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Int J Mol Sci. 2014 Apr 11;15(4):6224-40. doi: 10.3390/ijms15046224.

Substance P activates the Wnt signal transduction pathway and enhances the differentiation of mouse preosteoblastic MC3T3-E1 cells.

Author information

1
Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, Guangdong, China. meigang622@outlook.com.
2
Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, Guangdong, China. qvbghdkk@163.com.
3
Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, Guangdong, China. fusu321789@gmail.com.
4
Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, Guangdong, China. smilefish_2009@163.com.
5
Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, Guangdong, China. zhoujian321789@gmail.com.
6
Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, Guangdong, China. zhaoyan2016@gmail.com.
7
Department of Orthopaedic, Wuzhou Red Cross Hospital, Wuzhou 543002, Guangxi, China. tyh2150@gmail.com.
8
School of Engineering and Materials Science, Queen Mary University of London, Mile End, London E1 4NS, UK. ctwz198412@googlemail.com.
9
Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, Guangdong, China. jindanankle@gmail.com.

Abstract

Recent experiments have explored the impact of Wnt/β-catenin signaling and Substance P (SP) on the regulation of osteogenesis. However, the molecular regulatory mechanisms of SP on the formation of osteoblasts is still unknown. In this study, we investigated the impact of SP on the differentiation of MC3T3-E1 cells. The osteogenic effect of SP was observed at different SP concentrations (ranging from 10⁻¹⁰ to 10⁻⁸ M). To unravel the underlying mechanism, the MC3T3-E1 cells were treated with SP after the pretreatment by neurokinin-1 (NK1) antagonists and Dickkopf-1 (DKK1) and gene expression levels of Wnt/β-catenin signaling pathway components, as well as osteoblast differentiation markers (collagen type I, alkaline phosphatase, osteocalcin, and Runx2), were measured using quantitative polymerase chain reaction (PCR). Furthermore, protein levels of Wnt/β-catenin signaling pathway were detected using Western blotting and the effects of SP, NK1 antagonist, and DKK1 on β-catenin activation were investigated by immunofluorescence staining. Our data indicated that SP (10⁻⁹ to 10⁻⁸ M) significantly up-regulated the expressions of osteoblastic genes. SP (10⁻⁸ M) also elevated the mRNA level of c-myc, cyclin D1, and lymphocyte enhancer factor-1 (Lef1), as well as c-myc and β-catenin protein levels, but decreased the expression of Tcf7 mRNA. Moreover, SP (10-8 M) promoted the transfer of β-catenin into nucleus. The effects of SP treatment were inhibited by the NK1 antagonist and DKK1. These findings suggest that SP may enhance differentiation of MC3T3-E1 cells via regulation of the Wnt/β-catenin signaling pathway.

PMID:
24733069
PMCID:
PMC4013624
DOI:
10.3390/ijms15046224
[Indexed for MEDLINE]
Free PMC Article

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