NPY and BMSCs
BMSCs, isolated from bone marrow, are termed as a “fibroblast-like” osteogenic cell population. With plastic-adherent culture characteristics, BMSCs express specific surface antigens CD105 and CD90 (> 95%) but not CD45 or CD34 (< 2%), and are capable of differentiating into osteoblasts, chondrocytes, and adipocytes[19,46]. As BMSCs play a vital role in repairing musculoskeletal tissues, fracture healing and spinal injury regeneration can be accelerated following application of BMSCs. Local injection of BMSCs has also yielded promising results in the treatment of bone nonunion and bone defect.
Currently, conflicting results still exist regarding the potential role of NPY in proliferation of BMSCs[8,36,42]. Our previous study revealed that NPY can enhance proliferation and prevent apoptosis of BMSCs in a concentration-dependent manner by activating the Wnt/β-catenin signaling pathway. Such NPY-induced activities were partially blocked by PD160170, a Y1R antagonist, implying that NPY-induced proliferative and anti-apoptotic effects of BMSCs may be partially achieved through Y1R. Another study also found that NPY is able to stimulate proliferation of BMSCs derived from rats of different ages, and this capacity was blocked by Y5R antagonist, demonstrating an inhibitory role of Y5R in the proliferation of rat BMSCs. In addition, Igura et al found that NPY increased the proliferation of BMSCs of transgenic overexpression of Y5R in the elder rats by activating extracellular signal-regulated kinase 1/2 (Erk1/2) pathways. Aside from BMSCs, NPY can also promote the proliferation of human embryonic stem cells, which was achieved via NPY/Y1R/Y5R by activation of ERK1/2 pathways. However, one study failed to find a promotive role of NPY on BMSC proliferation. Lee et al reported that BMSCs isolated from Y1R (-/-) mice formed a greater number and larger size of colonies than the WT controls, implying that NPY may inhibit BMSC proliferation.
Apart from its potential influences on BMSC proliferation, NPY can also facilitate the migration of BMSCs by upregulated expression of CXC chemokin receptor 4, which is in accordance with the finding of our study that NPY therapy significantly increased the total migration distance and speed of BMSCs.
In addition to the controversy regarding the effect of NPY on the proliferation of BMSCs, the potential activities of NPY on osteogenic differentiation of BMSCs and the underlying mechanisms are also in debate. Some studies found that NPY stimulated the differentiation of BMSCs into osteoblasts, which was supported by upregulating the expression of alkaline phosphatase (ALP), collagen type I (COL-I), osteocalcin (OCN), and runt-related transcription factor 2 (Runx2) through the Wnt signaling pathway[51,52]. Other studies reported that NPY inhibited osteogenic differentiation of BMSCs, evidenced by the findings of decreased ALP and OCN expression, and reduced mineralization of BMSCs as well. Besides, another study also found the inhibitory effect of NPY on isoprenaline-induced differentiation into osteoblasts from BMSCs. Interestingly, BMSCs isolated from NPY (-/-) mice display an increased ability in osteogenic differentiation, which was confirmed by increases in ALP activity, OCN gene expression, and mineralization. These diverse outcomes can be explained by the fact that NPY-induced osteogenic differentiation of BMSCs may be achieved via auto-regulation mechanisms.
Growing evidence has revealed that auto-regulation mechanisms of NPY may correlate to the plasticity of its receptors. It has been noted that NPY led to upregulated expression of Y1R throughout BMSC osteogenic differentiation[36,38,41]. In a recent study, Wee et al found that during osteogenic differentiation of BMSCs in the WT mice, the expression of Y1R was increased while NPY was decreased. However, as in NPY (-/-) mice, Y1R expression level did not alter during differentiation, demonstrating that increased Y1R expression during BMSC differentiation may be assisted or induced by NPY. Outcomes of NPY or its receptor gene knockout animal models revealed that the presence of functional Y1R directly hindered the osteogenesis of BMSCs or bone cells, as evidenced by the finding that BMSCs isolated from Y1R (-/-) mice displayed an increased capacity of osteogenic differentiation. An in vitro experiment showed that blockade of Y1R by PD160170 facilitated osteogenic differentiation of BMSCs. On the contrary, Dong et al reported that melatonin can upregulate the expression of NPY and Y1R, and promote MSC osteoblastic differentiation. Apart from BMSCs, lack of Y1R also promoted differentiation of mesenchymal progenitor cells and activated mature osteoblasts. Yahara et al also noticed that inhibition of Y1R increased the ALP activity and mineralization in mouse pre-osteoblast MC3T3-E1 cells.
Aside Y1R, Y2R may also participate in osteogenic differentiation of BMSCs, which is supported by the finding that BMSCs, treated with either NPY1–36 (universal Y2R agonist) or PYY3-36 (Y2R preferring agonist), revealed significantly elevated levels of ALP activity and OCN expression. Such effects of NPY1-36 were blocked by a Y2R antagonist, BII0246, and a marked decrease of Y1R protein level was also found following treatment with exogenous PYY3–36 or NPY1–36.
During osteogenic differentiation of BMSCs, potential interactions may exist between Y1R and Y2R[42,43]. By detecting the NPY ligand-receptor system in BMSCs derived from rats of different ages, Igura et al found that, although NPY expression increased with age, Y1R protein was upregulated whereas Y2R protein was downregulated with an increase in age. In addition, Lundberg et al found that the lack of Y2R signaling mediated downregulation of Y1R in BMSCs, which was proved by the finding of decreased expression of Y1R in BMSCs from Y2R (-/-) mice. They inferred that downregulation of Y1R is possibly due to the lack of feedback inhibition of NPY release and resulted in elevated levels of NPY, which in turn caused subsequent overstimulation of Y1R, following desensitization and downregulation of the Y1R population. Moreover, alternation of Y1R expression may have different effects on NPY. The precursor osteogenic cells (e.g., BMSCs and osteoblast precursor cells), which are able to inhibit osteogenic differentiation through Y1R, have low expression levels of Y1R. However, in osteogenic conditions (e.g., osteogenic induction medium), Y1R expression in these cells is increased, with Y2R expression decreased.
In addition to Y1R and Y2R, y6R may also be involved in the regulation of BMSC differentiation into osteoblasts. BMSCs isolated from y6R (-/-) mice showed significant reductions of ALP, osterix, and mineralizing surface, implying possibly opposite roles of Y6R to those of Y1R and Y2R in BMSC differentiation and activities.
Currently, some studies also reported potential mechanisms of NPY in mediation of BMSC osteogenic differentiation from another perspectives. Gu et al indicated that NPY can directly promote the osteogenic differentiation of MSCs by upregulating Runx2. Ma et al reported that the anabolic activity of osteoblasts treated with NPY may also enhance the gap junction intercellular communication (GJIC). Tang et al noticed that the levels of NPY and p-ERK1/2 in fracture model rats were significantly higher than those in the controls. They also found that use of BIBP3226, a Y1R antagonist, inhibited the fracture healing process by downregulating the p-ERK expression in the fracture site, indicating that the ERK pathway may participate in the NPY-induced effects on fracture healing. Additionally, Sharma et al also observed that activation of ERK could facilitate osteogenic differentiation of human MSCs.
In summary, considering the still existing controversies regarding the role of NPY in BMSCs and still not-well-understood mechanisms, future more studies are warranted to provide more definitive evidence regarding the links between NPY/NPY receptors and BMSCs.
NPY and HSCs
HSCs, or hematopoietic stem/progenitor cells, responsible for regeneration and repopulation of all blood cell lineages, contact osteoblasts in endosteal microenvironments or sinusoidal endothelium. NPY receptors Y1, Y2, Y4, and Y5 were found to be highly expressed in HSCs, and NPY plays a crucial role in the proliferation and mobilization of HSCs[59,60].
Growing evidence has suggested that NPY acts directly or indirectly in the regulation of HSC proliferation. NPY can directly inhibit the proliferation of HSCs in cell cultures, as confirmed by an increased number of HSCs in G0 phase, with decreased numbers of cells in S and G2/M phases. Besides, the number of HSCs in NPY (-/-) mice is decreased, suggesting that NPY may protect HSCs in the bone marrow microenvironment[9,61].
In addition to potential influences on HSC proliferation, NPY and its receptors are also involved in regulating the survival and mobilization of HSCs. It was reported that NPY/Y1R can improve the survival and apoptosis of HSCs, which maintains the survival of nestin+ cells, and thus be linked to retention of HSCs. NPY-induced biological effects on HSCs, osteoblasts, and osteoclasts play an important role in the process of fracture healing. Nonetheless, detailed mechanisms of such effects remain largely unclear. Several studies explored the underlying mechanisms from different perspectives. Park et al indicated that the effect of NPY on HSC mobilization might be achieved by increasing the expression levels of matrix metalloproteinase 9 (MMP-9) in osteoblasts via Y1R, which was strengthened by the finding that NPY failed to display a positive activity on HSC mobilization in mice with Y1R (-/-) in osteoblasts. Singh et al reported that NPY3-36, a agonist of Y2R and Y5R, facilitated the mobilization of HSCs to the peripheral blood, whereas selective Y2R and Y5R antagonists hindered such activity. It has been noted that NPY can be also secreted by HSCs, indicating that HSCs may exert regulatory feedback on itself by releasing NPY. In ovariectomized mice, NPY therapy could help reduce the bone loss due to HSC mobilization, and result in an increased number of osteoblasts and a decreased number of osteoclasts. Using an ovariectomy-induced osteoporosis mouse model, Park et al also found that NPY-based recombinant peptides could relieve ovariectomy-induced bone loss and may be used for osteoporosis treatment.
In summary, current evidence suggests that NPY is able to induce the rapid mobilization of HSCs into the peripheral blood, and increase the number of osteoblasts. Therefore, it is reasonable to believe that a comprehensive understanding of the role of NPY in HSCs may pave the way for its future clinical applications.