Substance P Activates the Wnt Signal Transduction Pathway and Enhances the Differentiation of Mouse Preosteoblastic MC3T3-E1 Cells

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.

Potential of delphinidin-3-rutinoside extracted from Solanum melongena L. as promoter of osteoblastic MC3T3-E1 function and antagonist of oxidative damage

“…We can only speculate that D3R could increase osteoblast differentiation by activating the Wnt/β-catenin signalling pathway, since we have only data regarding the D3R-induced upregulation of β-catenin mRNA and not β-catenin protein levels. This is a limitation of our study since it has been reported that the amount of βcatenin protein levels depends on the state of β-catenin destruction complex rather than the expression of β-catenin mRNA [46]. Considering the complexity of the Wnt/ β-catenin signalling pathway, further studies will be required to examine the effects of D3R on β-catenin protein levels both in the cytoplasm and in the nucleus and its ability to inactivate the destruction complex leading to GSK-3β phosphorylation and inactivation.…”

Protective Effect of Neuropeptide Substance P on Bone Marrow Mesenchymal Stem Cells against Apoptosis Induced by Serum Deprivation

“…The canonical Wnt pathway has been activated by SP in order to promote the proliferation and differentiation of BMSCs [ 16 , 34 , 35 ]. This current study illustrated the active canonical Wnt pathway could be induced by SP in the serum-deprived BMSCs.…”

Short Anabolic Peptides for Bone Growth

“…Another study demonstrated SP's in vitro anabolic effect on mouse pre‐OB MC3T3‐E1 cell line whereby it upregulated the expressions of collagen type 1, ALP, Runx2 and osteocalcin at day 4. This effect was shown to involve the activation of Wnt/β‐catenin signalling pathway . Recently, Fu et al.…”

“…This effect was shown to involve the activation of Wnt/β-catenin signalling pathway. 218 Recently, Fu et al demonstrated that SP (10 −5 mM) promotes differentiation and migration capability of rat bone marrow MSCs and activates r AMSO, CORNISH, AND BRIMBLE BMP-2 expression in OBs. 219 The peptide also exerts an inhibitory effect on serum deprivationinduced apoptosis in bone marrow MSCs.…”