Int J Stem Cells.  2023 May;16(2):180-190. 10.15283/ijsc22053.

The Role of SDF-1α-CXCR4/CXCR7 in Migration of Human Periodontal Ligament Stem Cells

• Xu J
• Yang F
• Luo S
• Gao Y
• Huang D
• Zhang L

• KMID: 2542831
• DOI: http://doi.org/10.15283/ijsc22053

Abstract

Background and Objectives
Regenerative endodontic procedures (REPs) are a research hotspot in the endodontic field. One of the biggest problems of REPs is that it is difficult to realize regeneration of pulp-dentin complex and functional reconstruction. The reason is still not clear. We hypothesize that the migration may be different in different dental stem cells. Periodontal ligament stem cells (PDLSCs) may migrate faster than stem cells of apical papilla (SCAPs), differentiating into cementum-like tissue, bone-like tissue and periodontal ligament-like tissue and, finally affecting the outcomes of REPs. Hence, this study aimed to explore the mechanism that regulates the migration of PDLSCs.
Methods and Results
After isolating and culturing PDLSCs and SCAPs from human third molars, we compared the migration of PDLSCs and SCAPs. Then we investigated the role of SDF-1α-CXCR4/CXCR7 axis in PDLSC migration. We further investigated the impact of Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) on PDLSC migration and the potential mechanism. PDLSCs showed better migration under both noninflammatory and inflammatory conditions than SCAPs. SDF-1α can promote the migration of PDLSCs by elevating the expression of CXCR4 and CXCR7, increasing the interaction between them, promoting expression of β-arrestin1 and activating the ERK signaling pathway. P. gingivalis LPS can promote the migration of PDLSCs toward SDF-1α through increasing the expression of CXCR4 via the NF-κB signaling pathway, promoting the expression of β-arrestin1, and activating the ERK signaling pathway.
Conclusions
This study helped elucidate the potential reason for the difficulty in forming pulp-dentin complex.

Keyword

Regenerative endodontic procedures; Periodontal ligament stem cells; Migration; CXCR4; CXCR7

Figure

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  Fig. 1 Migration of PDLSCs and SCAPs under normal and inflammation condition. (A∼E) SDF-1α improves the migration of PDLSCs and SCAPs in normal condition. The migrated PDLSCs cells were much more than SCAPs regardless of stimulated with SDF-1α or not. (F∼N) LPS improved the migration of PDLSCs and SCAPs in concentration-depended manner. The migrated PDLSCs cells were much more than SCAPs regardless of the LPS concentration. ****Indicates p<0.0001.

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  Fig. 2 CXCR4 and CXCR7 is related to migration of PDLSCs to SDF-1α. (A, B) qPCR assay revealed that SDF-1α increased the gene expression of CXCR4 and CXCR7 in PDLSCs. (C∼E) WB results revealed that that SDF-1α increases the expression of CXCR4 and CXCR7 protein. (F) CXCR4 interacts with CXCR7, which is improved by SDF-1α stimulation according to results of co-Immunoprecipitation assay. (G∼J) The siRNA of CXCR4 and CXCR7 can successfully suppress the expression of CXCR4 and CXCR7 according to WB results. (K∼N) Both siCXCR4 and siCXCR7 treatment suppress the migration of PDLSCs. *Indicates p<0.05, **Indicates p<0.01, ***Indicates p<0.001, ****Indicates p<0.0001.

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  Fig. 3 ERK, AKT signal pathway and β-arrestin1 regulates the migration of PDLSCs towards SDF-1α. (A∼D) SDF-1α elevated the phos-phorylation level of ERK1/2 and AKT, as well as β-arrestin1. (Ea, b) The inhibitor of ERK can successfully suppress the expression of p-ERK. (Ia, b) The inhibitor of AKT can successfully suppress the expression of p-AKT. (Ma, b) The siRNA of ARRB1 can successfully suppress the expression of β-arrestin1 according to WB results. (F∼H, J∼L, N∼P) ERK inhibitor, AKT inhibitor treatment and siRNA of ARRB1 treatment suppressed the migration of PDLSCs. (Q∼T) SDF-1α activated the ERK and AKT signal pathway, improved the expression of β-arrestin1. Treatment of siRNA of CXCR4 and CXCR7 reversed this effect. (U∼W) Treatment of ERK inhibitor decreased the expression of p-ERK, increased the expression of β-arrestin1. (X, Y) Treatment of siRNA of β-arrestin1 decreased the expression of p-ERK. *Indicates p<0.05, **Indicates p<0.01, ***Indicates p<0.001, ****Indicates p<0.0001.

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  Fig. 4 CXCR4 expression is higher in inflammation condition. (Aa∼c) Representative images of apical lesion tissues and normal tissues (gingiva tissue and apical papilla) by hematoxylin and eosin staining. (Ad∼f) There was no positive expression of CXCR4 or CXCR7 in NC groups by immunohistochemistry staining. (Ag∼i) Much more fibroblast-like cells with positive expression of CXCR4 were presented in apical lesion tissue, comparing with normal gingiva tissue and apical papilla. (Aj∼l) CXCR7 were positively expressed in few cells in all tissues which mainly surround vessels. Almost no differences were shown in apical lesion tissues and normal tissues. (B, C) Expression of CXCR4 was higher in INFLAMMATION group while the expression of CXCR7 were nearly the same in two groups. (D, E) LPS treatment increased the CXCR4 mRNA expression in PDLSCs while almost did not affect the CXCR7 mRNA expression. (F∼I) LPS increased the CXCR4 protein expression in PDLSCs in time dependent pattern, while almost did not affect the CXCR7 protein expression. *Indicates p<0.05, ****Indicates p<0.0001.

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  Fig. 5 P. gingivalis LPS can promote migration of PDLSC towards SDF-1α through increasing expression of CXCR4 by NF-κB signaling pathway, promoting expression of β-arrestin1, and then activating ERK signaling pathway. (A, B) LPS improved the p-P65 expression and activated the NF-κB signal pathway, which was reversed by NF-κB inhibitor treatment. (C∼F) LPS improved the migration of PDLSCs, which was reversed by NF-κB inhibitor treatment. (G∼I) LPS improved the CXCR4 expression and this effect was suppressed by NF-κB signal inhibitor treatment, while LPS and NF-κB signal inhibitor treatment almost did not affect the expression of CXCR7. (J∼M) LPS improved the expression of p-AKT, p-ERK and β-arrestin1. The NF-κB inhibitor treatment suppressed the effect of LPS. *Indicates p<0.05, **Indicates p<0.01, ***Indicates p<0.001, ****Indicates p<0.0001.

Reference

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