM) in the local inflammatory microenvironment. ⁵⁴ Hao and colleagues constructed a model of OTM, and showed that mechanical stimulation could induce the proliferation of macrophages in mice, and increase Piezo1 expression in BMDMs. ⁵⁵ Piezo1 inhibitors or knocking out Piezo1 could reduce macrophage proliferation, thereby delaying OTM and weakening bactericidal activity. Studies have shown that Cyclin D1 (Ccnd1), which is an essential protein that regulates the cell cycle, can be activated by the Piezo1-AKT-GSK3β cascade to exert biological effects. Potential downstream target factors of this pathway promote the anti-inflammatory effects of macrophages by enhancing Rb phosphorylation.^55,56 Hao examined transgenic mice with Ccnd1 knockout and reported that Ccnd1 knockout could reduce macrophage activation and Rb phosphorylation induced by mechanical stimulation, inhibit the proliferation of periodontal macrophages, and delay OTM. Ccnd1 overexpression significantly stimulates macrophage proliferation, reversing the inhibition of macrophage bactericidal ability induced by the Piezo1 antagonist GsMTx4. When BMDMs are subjected to mechanical stimulation, Piezo1 activation leads to Ca²⁺ influx, significantly increasing intracellular Ca²⁺ concentrations and activating the downstream AKT-GSK3β-Ccnd1 axis to promote macrophage proliferation, which is a crucial process by which macrophages exert anti-inflammatory effects during OTM (Figure 1).

Piezo1-YAP

Recent studies have shown that immune regulation is mainly mediated by the polarization of macrophages and the secretion of cytokines, which may be a mechanism of bone formation and angiogenesis. ⁵⁷ In orthopaedic surgery, the implantation of biomaterials causes macrophage polarization, thereby inducing a continuous innate immune response that ranges from inflammation to tissue proliferation to tissue maturation.^58,59 Research indicates that M1 macrophages can produce vascular endothelial growth factor (VEGF) and promote vascular growth. ⁶⁰ M2 macrophages secrete platelet-derived growth factor-BB (PDGF-BB) and bone morphogenetic protein-2 (BMP-2), which contribute to vascular maturation, stability, and osteogenesis. ⁶¹ luo's research showed that bone implants could polarize macrophages into proinflammatory (M1) phenotypes initially and into anti-inflammatory (M2) phenotypes in the later stages of development, thus promoting the formation of mature bone to a greater extent. ⁶⁰ An increase in the number of anti-inflammatory macrophages (more M2 macrophages than M1macrophages) can ultimately alleviate the inflammatory response. ⁶²

In the resting state, large tumour suppressor factor 1/2 (LATS1/2) in macrophages can regulate Yes-associated protein (YAP) phosphorylation and inhibit its biological activity. When cells are stimulated, YAP can undergo dephosphorylation and be transported to the nucleus, where it regulates macrophage participation in the inflammatory response. ⁶³ Tang and colleagues discovered that pretreating macrophages with Ti2448(Ti-24Nh4Zr-8Sn, a novel biomedical metal with biomimetic characteristics of the human skeleton) could enhance angiogenesis and osteogenic differentiation by increasing the levels of PDGF-BB and BMP-2. ⁶⁴ Thus, Ti2448 was confirmed to promote angiogenesis and osteogenic differentiation via Piezo1-YAP signalling during macrophage polarization and associated cytokine secretion. Immunofluorescence analysis and immunoblotting revealed that Piezo1 and YAP were highly expressed in macrophages. This study suggests that when the Piezo1 channel on the surface of macrophages is stimulated by implantable materials, YAP phosphorylation is inhibited by LATS1/2 in the cytoplasm, and YAP undergoes dephosphorylation and transport to the nucleus, thereby facilitating the polarization of M1 macrophages into M2-type macrophages, which release PDGF-BB and BMP-2 to regulate inflammation and promote angiogenesis and osteogenesis (Figure 1).

Piezo1-JNK1-mTOR

Macrophages or microglia in the nervous system can trigger a robust immune response after being stimulated by LPS. Liu and colleagues examined high glucose-induced microglia and found that in the absence of GsMTx4, higher glucose concentrations significantly inhibited microglial inflammatory reactions. ³² High concentrations of GsMTx4(≥60 mM) significantly increased inflammatory mediators. ³² As the concentration of glucose increase, the effect of GsMTx4. JNK1(c-Jun N-terminal kinase 1, which is a member of the mitogen activated protein kinase superfamily) and mTOR(mammalian target of rapamycin, an atypical serine/threonine protein kinase) are important for the survival, proliferation, migration and differentiation of cells.^65–67 liu examined the role of JNK1 and mTOR in Ca²⁺ -dependent downstream signalling mediated by Piezo1. The results indicated that cellular anabolism was reduced by increasing glucose concentrations, intracellular Ca²⁺ levels were increased, and JNK1 and mTOR expression was gradually reduced. When GsMTx4 was used, the expression of JNK1 and mTOR increased, which could counteract the combined effects of high glucose and LPS. ³² Therefore, inhibiting the Piezo1 channel and reducing Ca²⁺ influx can reduce high glucose-induced cytotoxicity and reverse the microglia-mediated inflammatory response through the JNK1-mTOR signalling pathway (Figure 1).

Aerobic glycolysis pathway

Aerobic glycolysis is the process of converting glucose into pyruvate. Suppressing this process can influence the function of macrophages, such as cytokine secretion and phagocytosis.^68,69 Several studies have shown that the activation of M1 macrophages can lead to the transformation of metabolism from oxidative phosphorylation to aerobic glycolysis, ultimately leading to inflammatory reactions.^70,71 When the microenvironment of macrophages changes, Piezo1 on the surface is activated to transform mechanical force into Ca²⁺ influx and intracellular Ca²⁺ overload, causing changes in the immune response and metabolic state.^72,73 leng and colleagues used calcium-free medium to drain the extracellular calcium ions of different BMDMs and simultaneously administered Yoda1. ⁷⁴ The results indicated that the total levels of HIF1a (the main transcription factor that regulates the expression of glycolytic genes.) and nuclear HIF1a protein in macrophages were decreased. In contrast, the levels of aerobic glycolysis and inflammatory factor secretion decreased, resulting in a similar effect on Piezo1-deficient BMDMs and suggesting that HIF1a plays a key role in glycolysis induced by Ca²⁺.

Previous studies have confirmed that the downstream CaMKII signalling pathway induced by Piezo1 activation is involved in maintaining the stability of HIF1a.^75,76 leng and colleagues reported that LPS stimulation could significantly promote CaMKII phosphorylation and increase HIF1a levels in normal BMDMs, while treatment with KN93 (an CaMKII enzyme activity inhibitor) produced the opposite results. ⁷⁴ Blocking the translation of HIF1a in BMDMs with siHIF1a abolished the differences between normal BMDMs and Piezo1-deficient BMDMs. ⁷⁷ The results showed that when the cellular microenvironment changed, Piezo1 was activated to increase Ca²⁺ influx. Through the Ca²⁺-CaMKII -HIF1a axis, HIF1a is transferred to the nucleus, where it initiates the transcription of several glycolytic genesand ultimately leads to phagocytosis and sterilization (Figure 1).

Summary and future outlooks

As a highly evolutionarily conserved mechanically gated cation channel, Piezo1 plays a key role in various physiological and pathological processes, especially the inflammatory response. In addition to macrophage-related signalling pathways, some scientists have found that mechanical activation of Piezo1 in lung epithelial cells can trigger a cascade reaction of a disintegrin and a metalloproteinase 10/17, producing surface molecules associated with inflammatory responses, such as epithelial growth factor (amphiregulin) and tumour necrosis factor (TNF), which leads to inflammatory reactions. ⁷⁸ In summary, piezo1 is widely distributed. When the body is injured, Piezo1 converts mechanical injury signals into inflammatory cascades within macrophages, activating multiple signalling pathways to drive inflammation. Therefore, regulating the abnormal activity of Piezo1 is a novel and highly promising treatment approach for inflammatory diseases. Research has shown that Piezo1 can serve as a target for treating mechanical stress-related immune inflammatory diseases and is a powerful means of preventing inflammation. However, the current level of research on the structure of the Piezo1 protein is limited, its pharmacological effects are limited by low-affinity drugs with poor solubility and stability, such as Yoda1 and GsMTx4, and there is a lack of effective clinical compounds to verify its molecular mechanisms and therapeutic effects. ⁴² In addition, due to the diverse biological functions of Piezo1 in different tissues and organs,determining how to achieve the ideal therapeutic effect while avoiding potential toxic side effects is a major challenge for researchers.

In brief, the relationship between Piezo1, which is a mechanical signalling receptor, and inflammation is precise and complex. Because Piezo1 has been known for only approximately 10 years and its specific structure and physiological mechanisms have not yet been fully elucidated, researchers should focus on clarifying the structural basis of Piezo1, especially its dynamic characteristics and basic biological functions. From a long-term perspective, finding new Piezo1 specific activators and inhibitors is crucial for preventing and treating inflammatory diseases. ³ Undoubtedly, the pharmacology of Piezo1 has good application prospects. Research on the clinical applications of Piezo1 drugs is bound to be full of hope and other challenges.