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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.

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Abbreviations

PDL:

Periodontal ligament

PHT:

Phenytoin

EdU:

5-Ethynyl-2′-deoxyuridine

PMSF:

Phenylmethane sulfonyl fluoride

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene fluoride

TBS-T:

Triethanolamine buffered saline solution

HRP:

Horseradish peroxidase

ECL:

Enhanced chemiluminescent

BCIP/NBT:

5-Bromo-4-chloro-3-inodlyl-phosphate/Nitro-blue-tetrazolium

References

  1. Bharathi Mohan, P., U. K. Chapa, R. K. Chittoria, V. Chavan, A. Aggarwal, S. Gupta, C. L. Reddy, I. Pathan, and S. Koliyath. Role of phenytoin in diabetic foot ulcers. J. Cutan. Aesthet. Surg. 13:222–225, 2020.

    Google Scholar 

  2. Chen, L., L. Zheng, J. Jiang, J. Gui, L. Zhang, Y. Huang, X. Chen, J. Ji, and Y. Fan. Calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization via the mitogen-activated protein kinase pathway in human dental pulp stem cells. J. Endod. 42:1355–1361, 2016.

    Google Scholar 

  3. Chow, K. M., and C. C. Szeto. Cerebral atrophy and skull thickening due to chronic phenytoin therapy. Can. Med. Assoc. J. 176:321–323, 2007.

    Google Scholar 

  4. Correa, J. D., C. M. Queiroz-Junior, J. E. Costa, A. L. Teixeira, and T. A. Silva. Phenytoin-induced gingival overgrowth: a review of the molecular, immune, and inflammatory features. ISRN Dent. 2011:497850, 2011.

    Google Scholar 

  5. Doshi, A., J. W. McAuley, and D. N. Tatakis. Topical phenytoin effects on palatal wound healing. J Periodontol. 92(3):409–418, 2020.

    Google Scholar 

  6. Gu, H., Z. Huang, X. Yin, J. Zhang, L. Gong, J. Chen, K. Rong, J. Xu, L. Lu, and L. Cui. Role of c-Jun N-terminal kinase in the osteogenic and adipogenic differentiation of human adipose-derived mesenchymal stem cells. Exp. Cell Res. 339:112–121, 2015.

    Google Scholar 

  7. Hains, B. C., C. Y. Saab, A. C. Lo, and S. G. Waxman. Sodium channel blockade with phenytoin protects spinal cord axons, enhances axonal conduction, and improves functional motor recovery after contusion SCI. Exp. Neurol. 188:365–377, 2004.

    Google Scholar 

  8. Hassell, T. M., R. C. Page, A. S. Narayanan, and C. G. Cooper. Diphenylhydantoin (dilantin) gingival hyperplasia: drug-induced abnormality of connective tissue. Proc. Natl. Acad. Sci. USA. 73:2909–2912, 1976.

    Google Scholar 

  9. Hesselink, J. M. K., and D. J. Kopsky. Phenytoin: 80 years young, from epilepsy to breast cancer, a remarkable molecule with multiple modes of action. J. Neurol. 264:1617–1621, 2017.

    Google Scholar 

  10. Hu, L., Y. Liu, and S. Wang. Stem cell-based tooth and periodontal regeneration. Oral Dis. 24:696–705, 2018.

    Google Scholar 

  11. Huang, C., Z. Rajfur, C. Borchers, M. D. Schaller, and K. Jacobson. JNK phosphorylates paxillin and regulates cell migration. Nature. 424:219–223, 2003.

    Google Scholar 

  12. Ikedo, D., K. Ohishi, N. Yamauchi, M. Kataoka, J. Kido, and T. Nagata. Stimulatory effects of phenytoin on osteoblastic differentiation of fetal rat calvaria cells in culture. Bone. 25:653–660, 1999.

    Google Scholar 

  13. Isaka, J., A. Ohazama, M. Kobayashi, C. Nagashima, T. Takiguchi, H. Kawasaki, T. Tachikawa, and K. Hasegawa. Participation of periodontal ligament cells with regeneration of alveolar bone. J. Periodontol. 72:314–323, 2001.

    Google Scholar 

  14. Janakiram, C., and B. A. Dye. A public health approach for prevention of periodontal disease. Periodontol. 2000(84):202–214, 2020.

    Google Scholar 

  15. Johnson, G. L., and R. Lapadat. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science. 298:1911–1912, 2002.

    Google Scholar 

  16. Kataoka, M., J. Kido, Y. Shinohara, and T. Nagata. Drug-induced gingival overgrowth—a review. Biol. Pharm. Bull. 28:1817–1821, 2005.

    Google Scholar 

  17. Kato, T., N. Okahashi, S. Kawai, T. Kato, H. Inaba, I. Morisaki, and A. Amano. Impaired degradation of matrix collagen in human gingival fibroblasts by the antiepileptic drug phenytoin. J. Periodontol. 76:941–950, 2005.

    Google Scholar 

  18. Kato, T., N. Okahashi, T. Ohno, H. Inaba, S. Kawai, and A. Amano. Effect of phenytoin on collagen accumulation by human gingival fibroblasts exposed to TNF-alpha in vitro. Oral Dis. 12:156–162, 2006.

    Google Scholar 

  19. Kim, H. Y., S. Y. Park, and S. Y. Choung. Enhancing effects of myricetin on the osteogenic differentiation of human periodontal ligament stem cells via BMP-2/Smad and ERK/JNK/p38 mitogen-activated protein kinase signaling pathway. Eur. J. Pharmacol. 834:84–91, 2018.

    Google Scholar 

  20. Klemke, R. L., S. Cai, A. L. Giannini, P. J. Gallagher, P. de Lanerolle, and D. A. Cheresh. Regulation of cell motility by mitogen-activated protein kinase. J. Cell Biol. 137:481–492, 1997.

    Google Scholar 

  21. Li, L., M. X. Han, S. Li, Y. Xu, and L. Wang. Hypoxia regulates the proliferation and osteogenic differentiation of human periodontal ligament cells under cyclic tensile stress via mitogen-activated protein kinase pathways. J. Periodontol. 85:498–508, 2014.

    Google Scholar 

  22. Lopez-Gonzalez, M. J., E. Luis, O. Fajardo, V. Meseguer, K. Gers-Barlag, S. Ninerola, and F. Viana. TRPA1 channels mediate human gingival fibroblast response to phenytoin. J. Dent. Res. 96:832–839, 2017.

    Google Scholar 

  23. Manokawinchoke, J., P. Sumrejkanchanakij, L. Boonprakong, P. Pavasant, H. Egusa, and T. Osathanon. NOTCH2 participates in Jagged1-induced osteogenic differentiation in human periodontal ligament cells. Sci. Rep. 10:13329, 2020.

    Google Scholar 

  24. Mohammed, F. H., M. A. Khajah, M. Yang, W. J. Brackenbury, and Y. A. Luqmani. Blockade of voltage-gated sodium channels inhibits invasion of endocrine-resistant breast cancer cells. Int. J. Oncol. 48:73–83, 2016.

    Google Scholar 

  25. Nakade, O., D. J. Baylink, and K. H. Lau. Phenytoin at micromolar concentrations is an osteogenic agent for human-mandible-derived bone cells in vitro. J. Dent. Res. 74:331–337, 1995.

    Google Scholar 

  26. Nikolov, A., N. Popovski, and I. Hristova. Collagenases MMP-1, MMP-13, and tissue inhibitors TIMP-1, TIMP-2: their role in healthy and complicated pregnancy and potential as preeclampsia biomarkers—a brief review. Appl. Sci. 10:7731, 2020.

    Google Scholar 

  27. Nunez, J., F. Vignoletti, R. G. Caffesse, and M. Sanz. Cellular therapy in periodontal regeneration. Periodontol. 2000(79):107–116, 2019.

    Google Scholar 

  28. Ohta, T., J. E. Wergedal, H. E. Gruber, D. J. Baylink, and K. H. Lau. Low dose phenytoin is an osteogenic agent in the rat. Calcif. Tissue Int. 56:42–48, 1995.

    Google Scholar 

  29. Olsen, C. M., E. T. Meussen-Elholm, L. S. Roste, and E. Tauboll. Antiepileptic drugs inhibit cell growth in the human breast cancer cell line MCF7. Mol. Cell Endocrinol. 213:173–179, 2004.

    Google Scholar 

  30. Patejdl, R., A. C. Leroux, and T. Noack. Phenytoin inhibits contractions of rat gastrointestinal and portal vein smooth muscle by inhibiting calcium entry. Neurogastroenterol. Motil. 27:1453–1465, 2015.

    Google Scholar 

  31. Patsalos, P. N., D. J. Berry, B. F. Bourgeois, J. C. Cloyd, T. A. Glauser, S. I. Johannessen, I. E. Leppik, T. Tomson, and E. Perucca. Antiepileptic drugs–best practice guidelines for therapeutic drug monitoring: a position paper by the subcommission on therapeutic drug monitoring, ILAE Commission on Therapeutic Strategies. Epilepsia. 49:1239–1276, 2008.

    Google Scholar 

  32. Sanchez, A. B., K. E. Medders, R. Maung, P. Sanchez-Pavon, D. Ojeda-Juarez, and M. Kaul. CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and L-type Ca2+ channels upstream of p38 MAPK. J. Neuroinflamm. 13:1–12, 2016.

    Google Scholar 

  33. Sano, M., N. Ohuchi, T. Inoue, K. Tono, T. Tachikawa, Y. Kizawa, and H. Murakami. Proliferative response to phenytoin and nifedipine in gingival fibroblasts cultured from humans with gingival fibromatosis. Fundam. Clin. Pharmacol. 18:465–470, 2004.

    Google Scholar 

  34. Sapna, G., S. Gokul, and K. Bagri-Manjrekar. Matrix metalloproteinases and periodontal diseases. Oral Dis. 20:538–550, 2014.

    Google Scholar 

  35. Schrader, L. A., S. G. Birnbaum, B. M. Nadin, Y. J. Ren, D. Bui, A. E. Anderson, and J. D. Sweatt. ERK/MAPK regulates the Kv4.2 potassium channel by direct phosphorylation of the pore-forming subunit. Am. J. Physiol. 290:C852–C861, 2006.

    Google Scholar 

  36. Subramaniam, T., M. B. Fauzi, Y. Lokanathan, and J. X. Law. The role of calcium in wound healing. Int. J. Mol. Sci. 22:6486, 2021.

    Google Scholar 

  37. Takeuchi, R., H. Matsumoto, Y. Akimoto, and A. Fujii. Inhibition of G(1) cell cycle arrest in human gingival fibroblasts exposed to phenytoin. Fundam. Clin. Pharmacol. 28:114–119, 2014.

    Google Scholar 

  38. Tang, M., Z. Peng, Z. Mai, L. Chen, Q. Mao, Z. Chen, Q. Chen, L. Liu, Y. Wang, and H. Ai. Fluid shear stress stimulates osteogenic differentiation of human periodontal ligament cells via the extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling pathways. J. Periodontol. 85:1806–1813, 2014.

    Google Scholar 

  39. Teo, S. Y., M. Y. Yew, S. Y. Lee, M. J. Rathbone, S. N. Gan, and A. G. A. Coombes. In vitro evaluation of novel phenytoin-loaded alkyd nanoemulsions designed for application in topical wound healing. J. Pharm. Sci. 106:377–384, 2017.

    Google Scholar 

  40. Tomokiyo, A., N. Wada, and H. Maeda. Periodontal ligament stem cells: regenerative potency in periodontium. Stem Cells Dev. 28:974–985, 2019.

    Google Scholar 

  41. Trubiani, O., G. D. Marconi, S. D. Pierdomenico, A. Piattelli, F. Diomede, and J. Pizzicannella. Human oral stem cells, biomaterials and extracellular vesicles: a promising tool in bone tissue repair. Int. J. Mol. Sci. 20:4987, 2019.

    Google Scholar 

  42. Vijayasingham, S. M., P. J. Dykes, and R. Marks. Phenytoin has little effect on in-vitro models of wound healing. Br. J. Dermatol. 125:136–139, 1991.

    Google Scholar 

  43. Wittmack, E. K., A. M. Rush, A. Hudmon, S. G. Waxman, and S. D. Dib-Hajj. Voltage-gated sodium channel Na(v)1.6 is modulated by p38 mitogen-activated protein kinase. J. Neurosci. 25:6621–6630, 2005.

    Google Scholar 

  44. Yamamoto, T., Y. Ugawa, M. Kawamura, K. Yamashiro, S. Kochi, H. Ideguchi, and S. Takashiba. Modulation of microenvironment for controlling the fate of periodontal ligament cells: the role of Rho/ROCK signaling and cytoskeletal dynamics. J. Cell Commun. Signal. 12:369–378, 2018.

    Google Scholar 

  45. Yu, M. J., P. S. Yang, and S. H. Ge. Biological effects of phenytoin on cultured human periodontal ligament fibroblasts in vitro. Hua Xi Kou Qiang Yi Xue Za Zhi. 26:215–218, 2008.

    Google Scholar 

  46. Zheng, L., Y. Huang, W. Song, X. Gong, M. Liu, X. Jia, G. Zhou, L. Chen, A. Li, and Y. Fan. Fluid shear stress regulates metalloproteinase-1 and 2 in human periodontal ligament cells: involvement of extracellular signal-regulated kinase (ERK) and P38 signaling pathways. J. Biomech. 45:2368–2375, 2012.

    Google Scholar 

  47. Zheng, L. S., L. P. Chen, Y. C. Chen, J. P. Gui, Q. Li, Y. Huang, M. L. Liu, X. L. Jia, W. Song, J. Ji, et al. The effects of fluid shear stress on proliferation and osteogenesis of human periodontal ligament cells. J. Biomech. 49:572–579, 2016.

    Google Scholar 

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Acknowledgments

This work was supported by National Natural Science Foundation of China [Grant Nos. 11972067,11572030, 11120101001], National Key R&D Program of China [2017YFC0108505]; the Fundamental Research Funds for the Central Universities; the 111 Project [Grant Number B13003].

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Correspondence to Lisha Zheng or Yubo Fan.

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Jing Na, Lisha Zheng, Lijuan Wang, Qiusheng Shi, Nan Liu, Yuwei Guo and Yubo Fan declare that they have no conflict of interest.

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All human subjects research was carried out in accordance with the Declaration of Helsinki and approved by Beihang University Ethics Committee. This study did not involve any animal research.

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Na, J., Zheng, L., Wang, L. et al. Phenytoin Regulates Migration and Osteogenic Differentiation by MAPK Pathway in Human Periodontal Ligament Cells. Cel. Mol. Bioeng. 15, 151–160 (2022). https://doi.org/10.1007/s12195-021-00700-0

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Keywords

  • Human periodontal ligament cells
  • Phenytoin
  • Migration
  • Osteogenic differentiation
  • ALP
  • MAPK