Neuropeptide Y stimulates osteoblastic differentiation and VEGF expression of bone marrow mesenchymal stem cells related to canonical Wnt signaling activating in vitro
Introduction
Bone is plentifully innervated by small-diameter sensory nerves distributed in bone marrow, where the osteogenic differentiation process of bone marrow mesenchymal stem cells (BMSCs) produces mature bone tissue as well as vascular canals (Imai and Matsusue, 2002, Mach et al., 2002) and plays a vital role in promoting fracture healing by providing blood. Neuropeptides such as calcitonin gene-related peptide (CGRP), substance P (SP) and neuropeptide Y (NPY) are secreted from the peripheral sensory nervous system and participate in various cellular processes such as proliferation, differentiation, inflammation and apoptosis (Mapp et al., 2012, Liu et al., 2008, Decressac et al., 2009). Our previous studies observed that substance P could regulate BMSC proliferation, osteogenic differentiation, apoptosis and vascularization, thus promoting fracture healing (Fu et al., 2014, Mei et al., 2013). The interaction between NPY and sex steroid pathways was recently identified as a novel mechanism for neural signals in bone homeostasis (Allison et al., 2009). In addition, tissue-engineered bone implanted with vascular bundles showed significant increases in the expression of NPY-1R (Chen et al., 2010). Furthermore, bone mass was elevated in Y1 (−/−) mice with increasing bone formation, indicating that NPY-1R signaling inhibited bone formation (Baldock et al., 2007). These findings suggested that NPY was directly involved in the regulation of bone repair while playing a critical role in the metabolism of bone formation. NPY expression in cells of the osteoblast lineage inhibited osteoblast differentiation by acting through the NPY-1R (Igwe et al., 2009). Moreover, NPY inhibited the proliferation and differentiation of BMSCs as well as the activity of mature osteoblasts via the NPY-1R (Lee et al., 2010). However, the stimulatory action of NPY on cell viability of human osteoblasts was greater than that of other neuropeptides (Ma et al., 2013). Furthermore, NPY was reported to promote the proliferation of BMSCs through rejuvenating their growth characteristics (Igura et al., 2011). Therefore, the role of NPY in regulating bone homeostasis remains controversial and incompletely understood. Studying the unknown in vitro function of NPY in regulating osteogenic activity and angiogenic capacity of BMSCs contributes to revealing the physiology of sensory nerves during fracture healing.
The canonical Wnt signaling pathway is vital to osteogenesis (Rawadi and Roman-Roman, 2005). The Wnt/β-catenin pathway is activated when a Wnt ligand binds to the 7-pass transmembrane Frizzled (Fz) receptor and its coreceptors low-density lipoprotein receptor-related protein 5 and 6 (LRP5/6). Signals are generated through proteins such as Axin, disrupting the protein complex and inhibiting the activity of GSK3, thus causing hypophosphorylation of its substrate, β-catenin. Stabilized β-catenin then accumulates in the cytosol and translocates to the nucleus, where this transcriptional coactivator interacts with T cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors to mediate many of the effects of Wnt on gene transcription (MacDonald et al., 2009). Several studies have proved that increasing Wnt signal activation stimulates bone formation, thus leading to increased bone mass (Babij et al., 2003, Kato et al., 2002, Krishnan et al., 2006, Morvan et al., 2006).
Regarding the roles of NPY and the Wnt signaling pathway in mediating osteogenesis, it is likely that the effect of NPY on promoting osteoblastic differentiation of BMSCs may be associated with the activity of the Wnt signaling pathway. This study first addressed whether NPY could promote the expression of osteogenesis markers, as well as the expression of vascular endothelial growth factor (VEGF). This study also evaluated the hypothesis that NPY stimulates BMSCs osteogenic activity via regulation of the Wnt signaling pathway and meanwhile increases the migration and the VEGF expression of BMSCs. The obtained results may aid in understanding the mechanism by which NPY regulates the process of fracture repair.
Section snippets
Cell culture
In each experiment, 6-week-old male rats were sacrificed by CO2 inhalation. Primary BMSCs were then obtained from the bone marrow of the rats' femoral and tibial medullary cavities by flushing with ice-cold L-Dulbecco's Modified Eagle's Medium (L-DMEM) (Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, Australia). The suspension of flushed marrow cells was passed repeatedly through a 22-gauge needle and filtered through a 100-μm cell strainer before culturing. The BMSCs
The role of NPY in stimulating BMSCs osteoblastic differentiation
To identify the osteogenic differentiation of BMSCs, cells were treated with alkaline phosphatase and alizarin red staining after being subjected to the differentiation medium. ALP activity and the presence of calcium deposits in the extracellular matrix indicate that BMSCs possess properties of osteogenic differentiation. The staining results in Fig. 1 demonstrate that the cytoplasm treated with NPY showed higher levels of ALP and alizarin red staining than the control group. NPY treatment (10−
Discussion
NPY at varying levels has previously been reported to play a critical role in the metabolism of bone formation (Baldock et al., 2007, Lee et al., 2010, Ma et al., 2013, Igura et al., 2011, Lundberg et al., 2007, Lee and Herzog, 2009). However, the effect of NPY on regulating osteogenic activity and angiogenic capacity of BMSCs and its mechanism remains controversial. First, the effects of NPY on BMSCs in vitro are not entirely identical to its effects in vivo. NPY has been reported to inhibit
Disclosure
Song Liu, Dan Jin and Jian-qun Wu are joint first authors.
Authors' contribution
Song Liu, Dan Jin and Jian-qun Wu contributed equally to this work.
Conflicts of interest
The authors declare that they have no conflict of interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (81171723).
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