Mechanical gated ion channel Piezo1: Function,and role in macrophage 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).

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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).