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Repeated lipopolysaccharide stimulation promotes cellular senescence in human dental pulp stem cells (DPSCs)

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Abstract

Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell (MSC) characterized by multi-lineage differentiation making it an attractive choice for tissue regeneration. However, before DPSCs can be used for cell-based therapy, we have to understand their biological properties in response to intrinsic and extrinsic stimuli such as lipopolysaccharide (LPS). DPSCs were therefore stimulated with LPS and senescence was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining, with cell number and cell-cycle arrest being examined by BrdU assay and flow cytometry, respectively. The morphology of DPSCs was characterized by their flat shape, increased size and increased SA-β-gal activity after repeated stimulation (3 or 6 times) with LPS. Reactive oxygen species (ROS) staining showed that the number of ROS-stained cells and the DCFH fluorescent level were higher in the LPS-treated DPSCs compared with those in the untreated DPSCs. Protein and mRNA expression levels of γ-H2A.X and p16INK4A were also increased in DPSCs with repeated LPS stimulation. We found that the LPS bound with Toll-like receptor 4 (TLR4) and that TLR4 signaling accounted for p16INK4A expression. Further results indicated that the senescence of DPSCs stimulated repeatedly with LPS was reversed by p16INK4A short interfering RNA. The DNA damage response and p16INK4A pathways might be the main mediators of DPSC senescence induced by repeated LPS stimulation. Thus, DPSCs tend to undergo senescence after repeated activation, implying that DPSC senescence starts after many inflammatory challenges. Ultimately, these findings should lead to a better understanding of DPSC-based clinical therapy.

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References

  • Alongi DJ, Yamaza T, Song Y, Fouad AF, Romberg EE, Shi S, Tuan RS, Huang GT (2010) Stem/progenitor cells from inflamed human dental pulp retain tissue regeneration potential. Regen Med 5:617–631

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Arora V, Arora P, Munshi AK (2009) Banking stem cells from human exfoliated deciduous teeth (SHED): saving for the future. J Clin Pediatr Dent 33:289–294

    PubMed  Google Scholar 

  • Arthur A, Rychkov G, Shi S, Koblar SA, Gronthos S (2008) Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells 26:1787–1795

    Article  CAS  PubMed  Google Scholar 

  • Beyne-Rauzy O, Recher C, Dastugue N, Demur C, Pottier G, Laurent G, Sabatier L, Mansat-De Mas V (2004) Tumor necrosis factor alpha induces senescence and chromosomal instability in human leukemic cells. Oncogene 23:7507–7516

    Article  CAS  PubMed  Google Scholar 

  • Botero TM, Son JS, Vodopyanov D, Hasegawa M, Shelburne CE, Nor JE (2010) MAPK signaling is required for LPS-induced VEGF in pulp stem cells. J Dent Res 89:264–269

    Article  CAS  PubMed  Google Scholar 

  • Canepa ET, Scassa ME, Ceruti JM, Marazita MC, Carcagno AL, Sirkin PF, Ogara MF (2007) INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions. IUBMB Life 59:419–426

    Article  CAS  PubMed  Google Scholar 

  • Collins CJ, Sedivy JM (2003) Involvement of the INK4a/Arf gene locus in senescence. Aging Cell 2:145–150

    Article  CAS  PubMed  Google Scholar 

  • Dierick JF, Eliaers F, Remacle J, Raes M, Fey SJ, Larsen PM, Toussaint O (2002) Stress-induced premature senescence and replicative senescence are different phenotypes, proteomic evidence. Biochem Pharmacol 64:1011–1017

    Article  CAS  PubMed  Google Scholar 

  • Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O et al (1995) A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92:9363–9367

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Feng X, Feng G, Xing J, Shen B, Li L, Tan W, Xu Y, Liu S, Liu H, Jiang J, Wu H, Tao T, Gu Z (2013) TNF-alpha triggers osteogenic differentiation of human dental pulp stem cells via the NF-kappaB signaling pathway. Cell Biol Int 37:1267–1275

    Google Scholar 

  • Gandia C, Arminan A, Garcia-Verdugo JM, Lledo E, Ruiz A, Minana MD, Sanchez-Torrijos J, Paya R, Mirabet V, Carbonell-Uberos F, Llop M, Montero JA, Sepulveda P (2008) Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce infarct size in rats with acute myocardial infarction. Stem Cells 26:638–645

    Article  PubMed  Google Scholar 

  • Goossens V, De Vos K, Vercammen D, Steemans M, Vancompernolle K, Fiers W, Vandenabeele P, Grooten J (1999) Redox regulation of TNF signaling. Biofactors 10:145–156

    Article  CAS  PubMed  Google Scholar 

  • Gronthos S, Mankani M, Brahim J, Robey PG, Shi S (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 97:13625–13630

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, DenBesten P, Robey PG, Shi S (2002) Stem cell properties of human dental pulp stem cells. J Dent Res 81:531–535

    Article  CAS  PubMed  Google Scholar 

  • Gu Z, Cao X, Jiang J, Li L, Da Z, Liu H, Cheng C (2012) Upregulation of p16INK4A promotes cellular senescence of bone marrow-derived mesenchymal stem cells from systemic lupus erythematosus patients. Cell Signal 24:2307–2314

    Article  CAS  PubMed  Google Scholar 

  • Huang GT, Gronthos S, Shi S (2009) Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res 88:792–806

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Iohara K, Zheng L, Wake H, Ito M, Nabekura J, Wakita H, Nakamura H, Into T, Matsushita K, Nakashima M (2008) A novel stem cell source for vasculogenesis in ischemia: subfraction of side population cells from dental pulp. Stem Cells 26:2408–2418

    Article  PubMed  Google Scholar 

  • Janzen V, Forkert R, Fleming HE, Saito Y, Waring MT, Dombkowski DM, Cheng T, DePinho RA, Sharpless NE, Scadden DT (2006) Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature 443:421–426

    CAS  PubMed  Google Scholar 

  • Kim JC, Lee YH, Yu MK, Lee NH, Park JD, Bhattarai G, Yi HK (2012) Anti-inflammatory mechanism of PPARgamma on LPS-induced pulp cells: role of the ROS removal activity. Arch Oral Biol 57:392–400

    Article  CAS  PubMed  Google Scholar 

  • Kosar M, Bartkova J, Hubackova S, Hodny Z, Lukas J, Bartek J (2011) Senescence-associated heterochromatin foci are dispensable for cellular senescence, occur in a cell type- and insult-dependent manner and follow expression of p16(ink4a). Cell Cycle 10:457–468

    Article  CAS  PubMed  Google Scholar 

  • Ksiazek K (2009) A comprehensive review on mesenchymal stem cell growth and senescence. Rejuvenation Res 12:105–116

    Article  CAS  PubMed  Google Scholar 

  • Lavrovsky Y, Chatterjee B, Clark RA, Roy AK (2000) Role of redox-regulated transcription factors in inflammation, aging and age-related diseases. Exp Gerontol 35:521–532

    Article  CAS  PubMed  Google Scholar 

  • Lee YH, Kim GE, Cho HJ, Yu MK, Bhattarai G, Lee NH, Yi HK (2013) Aging of in vitro pulp illustrates change of inflammation and dentinogenesis. J Endod 39:340–345

    Article  PubMed  Google Scholar 

  • Lepperdinger G (2011) Inflammation and mesenchymal stem cell aging. Curr Opin Immunol 23:518–524

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ma D, Gao J, Yue J, Yan W, Fang F, Wu B (2012) Changes in proliferation and osteogenic differentiation of stem cells from deep caries in vitro. J Endod 38:796–802

    Article  PubMed  Google Scholar 

  • Mei C, Zheng F (2009) Chronic inflammation potentiates kidney aging. Semin Nephrol 29:555–568

    Article  CAS  PubMed  Google Scholar 

  • Nagaoka S, Tokuda M, Sakuta T, Taketoshi Y, Tamura M, Takada H, Kawagoe M (1996) Interleukin-8 gene expression by human dental pulp fibroblast in cultures stimulated with Prevotella intermedia lipopolysaccharide. J Endod 22:9–12

    Article  CAS  PubMed  Google Scholar 

  • Pierdomenico L, Bonsi L, Calvitti M, Rondelli D, Arpinati M, Chirumbolo G, Becchetti E, Marchionni C, Alviano F, Fossati V, Staffolani N, Franchina M, Grossi A, Bagnara GP (2005) Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp. Transplantation 80:836–842

    Article  PubMed  Google Scholar 

  • Rutherford RB, Gu K (2000) Treatment of inflamed ferret dental pulps with recombinant bone morphogenetic protein-7. Eur J Oral Sci 108:202–206

    Article  CAS  PubMed  Google Scholar 

  • Seoane M, Iglesias P, Gonzalez T, Dominguez F, Fraga M, Aliste C, Forteza J, Costoya JA (2008) Retinoblastoma loss modulates DNA damage response favoring tumor progression. PLoS One 3:e3632

    Article  PubMed Central  PubMed  Google Scholar 

  • Suematsu N, Tsutsui H, Wen J, Kang D, Ikeuchi M, Ide T, Hayashidani S, Shiomi T, Kubota T, Hamasaki N, Takeshita A (2003) Oxidative stress mediates tumor necrosis factor-alpha-induced mitochondrial DNA damage and dysfunction in cardiac myocytes. Circulation 107:1418–1423

    Article  CAS  PubMed  Google Scholar 

  • Wallace DC (2005) A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 39:359–407

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wang CY, Tani-Ishii N, Stashenko P (1997) Bone-resorptive cytokine gene expression in periapical lesions in the rat. Oral Microbiol Immunol 12:65–71

    Article  PubMed  Google Scholar 

  • Wang C, Jurk D, Maddick M, Nelson G, Martin Ruiz C, Zglinicki T von (2009) DNA damage response and cellular senescence in tissues of aging mice. Aging Cell 8:311–323

    Google Scholar 

  • Yang DG, Liu L, Zheng XY (2008) Cyclin-dependent kinase inhibitor p16(INK4a) and telomerase may co-modulate endothelial progenitor cells senescence. Ageing Res Rev 7:137–146

    Article  CAS  PubMed  Google Scholar 

  • Yoo J, Park SY, Kang SJ, Shim SI, Kim BK (2002) Altered expression of G1 regulatory proteins in human soft tissue sarcomas. Arch Pathol Lab Med 126:567–573

    CAS  PubMed  Google Scholar 

  • Yu HM, Zhao YM, Luo XG, Feng Y, Ren Y, Shang H, He ZY, Luo XM, Chen SD, Wang XY (2012) Repeated lipopolysaccharide stimulation induces cellular senescence in BV2 cells. Neuroimmunomodulation 19:131–136

    Article  CAS  PubMed  Google Scholar 

  • Yuan X, Zhou Y, Wang W, Li J, Xie G, Zhao Y, Xu D, Shen L (2013) Activation of TLR4 signaling promotes gastric cancer progression by inducing mitochondrial ROS production. Cell Death Dis 4:e794

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zhang W, Walboomers XF, Shi S, Fan M, Jansen JA (2006) Multilineage differentiation potential of stem cells derived from human dental pulp after cryopreservation. Tissue Eng 12:2813–2823

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Herbert BS, Rajashekhar G, Ingram DA, Yoder MC, Clauss M, Rehman J (2009) Premature senescence of highly proliferative endothelial progenitor cells is induced by tumor necrosis factor-alpha via the p38 mitogen-activated protein kinase pathway. FASEB J 23:1358–1365

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

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Authors and Affiliations

  1. Department of Stomatology, Affiliated Hospital of Nantong University, Nantong, China

    Xingmei Feng, Guijuan Feng, Jing Xing, Dan Huang & Xiaohui Lu

  2. Department of Nursing, The Second Affiliated Hospital of Nantong University, Nantong, China

    Biyu Shen

  3. Department of Rheumatology, Affiliated Hospital of Nantong University, Nantong, China

    Wei Tan & Zhifeng Gu

  4. Department of Immunology, Medical College, Nantong University, Nantong, China

    Tao Tao

  5. Department of Spine Surgery, The Second Affiliated Hospital of Nantong University, Nantong, China

    Jinlong Zhang

  6. Department of Hematology, Affiliated Hospital of Nantong University, Nantong, China

    Liren Li

Authors
  1. Xingmei Feng
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  2. Guijuan Feng
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  3. Jing Xing
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  4. Biyu Shen
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  5. Wei Tan
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  6. Dan Huang
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  7. Xiaohui Lu
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  8. Tao Tao
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  9. Jinlong Zhang
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  10. Liren Li
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  11. Zhifeng Gu
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Corresponding authors

Correspondence to Liren Li or Zhifeng Gu.

Additional information

Xingmei Feng and Guijuan Feng contributed equally to this work.

This work was supported by the Natural Science Foundation of China (grant no. 81172841, no. 81202368), the Natural Science Foundation of Jiangsu Colleges and Universities (grant no. 09KJB320010, 10KJB320012), the “Top Six Types of Talents” Financial Assistance of Jiangsu Province (grant no. 6, no. 7), the Jiangsu Province’s Outstanding Medical Academic Leader Program (LJ201135, LJ201136), the Science Foundation of Nantong City (grant no. HS2011054), a project from the Bureau of Jiangsu Province (Z2010005) and Preventive Medicine Projects from the Bureau of Jiangsu Province (Y2012083).

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Feng, X., Feng, G., Xing, J. et al. Repeated lipopolysaccharide stimulation promotes cellular senescence in human dental pulp stem cells (DPSCs). Cell Tissue Res 356, 369–380 (2014). https://doi.org/10.1007/s00441-014-1799-7

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  • DOI: https://doi.org/10.1007/s00441-014-1799-7

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