Mechanical memory based on chromatin and metabolism remodeling promotes proliferation and smooth muscle differentiation in mesenchymal stem cells.

Stem cells respond and remember mechanical cues from the microenvironment, which modulates their therapeutic effects. Chromatin organization and energy metabolism regulate the stem cell fate induced by mechanical cues. However, the mechanism of mechanical memory is still unclear. This study aimed to investigate the effects of mechanical amplitude, frequency, duration, and stretch cycle on mechanical memory in mesenchymal stem cells. It showed that the amplitude was the dominant parameter to the persistence of cell alignment. F-actin, paxillin, and nuclear deformation are more prone to be remolded than cell alignment. Stretching induces transcriptional memory, resulting in greater transcription upon subsequent reloading. Cell metabolism displays mechanical memory with sustained mitochondrial fusion and increased ATP production. The mechanical memory of chromatin condensation is mediated by histone H3 lysine 27 trimethylation, leading to much higher smooth muscle differentiation efficiency. Interestingly, mechanical memory can be transmitted based on direct cell-cell interaction, and stretched cells can remodel the metabolic homeostasis of static cells. Our results provide insight into the underlying mechanism of mechanical memory and its potential benefits for stem cell therapy.

Frizzled-9 triggers actin polymerization and activates mechano-transducer YAP to rescue simulated microgravity-induced osteoblast dysfunction.

Long-term spaceflight can result in bone loss and osteoblast dysfunction. Frizzled-9 (Fzd9) is a Wnt receptor of the frizzled family that is vital for osteoblast differentiation and bone formation. In the present study, we elucidated whether Fzd9 plays a role in osteoblast dysfunction induced by simulated microgravity (SMG). After 1-7 days of SMG, osteogenic markers such as alkaline phosphatase (ALP), osteopontin (OPN), and Runt-related transcription factor 2 (RUNX2) were decreased, accompanied by a decrease in Fzd9 expression. Furthermore, Fzd9 expression decreased in the rat femur after 3 weeks of hindlimb unloading. In contrast, Fzd9 overexpression counteracted the decrease in ALP, OPN, and RUNX2 induced by SMG in osteoblasts. Moreover, SMG regulated phosphorylated glycogen synthase kinase-3ß (pGSK3ß) and ß-catenin expression or sublocalization. However, Fzd9 overexpression did not affect pGSK3ß and ß-catenin expression or sublocalization induced by SMG. In addition, Fzd9 overexpression regulated protein kinase B also known as Akt and extracellular signal-regulated kinase (ERK) phosphorylation and induced F-actin polymerization to form the actin cap, press the nuclei, and increase nuclear pore size, thereby promoting the nuclear translocation of Yes-associated protein (YAP). Our study findings provide mechanistic insights into the role of Fzd9 in triggering actin polymerization and activating YAP to rescue SMG-induced osteoblast dysfunction and suggest that Fzd9 is a potential target to restore osteoblast function in individuals with bone diseases and after spaceflight.

Cuticular competing endogenous RNAs regulate insecticide penetration and resistance in a major agricultural pest.

BACKGROUND: The continuously developing pesticide resistance is a great threat to agriculture and human health. Understanding the mechanisms of insecticide resistance is a key step in dealing with the phenomenon. Insect cuticle is recently documented to delay xenobiotic penetration which breaks the previous stereotype that cuticle is useless in insecticide resistance, while the underlying mechanism remains scarce. RESULTS: Here, we find the integument contributes over 40.0% to insecticide resistance via different insecticide delivery strategies in oriental fruit fly. A negative relationship exists between cuticle thickening and insecticide penetration in resistant/susceptible, also in field strains of oriental fruit fly which is a reason for integument-mediated resistance. Our investigations uncover a regulator of insecticide penetration that miR-994 mimic treatment causes cuticle thinning and increases susceptibility to malathion, whereas miR-994 inhibitor results in opposite phenotypes. The target of miR-994 is a most abundant cuticle protein (CPCFC) in resistant/susceptible integument expression profile, which possesses capability of chitin-binding and influences the cuticle thickness-mediated insecticide penetration. Our analyses find an upstream transcriptional regulatory signal of miR-994 cascade, long noncoding RNA (lnc19419), that indirectly upregulates CPCFC in cuticle of the resistant strain by sponging miR-994. Thus, we elucidate the mechanism of cuticular competing endogenous RNAs for regulating insecticide penetration and demonstrate it also exists in field strain of oriental fruit fly. CONCLUSIONS: We unveil a regulatory axis of lnc19419 ~ miR-994 ~ CPCFC on the cuticle thickness that leads to insecticide penetration resistance. These findings indicate that competing endogenous RNAs regulate insecticide resistance by modulating the cuticle thickness and provide insight into the resistance mechanism in insects.

Fluid shear stress promotes periodontal ligament cells proliferation via p38-AMOT-YAP.

Periodontal ligament (PDL) cells are a promising tool for periodontal regeneration therapy. Achieving a sufficient number of PDL cells is essential to PDL regeneration. In our study, appropriate flow shear stress (FSS, 1-6 dyn/cm2) promotes the proliferation of PDL cells. FSS remodels cytoskeleton and focal adhesion in a duration-dependent manner. FSS induces PDL cells to form the actin cap within 10 min, flattens the nuclei, and increases the nuclear pore size, which promotes nuclear translocation of Yes-associated protein (YAP). FSS activates p38, which plays a dual function in YAP regulation. p38 regulates the phosphorylation of Akt and cofilin, as well as induced F-actin polymerization to induce YAP activity. In addition, p38 inhibits pLATS and consecutively regulates angiomotin (AMOT) and YAP phosphorylation. AMOT competitively binds to F-actin and YAP to participate in FSS-mediated YAP nuclear translocation and cell proliferation. Taken collectively, our results provide mechanistic insights into the role of p38-AMOT-YAP in FSS-mediated PDL cells proliferation and indicate potential applications in dental regenerative medicine.

Wnt/ß-catenin plays a dual function in calcium hydroxide induced proliferation, migration, osteogenic differentiation and mineralization in vitro human dental pulp stem cells.

AIM: Calcium hydroxide is the gold standard material for pulp capping and has been widely used in clinical dentistry. Calcium hydroxide promotes proliferation, migration and osteogenic differentiation of dental pulp stem cells (DPSCs). However, the underlying mechanism is not clear. Our study investigated the role of Wnt/ß-catenin pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation and mineralization of human DPSCs. METHODOLOGY: Protein and gene expression was detected by western blot (WB), immunofluorescence staining and quantitative real-time PCR (qPCR). Cell viability was analysed using the Cell Counting Kit-8 (CCK-8) assay. Wound-healing assay was used to analyse cell migration. The expression of alkaline phosphatase (ALP) was detected using ALP staining. Mineralization was analysed by alizarin red staining. RESULTS: Calcium hydroxide increased the protein expression of phosphorylated-GSK3ß/GSK3ß, ß-catenin and the gene expression of LEF-1. Inhibition of Wnt/ß-catenin abolished calcium hydroxide-induced proliferation and migration of DPSCs in 24 h. However, incubation with calcium hydroxide for 7 days and 14 days reduced Wnt/ß-catenin signalling. Inhibition of Wnt/ß-catenin promoted calcium hydroxide-induced osteogenic differentiation and mineralization in DPSCs. CONCLUSION: Wnt/ß-catenin pathway plays a dual role in calcium hydroxide-regulated DPSC behaviour. Incubation with calcium hydroxide promoted rapid proliferation and migration of DPSCs, while prolonged incubation negatively regulated osteogenic differentiation and mineralization.

[Differentiation of stem cells regulated by biophysical cues].

Stem cells have been regarded with promising application potential in tissue engineering and regenerative medicine due to their self-renewal and multidirectional differentiation abilities. However, their fate is relied on their local microenvironment, or niche. Recent studied have demonstrated that biophysical factors, defined as physical microenvironment in which stem cells located play a vital role in regulating stem cell committed differentiation. In vitro, synthetic physical microenvironments can be used to precisely control a variety of biophysical properties. On this basis, the effect of biophysical properties such as matrix stiffness, matrix topography and mechanical force on the committed differentiation of stem cells was further investigated. This paper summarizes the approach of mechanical models of artificial physical microenvironment and reviews the effects of different biophysical characteristics on stem cell differentiation, in order to provide reference for future research and development in related fields.

Knockdown of a ß-Adrenergic-Like Octopamine Receptor Affects Locomotion and Reproduction of Tribolium castaneum.

The neurohormone octopamine regulates many crucial physiological processes in insects and exerts its activity via typical G-protein coupled receptors. The roles of octopamine receptors in regulating behavior and physiology in Coleoptera (beetles) need better understanding. We used the red flour beetle, Tribolium castaneum, as a model species to study the contribution of the octopamine receptor to behavior and physiology. We cloned the cDNA of a ß-adrenergic-like octopamine receptor (TcOctß2R). This was heterologously expressed in human embryonic kidney (HEK) 293 cells and was demonstrated to be functional using an in vitro cyclic AMP assay. In an RNAi assay, injection of dsRNA demonstrated that TcOctß2R modulates beetle locomotion, mating duration, and fertility. These data present some roles of the octopaminergic signaling system in T. castaneum. Our findings will also help to elucidate the potential functions of individual octopamine receptors in other insects.

Influence of Micropatterning on Human Periodontal Ligament Cells' Behavior.

The periodontal ligament (PDL) is highly ordered connective tissue located between the alveolar bone and cementum. An aligned and organized architecture is required for its physiological function. We applied micropatterning technology to arrange PDL cells in 10- or 20-µm-wide extracellular protein patterns. Cell and nuclear morphology, cytoskeleton, proliferation, differentiation, and matrix metalloproteinase system expression were investigated. Micropatterning clearly elongated PDL cells with a low cell-shape index and low spreading area. The nucleus was also elongated as nuclear height increased, but the nuclear volume remained intact. The cytoskeleton was rearranged to form prominent bundles at cells' peripheral regions. Moreover, proliferation was promoted by 10- and 20-µm micropatterning. Osteogenesis and adipogenesis were each inhibited, but micropatterning increased PDL cells' stem cell markers. ß-catenin was expelled to cytoplasm. YAP/TAZ nuclear localization and activity both decreased, which might indicate their role in micropatterning-regulated differentiation. Collagen Ι expression increased in micropatterned groups. It might be due to the decreased expression of matrix metalloproteinase-1, 2 and the tissue inhibitor of metalloproteinase-1 gene expression elevation in micropatterned groups. The findings of this study provide insight into the effects of a micropatterned surface on PDL cell behavior and may be applicable in periodontal tissue regeneration.

Structural insight into the autoinhibition mechanism of AMP-activated protein kinase.

The AMP-activated protein kinase (AMPK) is characterized by its ability to bind to AMP, which enables it to adjust enzymatic activity by sensing the cellular energy status and maintain the balance between ATP production and consumption in eukaryotic cells. It also has important roles in the regulation of cell growth and proliferation, and in the establishment and maintenance of cell polarity. These important functions have rendered AMPK an important drug target for obesity, type 2 diabetes and cancer treatments. However, the regulatory mechanism of AMPK activity by AMP binding remains unsolved. Here we report the crystal structures of an unphosphorylated fragment of the AMPK alpha-subunit (KD-AID) from Schizosaccharomyces pombe that contains both the catalytic kinase domain and an autoinhibitory domain (AID), and of a phosphorylated kinase domain from Saccharomyces cerevisiae (Snf1-pKD). The AID binds, from the 'backside', to the hinge region of its kinase domain, forming contacts with both amino-terminal and carboxy-terminal lobes. Structural analyses indicate that AID binding might constrain the mobility of helix alphaC, hence resulting in an autoinhibited KD-AID with much lower kinase activity than that of the kinase domain alone. AMP activates AMPK both allosterically and by inhibiting dephosphorylation. Further in vitro kinetic studies demonstrate that disruption of the KD-AID interface reverses the autoinhibition and these AMPK heterotrimeric mutants no longer respond to the change in AMP concentration. The structural and biochemical data have shown the primary mechanism of AMPK autoinhibition and suggest a conformational switch model for AMPK activation by AMP.

Extracellular matrix stiffness as an energy metabolism regulator drives osteogenic differentiation in mesenchymal stem cells.

The biophysical factors of biomaterials such as their stiffness regulate stem cell differentiation. Energy metabolism has been revealed an essential role in stem cell lineage commitment. However, whether and how extracellular matrix (ECM) stiffness regulates energy metabolism to determine stem cell differentiation is less known. Here, the study reveals that stiff ECM promotes glycolysis, oxidative phosphorylation, and enhances antioxidant defense system during osteogenic differentiation in MSCs. Stiff ECM increases mitochondrial fusion by enhancing mitofusin 1 and 2 expression and inhibiting the dynamin-related protein 1 activity, which contributes to osteogenesis. Yes-associated protein (YAP) impacts glycolysis, glutamine metabolism, mitochondrial dynamics, and mitochondrial biosynthesis to regulate stiffness-mediated osteogenic differentiation. Furthermore, glycolysis in turn regulates YAP activity through the cytoskeletal tension-mediated deformation of nuclei. Overall, our findings suggest that YAP is an important mechanotransducer to integrate ECM mechanical cues and energy metabolic signaling to affect the fate of MSCs. This offers valuable guidance to improve the scaffold design for bone tissue engineering constructs.

Early Predicting Osteogenic Differentiation of Mesenchymal Stem Cells Based on Deep Learning Within One Day.

Osteogenic differentiation of mesenchymal stem cells (MSCs) is proposed to be critical for bone tissue engineering and regenerative medicine. However, the current approach for evaluating osteogenic differentiation mainly involves immunohistochemical staining of specific markers which often can be detected at day 5-7 of osteogenic inducing. Deep learning (DL) is a significant technology for realizing artificial intelligence (AI). Computer vision, a branch of AI, has been proved to achieve high-precision image recognition using convolutional neural networks (CNNs). Our goal was to train CNNs to quantitatively measure the osteogenic differentiation of MSCs. To this end, bright-field images of MSCs during early osteogenic differentiation (day 0, 1, 3, 5, and 7) were captured using a simple optical phase contrast microscope to train CNNs. The results showed that the CNNs could be trained to recognize undifferentiated cells and differentiating cells with an accuracy of 0.961 on the independent test set. In addition, we found that CNNs successfully distinguished differentiated cells at a very early stage (only 1 day). Further analysis showed that overall morphological features of MSCs were the main basis for the CNN classification. In conclusion, MSCs differentiation detection can be achieved early and accurately through simple bright-field images and DL networks, which may also provide a potential and novel method for the field of cell detection in the near future.

Coupling of Perinuclear Actin Cap and Nuclear Mechanics in Regulating Flow-Induced Yap Spatiotemporal Nucleocytoplasmic Transport.

Mechanical forces, including flow shear stress, govern fundamental cellular processes by modulating nucleocytoplasmic transport of transcription factors like Yes-associated Protein (YAP). However, the underlying mechanical mechanism remains elusive. In this study, it is reported that unidirectional flow induces biphasic YAP transport with initial nuclear import, followed by nuclear export as actin cap formation and nuclear stiffening. Conversely, pathological oscillatory flow induces slight actin cap formation, nuclear softening, and sustained YAP nuclear localization. To elucidate the disparately YAP spatiotemporal distribution, a 3D mechanochemical model is developed, which integrates flow sensing, cytoskeleton organization, nucleus mechanotransduction, and YAP transport. The results unveiled that despite the significant localized nuclear stress imposed by the actin cap, its inherent stiffness counteracts the dispersed contractile stress exerted by conventional fibers on the nuclear membrane. Moreover, alterations in nuclear stiffness synergistically regulate nuclear deformation, thereby governing YAP transport. Furthermore, by expanding the single-cell model to a collective vertex framework, it is revealed that the irregularities in actin cap formation within individual cells have the potential to induce topological defects and spatially heterogeneous YAP distribution in the cellular monolayer. This work unveils a unified mechanism of flow-induced nucleocytoplasmic transport, providing a linkage between transcription factor localization and mechanical stimulation.

Involvement of large conductance Ca(2+)-activated K (+) channel in laminar shear stress-induced inhibition of vascular smooth muscle cell proliferation.

The large conductance Ca(2+)-activated K(+) (BK(Ca)) channel in vascular smooth muscle cell (VSMC) is an important potassium channel that can regulate vascular tone. Recent work has demonstrated that abnormalities in BK(Ca) channel function are associated with changes in cell proliferation and the onset of vascular disease. However, until today there are rare reports to show whether this channel is involved in VSMC proliferation in response to fluid shear stress (SS). Here we investigated a possible role of BK(Ca) channel in VSMC proliferation under laminar SS. Rat aortic VSMCs were plated in parallel-plate flow chambers and exposed to laminar SS with varied durations and magnitudes. VSMC proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA) expression and DNA synthesis. BK(Ca) protein and gene expression was determined by flow cytometery and RT-PCR. The involvement of BK(Ca) in SS-induced inhibition of proliferation was examined by BK(Ca) inhibition using a BK(Ca) specific blocker, iberiotoxin (IBTX), and by BK(Ca) transfection in BK(Ca) non-expressing CHO cells. The changes in [Ca(2+)](i) were determined using a calcium-sensitive dye, fluo 3-AM. Membrane potential changes were detected with a potential-sensitive dye, DiBAC(4)(3). We found that laminar SS inhibited VSMC proliferation and stimulated BK(Ca) channel expression. Furthermore, laminar SS induced an increase in [Ca(2+)](i) and membrane hyperpolarization. Besides in VSMC, the inhibitory effect of BK(Ca) channel activity on cell proliferation in response to SS was also confirmed in BK(Ca)-transfected CHO cells showing a decline in proliferation. Blocking BK(Ca) channel reversed its inhibitory effect, providing additional support for the involvement of BK(Ca) in SS-induced proliferation reduction. Our results suggest, for the first time, that BK(Ca) channel mediates laminar SS-induced inhibition of VSMC proliferation. This finding is important for understanding the mechanism by which SS regulates VSMC proliferation, and should be helpful in developing strategies to prevent flow-initiated vascular disease formation.

Complete genomic sequence of a Muscovy duck-origin reticuloendotheliosis virus from China.

The complete proviral sequence of a Muscovy duck-origin reticuloendotheliosis virus (REV) associated with spontaneously occurring neoplastic disease in 2011 in Zhejiang province, China, was determined. Comparative sequence analyses indicate that the present REV is most closely related to the chicken-origin REV isolate HLJR0901 and the goose-origin isolate Goose/3410/06. These findings suggest that chickens or geese may transmit the REV to Muscovy ducks.

Pulse Capacitive Coupling Electric Field Regulates Cell Migration, Proliferation, Polarization, and Vascularization to Accelerate Wound Healing.

Objectives: Accelerating wound healing using continuous exogenous electrical stimulation is limited due to some serious side effects, including thermal damage. Many previous studies based on direct current contact stimulation may cause chemical burns or blisters, thereby increasing patients suffering. The aim of this study was to develop a safer and more convenient pulse capacitive coupling electrical field (PCCEF) stimulation to accelerate wound healing. Approach: A PCCEF-generating platform was self-designed to facilitate wound healing. The promoting effects and appropriate pulse width were explored by applying PCCEFs (54 mV/mm, 60 Hz) of different pulse widths to various cells involved in wound healing and mouse models for 2 h daily. Results: PCCEFs of ≥10 µs pulse width showed marked promotion of the migration and proliferation of human dermal fibroblasts and HaCaT cells, enhanced the M2-type polarization and YPA/TAZ expression of macrophages, and facilitated the wound healing of mouse models. Comprehensive histological results suggested that PCCEF of 100 µs pulse width exerted the most positive effects. Innovation: A safe and effective PCCEF was developed to promote wound healing, which prevented prolonged stimulation and averted direct contact. Conclusion: PCCEF accelerated wound healing, especially at the optimal 100 µs pulse width, and was expected to be translated to clinical application, helping alleviate patient suffering, while reducing side effects.

An Antenna-Abundant Glutathione S-Transferase BdGSTd8 Participates in Detoxification of Two Organophosphorus Insecticides in Bactrocera dorsalis (Hendel).

The oriental fruit fly, Bactrocera dorsalis, is a damaging insect pest for many vegetable and fruit crops that has evolved severe chemical insecticide resistance, including organophosphorus, neonicotinoid, pyrethroid, and macrolides. Hence, it is important to elucidate its detoxification mechanism to improve its management and mitigate resource destruction. Glutathione S-transferase (GST) is a critical secondary phase enzyme that plays multiple detoxification functions against xenobiotics. In this study, we identified several BdGSTs by characterizing their potential relationships with five insecticides using inducible and tissue-specific expression pattern analyses. We found that an antenna-abundant BdGSTd8 responded to four different classes of insecticides. Subsequently, our immunohistochemical and immunogold staining analysis further confirmed that BdGSTd8 was primarily located in the antenna. Our investigations also confirmed that BdGSTd8 possesses the capability to enhance cell viability by directly interacting with malathion and chlorpyrifos, which clarified the function of antenna-abundant GST in B. dorsalis. Altogether, these findings enrich our understanding of GST molecular characteristics in B. dorsalis and provide new insights into the detoxification of superfluous xenobiotics in the insect antenna.

Mechanically sensitive HSF1 is a key regulator of left-right symmetry breaking in zebrafish embryos.

The left-right symmetry breaking of vertebrate embryos requires nodal flow. However, the molecular mechanisms that mediate the asymmetric gene expression regulation under nodal flow remain elusive. Here, we report that heat shock factor 1 (HSF1) is asymmetrically activated in the Kupffer's vesicle of zebrafish embryos in the presence of nodal flow. Deficiency in HSF1 expression caused a significant situs inversus and disrupted gene expression asymmetry of nodal signaling proteins in zebrafish embryos. Further studies demonstrated that HSF1 is a mechanosensitive protein. The mechanical sensation ability of HSF1 is conserved in a variety of mechanical stimuli in different cell types. Moreover, cilia and Ca2+-Akt signaling axis are essential for the activation of HSF1 under mechanical stress in vitro and in vivo. Considering the conserved expression of HSF1 in organisms, these findings unveil a fundamental mechanism of gene expression regulation by mechanical clues during embryonic development and other physiological and pathological transformations.

A continuous spectrophotometric assay for mitogen-activated protein kinase kinases.

We describe a convenient and simple continuous spectrophotometric method for the determination of mitogen-activated protein kinase (MAPK) kinase activity with its protein substrate. The assay relies on the measurement of phosphoprotein product generated in the first step of the MAPK kinase reaction. Dephosphorylation of the phosphoprotein is coupled to a MAPK phosphatase to generate phosphate, which is then used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methyl purine ribonucleoside. Of the reaction products ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360nm relative to the nucleoside and, hence, provides a spectrophotometric signal that can be continuously followed. In the presence of excess phosphatase, the phosphorylated protein substrate molecules undergo dephosphorylation almost immediately after their formation; the steady-state use of the resultant inorganic phosphate is a reflection of the constant initial velocity of the exchange reaction. The validity of this method has been confirmed by using it to measure the activities of MEK1 (MAPK/ERK kinase 1) and MKK6 (MAPK kinase 6) toward their physiological substrates. Our findings of the MAPK kinases in the current study provide evidence that the substrate binding affinities of this subfamily of protein kinases are at the submicromolar concentration.

HDAC1 promotes the migration of human myeloma cells via regulation of the lncRNA/Slug axis.

Understanding the mechanisms underlying malignancy in myeloma cells is important for targeted treatment and drug development. Histone deacetylases (HDACs) can regulate the progression of various cancer types; however, their roles in myeloma are not well known. In the present study, the expression of class I HDACs in myeloma cells and tissues was evaluated. Furthermore, the effects of HDAC1 on the migration of myeloma cells and the associated mechanisms were investigated. Among the class I HDACs evaluated, HDAC1 was upregulated in both myeloma cells and tissues. Targeted inhibition of HDAC1 suppressed the migration of myeloma cells. Of the assessed transcription factors, small interfering (si)­HDAC1 decreased the expression of Slug. Overexpression of Slug reversed the si­HDAC1­mediated suppressed migration of myeloma cells. Mechanistically, the results revealed that HDAC1 regulated the mRNA stability of Slug, while it had no effect on its transcription or nuclear export. Furthermore, HDAC1 negatively regulated the expression of long non­coding RNA (lncRNA) NONHSAT113026, which could bind with the 3'­untranslated region of Slug mRNA to facilitate its degradation. The present study demonstrated that HDAC1 promoted the migration of human myeloma cells via regulation of lncRNA/Slug signaling.

Static magnetic field regulates proliferation, migration, and differentiation of human dental pulp stem cells by MAPK pathway.

Magnetic materials are now commonly used in dental clinics. These materials generally produce a static magnetic field (SMF). While it is known that SMF can affect cells' behaviors such as proliferation, migration, and differentiation, the mechanisms underlying these effects are still unclear. Our study investigates the role of the mitogen-activated protein (MAP) kinase pathway in SMF-induced proliferation, migration, osteogenic/odontogenic differentiation, and mineralization in human dental pulp stem cells (DPSCs). Human DPSCs were exposed to SMF of 1 mT and the phosphorylated MAP kinases were detected by Western blot analysis. Three MAP kinases inhibitors were pre-cultured with DPSCs and exposed to SMF for 24 h. Cell viability was analyzed using Cell Counting Kit-8. Cell migration was tested by a wound healing assay. Osteogenic/odontogenic differentiation was detected by ALP staining assay, ALP and DSPP Western blot analysis. Mineralization was studied by alizarin red staining analysis. SMF activated phosphorylation of c-Jun N-terminal kinase (JNK), P38 and extracellular signal-regulated kinase (ERK). The inhibition of JNK, P38, and ERK signaling decreased SMF-induced proliferation and migration. ERK and P38 play more important roles in SMF-induced ALP staining and protein expression. JNK was vital for SMF-induced DSPP expression. JNK, P38, and ERK all involved in SMF-mediated mineralization. Our study demonstrated that the MAPK pathway regulated SMF-induced proliferation, migration, osteogenic/odontogenic differentiation, and mineralization in human DPSCs.