Elsevier

Archives of Oral Biology

Volume 68, August 2016, Pages 1-8
Archives of Oral Biology

Endogenous hydrogen sulfide is involved in osteogenic differentiation in human periodontal ligament cells

https://doi.org/10.1016/j.archoralbio.2016.03.009Get rights and content

Highlights

  • CBS and CSE produce H2S in hPDLCs to promote their osteogenic differentiation.

  • Optimal concentrations of endogenous H2S must be maintained.

  • Excessive or deficient H2S levels temper the observed osteogenic effect.

  • These effects are exerted by activating the Wnt/β-catenin signaling cascade.

Abstract

Objective

Endogenous hydrogen sulfide (H2S) has recently emerged as an important intracellular gaseous signaling molecule within cellular systems. Endogenous H2S is synthesized from l-cysteine via cystathionine β-synthase and cystathionine γ-lyase and it regulates multiple signaling pathways in mammalian cells. Indeed, aberrant H2S levels have been linked to defects in bone formation in experimental mice. The aim of this study was to examine the potential production mechanism and function of endogenous H2S within primary human periodontal ligament cells (PDLCs).

Design

Primary human PDLCs were obtained from donor molars with volunteer permission. Immunofluorescent labeling determined expression of the H2S synthetase enzymes. These enzymes were inhibited with D,L-propargylglycine or hydroxylamine to examine the effects of H2S signaling upon the osteogenic differentiation of PDLCs. Gene and protein expression levels of osteogenic markers in conjunction with ALP staining and activity and alizarin red S staining of calcium deposition were used to assay the progression of osteogenesis under different treatment conditions. Cultures were exposed to Wnt3a treatment to assess downstream signaling mechanisms.

Results

In this study, we show that H2S is produced by human PDLCs via the cystathionine β-synthase/cystathionine γ-lyase pathway to promote their osteogenic differentiation. These levels must be carefully maintained as excessive or deficient H2S levels temper the observed osteogenic effect by inhibiting Wnt/β-catenin signaling.

Conclusions

These results demonstrate that optimal concentrations of endogenous H2S must be maintained within PDLCs to promote osteogenic differentiation by activating the Wnt/β-catenin signaling cascade.

Introduction

Periodontitis is a common chronic inflammatory disease leading to destruction of the supporting structures of the teeth including the alveolar bone, periodontal ligament and cementum (Trofin, Monsarrat, & Kemoun, 2013; Wolf et al., 2013). This degeneration can lead to early tooth loss in affected individuals (Preshaw et al., 2012, Reich and Hiller, 1993). Periodontal ligament (PDL) is a specialized connective tissue that maintains and supports teeth in situ (Nanci and Bosshardt, 2006, Seo et al., 2004). Current periodontal treatments rely in the main on the osteogenic differentiation of PDL, which is responsible for the regeneration of the adjacent periodontal structure (Acil et al., 2015, Sun and Liu, 2014). Periodontal ligament cells (PDLCs) are endogenous multipotential progenitors, which form a major cellular component of the PDL (Liu, Zha, Xuan, Xie, & Zhang, 2010; Wei, Wu, Ling, & Liu, 2008). The osteogenic differentiation of PDLCs is mediated through diverse signaling mechanisms by a vast array of signaling molecules, such as nitric oxide (Klein-Nulend, Semeins, Ajubi, Nijweide, & Burger, 1995), prostaglandin E1 (Bakker, Soejima, Klein-Nulend, & Burger, 2001; Jessop, Rawlinson, Pitsillides, & Lanyon, 2002), prostaglandin E2 (Ryder & Duncan, 2001)and mechano- and voltage-sensitive Ca2+ channels (Kolluru, Shen, Bir, & Kevil, 2013).

Hydrogen sulfide (H2S), as the most recently discovered gasotransmitter, following nitric oxide and carbon monoxide, has aroused considerable interest as a potential signaling molecule (Li, Rose, & Moore, 2011). In mammalian cells, H2S is endogenously synthesized from l-cysteine via the action of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). It has been suggested that H2S may regulate a variety of signaling pathways17 and abnormal metabolism links to a number of diseases, such as hypertension, coronary heart disease, inflammation (Kamoun, 2004). It has also been shown to be involved in the regulation of body temperature, metabolic levels (Lowicka & Beltowski, 2007; Zhang, Dong, & Chu, 2010) and the induction of apoptosis in PDLCs (Irie et al., 2009).

Recently the role of endogenous H2S in regulating ossification has generated a great deal of controversy. Whereas Koichiro et al. showed that H2S could transiently promote osteoclast differentiation through the up-regulation of RANKL expression in osteoblasts (Irie et al., 2009), Liu et al. revealed that H2S-deficient mice displayed an osteoporotic phenotype (Liu et al., 2014).

It is currently unknown whether PDLCs produce endogenous H2S or whether H2S can exert osteoblastic or osteoclastic effects within this cell type. In this study, we investigated the potential of primary human PDLCs to endogenously synthesize H2S and whether this occurs via the typical CBS/CSE pathway in this cell type. Additionally, we have attempted to clarify the signaling mechanism responsible for the osteogenic effects of H2S within PDLCs.

Section snippets

Materials and methods

The experiments were approved by the Medical Ethics Committee of Wenzhou Medical University and the Hospital of Stomatology. Informed written consent was obtained from all donors.

Morphology and identification of PDLCs

We assayed the morphology and expression profile of PDLC markers to assess the primary cell type and purity. Primary PDLCs attached to the plastic culture dishes and displayed the expected spindle-shaped fibroblast-like morphology (Fig. 1A and B). The cells stained positively for vimentin and negatively for cytokeratin indicating their mesodermal origin (Fig. 1C and D).

Effect of NaHS on cells viability

Following incubation of established PDLC cultures with NaHS for 24, 48 and 72 h, the viability of PDLCs in the presence or

Discussion

As one of the most common oral diseases, periodontitis can lead to alveolar bone deficiencies and early tooth loss (Zhu & Liang, 2015). PDLCs are able to differentiate into osteoblasts (Li & Zhang, 2015), and may thus play an important role in the regeneration of alveolar bone. In this study, we found that PDLCs expressed CBS and CSE, the two main synthetases of H2S in mammalian cells, and produced endogenous H2S. Additionally, when H2S synthesis was blocked in primary PDLC cultures, the

Conclusion

In summary, the present study has indicated that H2S can be endogenously synthesized by CBS and CSE, and plays an important role in osteogenic differentiation in hPDLCs via activation of Wnt/β-catenin signaling. These results indicate its therapeutic potential as a host modulatory agent in periodontitis.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (Grant No. 81200795), the Zhejiang Provincial Nature Science Foundation of China (Grant No. LY12H14003), the Zhejiang Provincial Medical and Health Science and Technology Plan of China (Grant No. 2015KYA149), and Medical Scientific Research Foundation of Zhejiang Province, China (Grant No. 2015ZA122).

References (31)

  • E. Zavaczki et al.

    Hydrogen sulfide inhibits the calcification and osteoblastic differentiation of vascular smooth muscle cells

    Kidney International

    (2011)
  • Y. Acil et al.

    Isolation, characterization and investigation of differentiation potential of human periodontal ligament cells and dental follicle progenitor cells and their response to BMP-7 in vitro

    Odontology

    (2015)
  • X. Jiang et al.

    GYY4137 promotes bone formation in a rabbit distraction osteogenesis model: a preliminary report

    Journal of Oral and Maxillofacial Surgery

    (2015)
  • P. Kamoun

    Endogenous production of hydrogen sulfide in mammals

    Amino Acids

    (2004)
  • D. Lanza et al.

    Impact of the uremic milieu on the osteogenic potential of mesenchymal stem cells

    PLoS One

    (2015)
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