TRPM7 kinase-mediated immunomodulation in macrophage plays a central role in magnesium ion-induced bone regeneration.
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ABSTRACT:
SUBMITTER: Qiao W
PROVIDER: S-EPMC8128914 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:TRPM7 is a Ca(2+)- and Mg(2+)-permeable cation channel that also contains a protein kinase domain. While there is general consensus that the channel is inhibited by free intracellular Mg(2+), the functional roles of intracellular levels of Mg.ATP and the kinase domain in regulating TRPM7 channel activity have been discussed controversially. To obtain insight into these issues, we have determined the effect of purine and pyrimidine magnesium nucleotides on TRPM7 currents and investigated the possible involvement of the channel's kinase domain in mediating them. We report here that physiological Mg.ATP concentrations can inhibit TRPM7 channels and strongly enhance the channel blocking efficacy of free Mg(2+). Mg.ADP, but not AMP, had similar, albeit smaller effects, indicating a double protection against possible Mg(2+) and Ca(2+) overflow during variations of cell energy levels. Furthermore, nearly all Mg-nucleotides were able to inhibit TRPM7 activity to varying degrees with the following rank in potency: ATP > TTP > CTP > or = GTP > or = UTP > ITP approximately free Mg(2+) alone. These nucleotides also enhanced TRPM7 inhibition by free Mg(2+), suggesting the presence of two interacting binding sites that jointly regulate TRPM7 channel activity. Finally, the nucleotide-mediated inhibition was lost in phosphotransferase-deficient single-point mutants of TRPM7, while the Mg(2+)-dependent regulation was retained with reduced efficacy. Interestingly, truncated mutant channels with a complete deletion of the kinase domain regained Mg.NTP sensitivity; however, this inhibition did not discriminate between nucleotide species, suggesting that the COOH-terminal truncation exposes the previously inaccessible Mg(2+) binding site to Mg-nucleotide binding without imparting nucleotide specificity. We conclude that the nucleotide-dependent regulation of TRPM7 is mediated by the nucleotide binding site on the channel's endogenous kinase domain and interacts synergistically with a Mg(2+) binding site extrinsic to that domain.
| S-EPMC2151514 | biostudies-literature
Project description:Bone grafts are commonly used for the treatment of critical sized bone defects. Since the supply of autologous bone is insufficient, allogeneic bone grafts have been used most of the time. However, the poor osteogenic property of allogeneic bone grafts after pretreatment results in delayed union, non-union, or even occasional deformity. Calcium phosphate cement (CPC) is one of the most promising bone filling materials due to its good biocompatibility and similar chemical components as natural bone. However, clinical applications of CPC were hampered by limited osteogenic effects, undesired immune response which results in resorption, and poor mechanical stability in vivo. Magnesium (Mg) has been proven to trigger bone regeneration through modulating cell behaviors of mesenchymal stem cells and macrophages significantly. Unfortunately, the degradation raters of pure Mg and Mg oxide are extremely fast, resulting in early collapse of Mg contained CPC. In this study, we developed a novel magnesium contained calcium phosphate bone cement (Mg-CPC), possessing long-term mechanical stability and osteogenic effects through sustained release of Mg. Furthermore, in vitro studies showed that Mg-CPC had no cytotoxic effects on hBMMSCs and macrophage RAW 264.7, and could enhance the osteogenic differentiation as determined by alkaline phosphate (ALP) activity and calcium nodule staining, as well as suppress the inflammatory as determined by expression of anti-inflammatory cytokine IL-1RA. We also found that Mg-CPC promoted new bone formation and bone maturation in vivo. These results suggest that Mg-CPC should be a good substitute material for bone grafts in clinical use.
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Project description:TRPM7 is an unusual bi-functional protein containing an ion channel covalently linked to a protein kinase domain. TRPM7 is implicated in regulating cellular and systemic magnesium homeostasis. While the biophysical properties of TRPM7 ion channel and its function are relatively well characterized, the function of the TRPM7 enzymatically active kinase domain is not understood yet. To investigate the physiological role of TRPM7 kinase activity, we constructed mice carrying an inactive TRPM7 kinase. We found that these mice were resistant to dietary magnesium deprivation, surviving three times longer than wild type mice; also they displayed decreased chemically induced allergic reaction. Interestingly, mutant mice have lower magnesium bone content compared to wild type mice when fed regular diet; unlike wild type mice, mutant mice placed on magnesium-depleted diet did not alter their bone magnesium content. Furthermore, mouse embryonic fibroblasts isolated from TRPM7 kinase-dead animals exhibited increased resistance to magnesium deprivation and oxidative stress. Finally, electrophysiological data revealed that the activity of the kinase-dead TRPM7 channel was not significantly altered. Together, our results suggest that TRPM7 kinase is a sensor of magnesium status and provides coordination of cellular and systemic responses to magnesium deprivation.
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Project description:Sick sinus syndrome and atrioventricular block are common clinical problems, often necessitating permanent pacemaker placement, yet the pathophysiology of these conditions remains poorly understood. Here we show that Transient Receptor Potential Melastatin 7 (TRPM7), a divalent-permeant channel-kinase of unknown function, is highly expressed in embryonic myocardium and sinoatrial node (SAN) and is required for cardiac automaticity in these specialized tissues. TRPM7 disruption in vitro, in cultured embryonic cardiomyocytes, significantly reduces spontaneous Ca(2+) transient firing rates and is associated with robust down-regulation of Hcn4, Cav3.1, and SERCA2a mRNA. TRPM7 knockdown in zebrafish, global murine cardiac Trpm7 deletion (KO(?MHC-Cre)), and tamoxifen-inducible SAN restricted Trpm7 deletion (KO(HCN4-CreERT2)) disrupts cardiac automaticity in vivo. Telemetered and sedated KO(?MHC-Cre) and KO(HCN4-CreERT2) mice show episodes of sinus pauses and atrioventricular block. Isolated SAN from KO(?MHC-Cre) mice exhibit diminished Ca(2+) transient firing rates with a blunted diastolic increase in Ca(2+). Action potential firing rates are diminished owing to slower diastolic depolarization. Accordingly, Hcn4 mRNA and the pacemaker current, I(f), are diminished in SAN from both KO(?MHC-Cre) and KO(HCN4-CreERT2) mice. Moreover, heart rates of KO(?MHC-Cre) mice are less sensitive to the selective I(f) blocker ivabradine, and acute application of the recently identified TRPM7 blocker FTY720 has no effect on action potential firing rates of wild-type SAN cells. We conclude that TRPM7 influences diastolic membrane depolarization and automaticity in SAN indirectly via regulation of Hcn4 expression.
| S-EPMC3740880 | biostudies-other
Project description:Biomaterial-based bone grafts are emerged as an effective strategy for the treatment of large bone defects, especially for the scaffolds with enhanced osteogenic and angiogenic bioactivities. However, most studies focused on the direct interactions between scaffolds and bone-related cells such as osteoblasts and endothelial cells, and ignored the effects of material-triggered immunomodulation and the subsequent immune-regulated bone regeneration process. In this study, we developed a silicate bioceramic (Sr2ZnSi2O7, SZS) scaffold with well-defined pore structures using a three-dimensional (3D) printing technique. The prepared scaffolds were biodegradable, and the released bioactive ions were beneficial for immunomodulation, which stimulated macrophages to release more pro-healing cytokines and less pro-inflammatory cytokines. The obtained scaffold/macrophage conditioned medium further promoted the proliferation and osteogenic differentiation of a murine preosteoblast cell line (MC3T3-E1), as well as the angiogenic activity of human umbilical vein endothelial cells (HUVECs). Moreover, the in vivo experiments of critical-sized calvarial defects in rats revealed that the 3D printed SZS scaffolds could facilitate more vascularized bone regeneration than the 3D printed β-tricalcium phosphate (β-TCP, a typical clinically used bioceramic) scaffolds, suggesting that the 3D-printed SZS scaffolds hold the potential as implantable biomaterials with favorable osteoimmunomodulation for bone repair.
| S-EPMC9523139 | biostudies-literature
Project description:Gelatin methacryloyl (GelMA) has been widely used in bone engineering. It can also be filled into the calvarial defects with irregular shape. However, lack of osteoinductive capacity limits its potential as a candidate repair material for calvarial defects. In this study, we developed an injectable magnesium-zinc alloy containing hydrogel complex (Mg-IHC), in which the alloy was fabricated in an atomization process and had small sphere, regular shape, and good fluidity. Mg-IHC can be injected and plastically shaped. After cross-linking, it contents the elastic modulus similar to GelMA, and has inner holes suitable for nutrient transportation. Furthermore, Mg-IHC showed promising biocompatibility according to our evaluations of its cell adhesion, growth status, and proliferating activity. The results of alkaline phosphatase (ALP) activity, ALP staining, alizarin red staining, and real-time polymerase chain reaction (PCR) further indicated that Mg-IHC could significantly promote the osteogenic differentiation of MC3T3-E1 cells and upregulate the genetic expression of collagen I (COL-I), osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2). Finally, after applied to a mouse model of critical-sized calvarial defect, Mg-IHC remarkably enhanced bone formation at the defect site. All of these results suggest that Mg-IHC can promote bone regeneration and can be potentially considered as a candidate for calvarial defect repairing.
| S-EPMC7726114 | biostudies-literature
Project description:Lymphocytes lacking the TRPM7 (transient receptor potential cation channel, subfamily M, member 7) dual function ion channel/protein kinase exhibit a unique phenotype: they are unable to proliferate in regular media, but proliferate normally in media supplemented with 10-15 mM extracellular Mg(2+). Here, we have analyzed the molecular mechanisms underlying this phenotype. We find that upon transition from proliferation-supporting Mg(2+)-supplemented media to regular media, TRPM7-deficient cells rapidly downregulate their rate of growth, resulting in a secondary arrest in proliferation. The downregulated growth rate of transitioning cells is associated with a deactivation of signaling downstream from phosphoinositide 3-kinase, and expression of constitutively active p110 phosphoinositide 3-kinase is sufficient to support growth and proliferation of TRPM7-deficient cells in regular media. Together, these observations indicate that TRPM7 channels are required for sustained phosphoinositide 3-kinase-dependent growth signaling and therefore, that TRPM7 is positioned alongside phosphoinositide 3-kinases as a central regulator of lymphocyte growth and proliferation.
| S-EPMC3199037 | biostudies-literature
Project description:Multiple local and systemic factors including inflammation influence bone regeneration. Several lines of evidence demonstrate that macrophages contribute to the immunological regulation of MSC and osteoblast function during bone regeneration. Recent studies demonstrate that macrophage polarization influences this regulatory process. In this manuscript, we investigated the paracrine functional role of naïve (M0), M1 and M2 polarized macrophage derived EVs in bone repair. Treatment of rat calvaria defects with no EVs, M0 EVs, M1 EVs, or M2 EVs revealed polarization-specific control of bone regeneration by macrophage EVs at 3 and 6 weeks. M0 and M2 EVs promoted repair/regeneration and M1 EVs inhibited bone repair. Pathway-specific studies conducted in cell culture showed that M1 EVs negatively regulated the BMP signaling pathway, specifically BMP2 and BMP9. In parallel, miRNA sequencing studies showed similar miRNA cargo in M0 and M2 EVs and different miRNA cargo in M1 EVs. Functional examination of M1 macrophage EV-enriched miR-155 demonstrated that miR-155 mimic treatment reduced MSC osteogenic differentiation as measured by reduced BMP2, BMP9 and RUNX2 expression when compared to controls. Conversely, treatment of MSCs with the M2 macrophage EV-enriched miR-378a mimic increased MSC osteoinductive gene expression when compared to controls. These functional studies implicate polarized macrophage EV miRNAs in the positive or negative regulation of bone regeneration that was observed in vivo. Overall, the results presented in this study indicate that macrophage polarization influences EV cargo and related EV function in the paracrine regulation of bone regeneration.
| S-EPMC8107826 | biostudies-literature
Project description:The seek of bioactive materials for promoting bone regeneration is a challenging and long-term task. Functionalization with inorganic metal ions or drug molecules is considered effective strategies to improve the bioactivity of various existing biomaterials. Herein, amorphous calcium magnesium phosphate (ACMP) nanoparticles and simvastatin (SIM)-loaded ACMP (ACMP/SIM) nanocomposites were developed via a simple co-precipitation strategy. The physiochemical property of ACMP/SIM was explored using transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction (XRD) and high-performance liquid chromatograph (HPLC), and the role of Mg2+ in the formation of ACMP/SIM was revealed using X-ray absorption near-edge structure (XANES). After that, the transformation process of ACMP/SIM in simulated body fluid (SBF) was also tracked to simulate and explore the in vivo mineralization performance of materials. We find that ACMP/SIM releases ions of Ca2+, Mg2+ and PO 4 3 - , when it is immersed in SBF at 37°C, and a phase transformation occurred during which the initially amorphous ACMP turns into self-assembled hydroxyapatite (HAP). Furthermore, ACMP/SIM displays high cytocompatibility and promotes the proliferation and osteogenic differentiation of MC3T3-E1 cells. For the in vivo studies, lamellar ACMP/SIM/Collagen scaffolds with aligned pore structures were prepared and used to repair a rat defect model in calvaria. ACMP/SIM/Collagen scaffolds show a positive effect in promoting the regeneration of calvaria defect after 12 weeks. The bioactive ACMP/SIM nanocomposites are promising as bone repair materials. Considering the facile preparation process and superior in vitro/vivo bioactivity, the as-prepared ACMP/SIM would be a potential candidate for bone related biomedical applications.
| S-EPMC8670301 | biostudies-literature
Project description:Isocitrate dehydrogenase kinase/phosphatase (AceK) is the founding member of the protein phosphorylation system in prokaryotes. Based on the novel and unique structural characteristics of AceK recently uncovered, we sought to understand its kinase reaction mechanism, along with other features involved in the phosphotransfer process. Herein we report density functional theory QM calculations of the mechanism of the phosphotransfer reaction catalysed by AceK. The transition states located by the QM calculations indicate that the phosphorylation reaction, catalysed by AceK, follows a dissociative mechanism with Asp457 serving as the catalytic base to accept the proton delivered by the substrate. Our results also revealed that AceK prefers a single Mg(2+)-containing active site in the phosphotransfer reaction. The catalytic roles of conserved residues in the active site are discussed.
| S-EPMC3747045 | biostudies-literature