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Mechanical force-promoted osteoclastic differentiation via periodontal ligament stem cell exosomal protein ANXA3.

ABSTRACT: Exosomes play a critical role in intracellular communication. The biogenesis and function of exosomes are regulated by multiple biochemical factors. In the present study, we find that mechanical force promotes the biogenesis of exosomes derived from periodontal ligament stem cells (PDLSCs) and alters the exosomal proteome profile to induce osteoclastic differentiation. Mechanistically, mechanical force increases the level of exosomal proteins, especially annexin A3 (ANXA3), which facilitates exosome internalization to activate extracellular signal-regulated kinase (ERK), thus inducing osteoclast differentiation. Moreover, the infusion of exosomes derived from PDLSCs into mice promotes mechanical force-induced tooth movement and increases osteoclasts in the periodontal ligament. Collectively, this study demonstrates that mechanical force treatment promotes the biogenesis of exosomes from PDLSCs and increases exosomal protein ANXA3 to facilitate exosome internalization, which activates ERK phosphorylation, thus inducing osteoclast differentiation. Our findings shed light on new mechanisms for how mechanical force regulates the biology of exosomes and bone metabolism.

SUBMITTER: Huang HM

PROVIDER: S-EPMC9391435 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Mechanical force-promoted osteoclastic differentiation via periodontal ligament stem cell exosomal protein ANXA3.

Huang Hua-Ming HM Han Chun-Shan CS Cui Sheng-Jie SJ Zhou Yi-Kun YK Xin Tian-Yi TY Zhang Ting T Zhu Song-Biao SB Zhou Yan-Heng YH Yang Rui-Li RL

Stem cell reports 20220721 8

Exosomes play a critical role in intracellular communication. The biogenesis and function of exosomes are regulated by multiple biochemical factors. In the present study, we find that mechanical force promotes the biogenesis of exosomes derived from periodontal ligament stem cells (PDLSCs) and alters the exosomal proteome profile to induce osteoclastic differentiation. Mechanistically, mechanical force increases the level of exosomal proteins, especially annexin A3 (ANXA3), which facilitates exo ...[more]

PMID: 35868309

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Project description:BackgroundThe preference for glucose oxidative mode has crucial impacts on various physiological activities, including determining stem cell fate. External mechanical factors can play a decisive role in regulating critical metabolic enzymes and pathways of stem cells. Periodontal ligament stem cells (PDLSCs) are momentous effector cells that transform mechanical force into biological signals during the reconstruction of alveolar bone. However, mechanical stimuli-induced alteration of oxidative characteristics in PDLSCs and the underlying mechanisms have not been fully elucidated.MethodsHerein, we examined the expression of LDH and COX4 by qRT-PCR, western blot, immunohistochemistry and immunofluorescence. We detected metabolites of lactic acid and reactive oxygen species for functional tests. We used tetramethylrhodamine methyl ester (TMRM) staining and a transmission electron microscope to clarify the mitochondrial status. After using western blot and immunofluorescence to clarify the change of DRP1, we further examined MFF, PINK1, and PARKIN by western blot. We used cyclosporin A (CsA) to confirm the regulation of mitophagy and ceased the stretching as a rescue experiment.ResultsHerein, we ascertained that mechanical force could increase the level of LDH and decrease the expression of COX4 in PDLSCs. Simultaneously, the yield of reactive oxygen species (ROS) in PDLSC reduced after stretching, while lactate acid augmented significantly. Furthermore, mitochondrial function in PDLSCs was negatively affected by impaired mitochondrial membrane potential (MMP) under mechanical force, and the augment of mitochondrial fission further induced PRKN-dependent mitophagy, which was confirmed by the rescue experiments via blocking mitophagy. As a reversible physiological stimulation, the anaerobic preference of PDLSCs altered by mechanical force could restore after the cessation of force stimulation.ConclusionsAltogether, our study demonstrates that PDLSCs under mechanical force preferred anaerobic oxidation induced by the affected mitochondrial dynamics, especially mitophagy. Our findings support an association between mechanical stimulation and the oxidative profile of stem cells, which may shed light on the mechanical guidance of stem cell maintenance and commitment, and lay a molecular foundation for periodontal tissue regeneration.

Project description:BackgroundAn increasing number of studies have shown that dysregulated miR-589-3p is associated with multiple diseases. However, the role of miR-589-3p in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) remains unknown. This study aimed to explore the biological function and potential molecular mechanism of miR-589-3p in osteogenic differentiation of PDLSCs.MethodsGSE159508 was downloaded from Gene Expression Omibus (GEO, http://www.ncbi.nlm.nih.gov/geo/ ). Differentially expressed miRNAs between osteogenic induction PDLSCs versus non-induction PDLSCs were obtained by R software. miR-589-3p mimic and miR-589-3p inhibitor and corresponding negative control were obtained and to identify the role of miR-589-3p in osteogenic differentiation of PDLSCs. ALP staining and ARS were used to evaluate ALP activity and mineralization, respectively. The targeted binding relationship between miR-589-3p and ATF1 was predicted and verified by target prediction analysis and dual-luciferase assay. Furthermore, the functional mechanism based on miR-589-3p and ATF1 in osteogenic differentiation of PDLSCs was further investigated through rescue experiments.ResultsAccording to the cut-off criteria with log 2 FC > 1.0 and P < 0.05, 514 differentially expressed miRNAs were identified between osteogenic induction and non-induction PDLSCs, including 309 upregulated miRNAs and 205 downregulated miRNAs. Compared with control PDLSCs, miR-589-3p expression level was notably increased in PDLSCs that underwent osteogenic induction. The overexpression of miR-589-3p promoted the cell viability of PDLSCs, while the low expression of miR-589-3p had the opposite effect. The dual luciferase reporter assay verified that ATF1 was proved to be a direct target of miR-589-3p in PDLSCs. And overexpressed miR-589-3p reduced the expression of ATF1. Overexpression of miR-589-3p enhanced the osteogenic capacity of PDLSCs, as demonstrated by increases in ALP activity, matrix mineralization, and RUNX2, OCN and OSX expression. In addition, the rescue experiments confirmed that overexpressed ATF1 restored the effects of overexpressed miR-589-3p on cell proliferation and osteogenic differentiation of PDLSCs.ConclusionmiR-589-3p could down-regulate the expression of ATF1, thereby promote the proliferation and osteogenic differentiation of PDLSCs. This finding may provide a new therapeutic target for molecular therapy of periodontitis.

Project description:Introduction and aimsSpecific circular RNAs (circRNAs) have been proven to play crucial roles in osteogenesis in vitro and in vivo. This study aims to identify a certain circRNA involved in the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and explore its regulatory role.MethodsThe expression of 5 candidate circRNAs (circ_0026344, circ_ACAP2, circ_0003764, circ_0008259, and circ_0060731) was detected by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) after PDLSCs were cultured in the osteogenic induction medium or medium supplemented with tumour necrosis factor-α (TNF-α, 10 ng/mL) for 3 and 7 days. The circRNA significantly decreased in both 3 and 7 days of osteogenic induction in PDLSCs and markedly increased in TNF-α-induced PDLSCs for 3 and 7 days screened. Identified circRNA was knocked down or overexpressed, and the effect on the osteogenic differentiation of PDLSCs was investigated by qRT-PCR, western blot, alkaline phosphatase (ALP) staining, and alizarin red S (ARS) staining. Cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were applied to detect the effect of the circRNA on the proliferation of PDLSCs.ResultsqRT-PCR results showed that the expression of circ_0003764 was significantly decreased when PDLSCs were cultured in the osteogenic induction medium for 3 or 7 days, whereas it was dramatically increased in TNF-α-induced PDLSCs. Knockdown of circ_0003764 promoted the expression of the osteogenesis-related genes (RUNX2, ALP, OCN) and proteins (RUNX2, OCN), enhanced the ALP activity, and elevated the mineralization by PDLSCs, as shown by ARS staining. However, with the overexpression of circ_0003764, the osteogenic differentiation capacity of PDLSCs was significantly reduced. The CCK-8 and EdU results indicated that circ_0003764 could inhibit the proliferation of PDLSCs.ConclusionCirc_0003764 is involved in the osteogenesis process and inhibits the osteogenic differentiation and proliferation of PDLSCs.Clinical relevanceThis study indicates that circ_0003764 can serve as a diagnostic and therapeutic target in bone regeneration-related diseases treated by PDLSCs-based tissue engineering.

Project description:Stromal cell-derived factor-1 (SDF-1) and Exendin-4 (EX-4) play beneficial roles in promoting periodontal ligament stem cells (PDLSCs) osteogenic differentiation, while the detailed mechanism has not been clarified. In this study, we aimed to evaluate the biological mechanism of SDF-1 and EX-4 alone or synergistic application in regulating PDLSCs differentiation by RNA-sequencing (RNA-seq). A total of 110, 116 and 109 differentially expressed genes (DEGs) were generated in osteogenic medium induced PDLSCs treated by SDF-1, EX-4, and SDF-1+EX-4, respectively. The DEGs in SDF-1 group were enriched in signal transduction related signaling pathways; the DEGs in EX-4 group were enriched in metabolism and biosynthesis-related pathways; and the DEGs generated in SDF-1+EX-4 group were mainly enriched in RNA polymerase II transcription, cell differentiation, chromatin organization, protein phosphorylation pathways. Based on Venn analysis, a total of 37 specific DEGs were identified in SDF-1+EX-4 group, which were mainly enriched in negative regulation of autophagy and cellular component disassembly signaling pathways. Short time-series expression miner (STEM) analysis grouped all expressed genes of PDLSCs into 49 clusters according to the dynamic expression patterns and 25 genes, including NRSN2, CHD9, TUBA1A, distributed in 10 gene clusters in SDF-1+EX-4 treated PDLSCs were significantly up-regulated compared with the SDF-1 and EX-4 alone groups. The gene set enrichment analysis indicated that SDF-1 could amplify the role of EX-4 in regulating varied signaling pathways, such as type II diabetes mellitus and insulin signaling pathways; while EX-4 could aggravate the effect of SDF-1 on PDLSCs biological roles via regulating primary immunodeficiency, tight junction signaling pathways. In summary, our study confirmed that SDF-1 and EX-4 combined application could enhance PDLSCs biological activity and promote PDLSCs osteogenic differentiation by regulating the metabolism, biosynthesis and immune-related signaling pathways.

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Mechanical force-promoted osteoclastic differentiation via periodontal ligament stem cell exosomal protein ANXA3.

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Mechanical force-promoted osteoclastic differentiation via periodontal ligament stem cell exosomal protein ANXA3.

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