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