No 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.

Substance P (SP) mediates multiple activities in various cell types, such as proliferation, anti-apoptotic response, and inflammation. We have investigated the effects of SP, NK1 antagonist and DKK1 on proliferation of bone marrow stromal stem cells (BMSCs), as well as the underlying mechanism. Isolated BMSCs were exposed to SP (10(-8)  M) (group A), SP + NK1 antagonist (1 µM) (group B), SP + DKK1 (0.2 µg/mL) (group C), or the same amount of PBS (group D). Expression of gene and protein of Wnt/β-catenin signalling was detected using quantitative PCR and western blotting. SP (10(-8)  M) significantly enhanced the proliferation of BMSCs and the number of viable cells was reduced by treatment with NK1 antagonist (1 µM) or DKK1 (0.2 µg/mL). SP also significantly increased the expression of C-myc mRNA, Lef1, β-catenin protein and C-myc protein, but decreased the expression of Tcf7 and p-β-catenin protein compared to group D. These roles of SP were inhibited by the NK1 antagonist and DKK1. Expression of CyclinD1 and β-catenin mRNAs, however, was not significantly influenced by SP, NK1 antagonist and DKK1. These findings suggest that SP enhances BMSC proliferation via regulation of the Wnt/β-catenin signalling pathway.