Dataset Information

Phenytoin Regulates Migration and Osteogenic Differentiation by MAPK Pathway in Human Periodontal Ligament Cells.

ABSTRACT:

Introduction

Periodontal healing requires an adequate number of periodontal ligament (PDL) cells to rebuild the impaired tissue. Phenytoin (PHT) has been reported to promote wound healing and extracellular matrix deposition, which indicates its promising application of periodontal healing. However, the effects of PHT on PDL cells behavior and the underlying mechanism are still unknown.

Methods

Human PDL cells were cultured and identified. 20-100 μg/mL PHT were used in our study. The proliferation of PDL cells was determined by the EdU assay. A wound healing assay was used to detect cell migration. Matrix metalloproteinase (MMP)-1, MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 expression were analyzed by real time-PCR. The protein expression of MMP-1 and phosphorylated mitogen-activated protein kinases (MAPKs) were detected by western blotting assay. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) staining.

Results

We found that 20-100 μg/mL of PHT did not affect PDL cells proliferation, whereas 50-100 μg/mL of PHT inhibited cell migration. The 50 or 100 μg/mL of PHT decreased the gene and protein expression of MMP-1, but increased the gene expression of TIMP-1. MMP-2 and TIMP-2 were not affected by 20-100 μg/mL of PHT. Further, 20-50 μg/mL of PHT increased ALP expression, but 100 μg/mL of PHT depressed ALP expression. The extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 were activated by PHT. JNK and ERK are involved in PHT-regulated migration. JNK plays an essential role in PHT-induced osteogenic differentiation.

Conclusions

MAPK pathway involved in PHT-regulated migration and osteogenic differentiation in human PDL cells.

SUBMITTER: Na J

PROVIDER: S-EPMC8761188 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Publications

Phenytoin Regulates Migration and Osteogenic Differentiation by MAPK Pathway in Human Periodontal Ligament Cells.

Na Jing J Zheng Lisha L Wang Lijuan L Shi Qiusheng Q Yang Zhijie Z Liu Nan N Guo Yuwei Y Fan Yubo Y

Cellular and molecular bioengineering 20210910 1

<h4>Introduction</h4>Periodontal healing requires an adequate number of periodontal ligament (PDL) cells to rebuild the impaired tissue. Phenytoin (PHT) has been reported to promote wound healing and extracellular matrix deposition, which indicates its promising application of periodontal healing. However, the effects of PHT on PDL cells behavior and the underlying mechanism are still unknown.<h4>Methods</h4>Human PDL cells were cultured and identified. 20-100 <i>μ</i>g/mL PHT were used in our s ...[more]

PMID: 35096190

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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:BackgroundHigh glucose-induced damage to the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) has long been a challenge to periodontal regeneration for diabetic individuals. Metformin is an anti-hyperglycemic drug that exhibits abundant biological activities associated with cell metabolism and downstream tissue regeneration. However, how metformin combats damage to PDLSC osteogenic differentiation under high glucose and the underlying mechanisms remain unknown.MethodsOsteogenic differentiation of PDLSCs was assessed by alkaline phosphatase (ALP) staining, ALP activity, Alizarin Red staining and quantitative assay, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. RNA-seq analysis was performed to screen target genes of metformin, and the effects of target genes were confirmed using lentivirus transfection. Western blot analysis was also used to detect the protein level of underlying signaling pathways.ResultsWe found that osteogenic differentiation of PDLSCs under high glucose was decreased, and metformin addition enhanced this capacity of differentiation. Furthermore, the results of RNA-seq analysis showed that natriuretic peptide receptor 3 (NPR3) was upregulated in PDLSCs under high glucose and downregulated after metformin addition. When the underlying pathways involved were investigated, we found that upregulation of NPR3 can compromise the metformin-enhanced PDLSC osteogenic differentiation and activate the MAPK pathway (especially the p38 MAPK and Erk1/2 pathway), and that inhibition of the NPR3-mediated p38 MAPK or Erk1/2 pathway enhanced the osteogenic differentiation of PDLSCs under high glucose.ConclusionsThe present study suggests that metformin may enhance the osteogenic differentiation of PDLSCs under high glucose via downregulation of NPR3 and inhibition of its downstream MAPK pathway. This is the first report identifying the involvement of NPR3-mediated MAPK pathway in the metformin-enhanced osteogenic differentiation, indicating that NPR3 antagonists, such as metformin, may be feasible therapeutics for periodontal tissue regeneration in diabetic individuals.

Project description:Mesenchymal stem cell (MSC)-mediated periodontal tissue regeneration is considered to be a promising method for periodontitis treatment. The molecular mechanism of functional regulation by MSCs remains unclear, thus limiting their application. Our previous study discovered that Periostin (POSTN) promoted the migration and osteogenic differentiation of periodontal ligament mesenchymal stem cells (PDLSCs), but it is still unclear whether POSTN is able to restore the regenerative potential of PDLSCs under inflammatory conditions. In this study, we investigated the effect of POSTN on PDLSCs under inflammatory conditions and its mechanism. PDLSCs were isolated from periodontal ligament tissue. TNF-α was used at 10 ng/mL to mimic inflammatory conditions. Lentivirus POSTN shRNA was used to knock down POSTN. Recombinant human POSTN (rhPOSTN) was used to stimulate PDLSCs. A scratch assay was used to analyse cell migration. Alkaline phosphatase (ALP) activity, Alizarin Red staining and expression of osteogenesis-related genes were used to investigate the osteogenic differentiation potential. Western blot analysis was used to detect the mitogen-activated protein kinases (MAPK) and AKT signalling pathways. After a 10 ng/mL TNF-α treatment, knockdown of POSTN impeded scratch closure, inhibited ALP activity and mineralization in vitro, and decreased expression of RUNX2, OSX, OPN and OCN in PDLSCs, while 75 ng/mL rhPOSTN significantly accelerated scratch closure, enhanced ALP activity and mineralization in vitro, and increased expression of RUNX2, OSX, OPN and OCN. In addition, knockdown of POSTN inhibited expression of phosphorylated c-Jun N-terminal kinase (p-JNK), while 75 ng/mL rhPOSTN increased expression of p-JNK in PDLSCs with TNF-α treatment. Furthermore, inhibition of JNK by its inhibitor SP600125 dramatically blocked POSTN-enhanced scratch closure, ALP activity and mineralization in PDLSCs. Our results revealed that POSTN might promote the migration and osteogenic differentiation potential of PDLSCs via the JNK pathway, providing insight into the mechanism underlying MSC biology under inflammatory conditions and identifying a potential target for improving periodontal tissue regeneration.

Dataset Information

Phenytoin Regulates Migration and Osteogenic Differentiation by MAPK Pathway in Human Periodontal Ligament Cells.

Introduction

Methods

Results

Conclusions

Publications

Phenytoin Regulates Migration and Osteogenic Differentiation by MAPK Pathway in Human Periodontal Ligament Cells.

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