p75 neurotrophin receptor regulates differential mineralization of rat ectomesenchymal stem cells

Abstract

Objectives: The aim of this study was to investigate whether p75NTR (p75 neurotrophin receptor) regulates differential mineralization capacity of rEMSCs (rat ectomesenchymal stem cells) and underlying mechanisms associated with Mage-D1 (melanoma-associated antigens-D1).

Materials and methods: Immunohistochemical staining of p75NTR in developing tooth germs was performed on E12.5d (embryonic 12.5 days) and E19.5d (embryonic 19.5 days). E12.5d EMSCs and E19.5d EMSCs were isolated in the same pregnant Sprague-Dawley rats from embryonic maxillofacial processes and tooth germs. p75NTR small-interfering RNA, p75NTR overexpression plasmid, Mage-D1 small-interfering RNA and recombined rat NGF were used to transfect cells.

Results: p75NTR was expressed in epithelial-mesenchymal interaction areas at E12.5d and E19.5d tooth germ development stages. E19.5d EMSCs had higher p75NTR expression levels and differential mineralization capacity but lower levels of cell proliferation. Under induction by mineralized culture medium, the potential of differential mineralization had identical trends in regulation of p75NTR in EMSCs; Mage-D1 did not fluctuate and TrkA was not expressed. Binding of p75NTR and Mage-D1 were detected. Mage-D1 knockdown significantly down-regulated expression of related genes, which NGF could not rescue.

Conclusion: p75NTR participated in tooth germ development stages and mediated differential mineralization of EMSCs. p75NTR played a critical role in regulating the potential of differential mineralization of EMSCs. Mage-D1 seemed to act as a bridge in the underlying mechanism of effects of p75NTR.

Figure 1

Immunohistochemistry staining for E12.5d and E19.5d tooth germ. (a) Haematoxylin and eosin (HE) staining for rat E12.5d tooth germ at bud stage. (b) At bud stage, p75NTR staining was detected in cells of mesenchyme (me) in dental epithelial‐mesenchymal interaction area, the condensed mesenchyme (cm) exhibited faint immunoreactivity, and staining was absent from dental epithelium (de). (c) Haematoxylin and eosin staining for rat E19.5d tooth germ at late bell stage. (d) At late bell stage, p75NTR staining was detected in cells of inner dental epithelial (ide), dental follicle (df) and dental papilla (p); the immunoreactivity in cells of outer dental epithelium (ode) was not detected. Scale bar represents 100 μm

Figure 2

The proliferation capacities of E12.5d EMSCs and E19.5d EMSCs. (a)The primary cells and the third passage (P3) cells of E12.5d and E19.5d EMSCs were cultured for 3 d. Scale bar represents 100 μm. (b) Representative images of colonies formed by E12.5d and E19.5d EMSCs at low seeding density (1×10³/plate) after 2 wk in culture. (c) The proliferation ratio of E12.5d and E19.5d EMSCs were assessed by CCK‐8 (cell counting kit‐8) cultured for 7 d (*P<.05)

Figure 3

Flow cytometry analysis of the expression of cell surface markers. These cell surface markers related to cranial neural crest originated (p75NTR) or mesenchymal (CD14, CD29, CD44, CD90, CD105, CD146 and CD166) or hematopoietic stem cells (CD45). (a–i) The cell surface markers of E12.5d EMSCs. (j–r) The cell surface markers of E19.5d EMSCs

Figure 4

The comparison of mineralized capacities of E12.5d and E19.5d EMSCs. (a) Under induction with mineralized culture medium for 7 d, ALP staining was used to detect their potential of differential mineralization. Scale bar represents 50 μm. (b) E12.5d EMSCs and E19.5d EMSCs were induced in mineralized induction medium for 21 d; Alizarin red staining was used to detect their mineralized nodules. Scale bar represents 150 μm. (c) The expression levels of p75NTR, Mage‐D1, ALP, Runx2, Osterix, Dlx5 and Msx2 were examined by real‐time PCR normalized to GAPDH. (d) Under induction with mineralized culture medium for 7 d, the expression levels of p75NTR Mage‐D1, Runx2, Col1, and TrkA were detected by Western blot analysis, GAPDH used as the reference gene. (*P<.05, **P<.01, ***P<.001, ns=no significant difference)

Figure 5

The expression level of mineralization‐related genes and binding of p75NTR and Mage‐D1 in E19.5d EMSCs during mineralized induction. (a) The expression levels of p75NTR, Mage‐D1, ALP, Runx2, Osterix, Dlx5 and Msx2 were examined by real‐time PCR normalized to GAPDH. (b) The expression levels of p75NTR, Mage‐D1, Runx2, Col1 and TrkA were detected by Western blot analysis during mineralized induction, GAPDH used as the reference gene. (c) Immunoblot analysis of p75NTR protein in cell lysates of E19.5d EMSCs immunoprecipitated with anti‐Mage‐D1 antibody. (d)Supernatant was collected at mineralized induction time points, and NGF was quantified by ELISA (*P<.05, **P<.01, ***P<.001, ns=no significant difference)

Figure 6

Regulation of p75NTR influent the mineralized capacity of E19.5d EMSCs. (a) Under induction with mineralized culture medium for 7 d, immunocytofluorescence staining of p75NTR and Mage‐D1 in transfection with p75NTR siRNA (p75siRNA group), negative control (NCon group), transfection with p75NTR overexpression plasmid pLJM1(pLJM1‐p75 group), empty plasmid (pLJM1 group) in E19.5d EMSCs; scale bar represents 25 μm. (b) The expression levels of p75NTR, Mage‐D1, ALP, Runx2, Osterix, Dlx5 and Msx2 were examined by real‐time PCR normalized to GAPDH. (c) Under induction with mineralized culture medium for 7 d, the expression levels of p75NTR Mage1‐D, Runx2, Col1 and TrkA were detected by Western blot analysis, GAPDH used as the reference gene. (d) Under induction with mineralized culture medium for 7 d, ALP staining was used to detect their potential of differential mineralization. Scale bar represents 50 μm. (e) Under induction with mineralized culture medium for 21 d, Alizarin red staining was used to detect their mineralized nodules. Scale bar represents 150 μm. (f, g) The third passage (P3) of pLJM1 and pLJM1‐p75 EMSCs, the cellular morphology of pLJM1‐p75 EMSCs was more regular spindly fibroblast‐like. Scale bar represents 50 μm. (h) The proliferation rate of pLJM1 and pLJM1‐p75 EMSCs were assessed by CCK‐8 (cell counting kit‐8) cultured for 7 d (*P<.05, **P<.01, ***P<.001, ns=no significant difference)

Figure 7

The mineralized capacity of E19.5d EMSCs is regulated by Mage‐D1 siRNA and NGF. (a) Under induction with mineralized culture medium for 7 d, negative control (NCon), Mage‐D1siRNA transfection (Mage‐D1siRNA), NGF treatment (NGF 100 ng/mL) and NGF treatment with Mage‐D1siRNA transfection (NGF+Mage‐D1siRNA) in E19.5d EMSCs, ALP staining was detected. Scale bar represents 50 μm. (b) Under induction with mineralized induction medium for 7 d, the expression levels of p75NTR, Runx2 and Col1 were detected by Western blot analysis, GAPDH used as the reference gene. (c) Immunoblot analysis of p75NTR protein in cell lysates of E19.5d EMSCs immunoprecipitated with anti‐Mage‐D1 antibody. (d) The expression levels of Mage‐D1, ALP, Runx2, Osterix, Dlx5 and Msx2 were examined by real‐time PCR normalized to GAPDH (*P<.05, **P<.01, ***P<.001, ns=no significant difference)