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. 2018 Oct 17:6:e5808.
doi: 10.7717/peerj.5808. eCollection 2018.

Sclerostin promotes human dental pulp cells senescence

Yanjing Ou  1 , Yi Zhou  1 , Shanshan Liang  1 , Yining Wang  1
Affiliations

Sclerostin promotes human dental pulp cells senescence

Yanjing Ou et al. PeerJ. .

Abstract

Background: Senescence-related impairment of proliferation and differentiation limits the use of dental pulp cells for tissue regeneration. Deletion of sclerostin improves the dentinogenesis regeneration, while its role in dental pulp senescence is unclear. We investigated the role of sclerostin in subculture-induced senescence of human dental pulp cells (HDPCs) and in the senescence-related decline of proliferation and odontoblastic differentiation.

Methods: Immunohistochemical staining and qRT-PCR analyses were performed to examine the expression pattern of sclerostin in young (20-30-year-old) and senescent (45-80-year-old) dental pulps. HDPCs were serially subcultured until senescence, and the expression of sclerostin was examined by qRT-PCR analysis. HDPCs with sclerostin overexpression and knockdown were constructed to investigate the role of sclerostin in HDPCs senescence and senescence-related impairment of odontoblastic differentiation potential.

Results: By immunohistochemistry and qRT-PCR, we found a significantly increased expression level of sclerostin in senescent human dental pulp compared with that of young human dental pulp. Additionally, elevated sclerostin expression was found in subculture-induced senescent HDPCs in vitro. By sclerostin overexpression and knockdown, we found that sclerostin promoted HDPCs senescence-related decline of proliferation and odontoblastic differentiation potential with increased expression of p16, p53 and p21 and downregulation of the Wnt signaling pathway.

Discussion: The increased expression of sclerostin is responsible for the decline of proliferation and odontoblastic differentiation potential of HDPCs during cellular senescence. Anti-sclerostin treatment may be beneficial for the maintenance of the proliferation and odontoblastic differentiation potentials of HDPCs.

Keywords: Human dental pulp cell; Sclerostin; Senescence; p16; p21; p53.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Expressions of sclerostin in senescent dental pulp, subculture-induced senescent HDPCs and lentiviral infected HDPCs.
(A) Immunohistochemical staining for sclerostin in young dental pulps; (B) immunohistochemical staining for sclerostin in senescent dental pulps (green arrows point to odontoblasts and red arrows point to dental pulp cells); d, dentin; pd, predentin; p, pulp; ob, odontoblast; scale bar = 20 μm. (C) qRT-PCR analyses of expression levels of sclerostin, p16, p53 and p21 in young and senescent dental pulps; (D) qRT-PCR analyses of expression levels of sclerostin, p16, p53 and p21 in subculture-induced senescent HDPCs. (E) qRT-PCR and (F) Western blot analyses of sclerostin expressions in HDPCs infected with control vector and SOST; (G) qRT-PCR and (H) Western blot analyses of sclerostin expression in HDPCs infected with sh-control vector and sh-SOST. **P < 0.01; ***P < 0.001.
Figure 2
Figure 2. Effects of sclerostin overexpression and knockdown on senescence and proliferation of HDPCs.
SA-β-Gal staining of Control (A) and SOST (B) HDPCS; (C) cell proliferation activity of Control and SOST HDPCs; (D) qRT-PCR analyses of expression levels of p16, p53 and p21 in Control and SOST HDPCs. SA-β-Gal staining of sh-Ctrl (E) and sh-SOST (F) HDPCs; (G) cell proliferation activity of sh-Ctrl and sh-SOST HDPCs; (H) qRT-PCR analyses of expression levels of p16, p53 and p21 in sh-Ctrl and sh-SOST HDPCs. *P < 0.05, **P < 0.01; ***P < 0.001.
Figure 3
Figure 3. Effects of sclerostin on odontoblastic differentiation of HDPCs.
(A) ALP staining and ALP activity of early-passaged Control and SOST HDPCs on day 7; (B) alizarin red staining for mineral nodule formation of early-passaged Control and SOST HDPCs on day 14; (C) qRT-PCR analyses of expression levels of odontoblastic markers in early-passaged Control and SOST HDPCs on day 7. (D) ALP staining and ALP activity of late-passaged sh-Ctrl and sh-SOST HDPCs on day 7; (E) alizarin red staining for mineral nodule formation of late-passaged sh-Ctrl and sh-SOST HDPCs on day 14; (F) qRT-PCR analyses of odontoblastic markers in late-passaged sh-Ctrl and sh-SOST HDPCs on day 7. *P < 0.05, **P < 0.01; ***P < 0.001.
Figure 4
Figure 4. Effect of sclerostin on Wnt/β-catenin pathway.
(A) Western blot analysis of p-β-catenin expression in early-passaged Control and SOST HDPCs; (B) Western blot analysis of p-β-catenin expression in late-passaged sh-Ctrl and sh-SOST HDPCs. *P < 0.05, **P < 0.01.
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