P2X7 nucleotide receptor signaling potentiates the Wnt/β-catenin pathway in cells of the osteoblast lineage

Abstract

The P2X7 and Wnt/β-catenin signaling pathways regulate osteoblast differentiation and are critical for the anabolic responses of bone to mechanical loading. However, whether these pathways interact to control osteoblast activity is unknown. The purpose of this study was to investigate the effects of P2X7 activation on Wnt/β-catenin signaling in osteoblasts. Using MC3T3-E1 cells, we found that combined treatment with Wnt3a and the P2X7 agonist 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) elicited more sustained β-catenin nuclear localization than that induced by Wnt3a alone. Wnt3a-induced increases in β-catenin transcriptional activity were also potentiated by treatment with BzATP. Consistent with involvement of P2X7, a high ATP concentration (1 mM) potentiated Wnt3a-induced β-catenin transcriptional activity, whereas a low concentration (10 μM) of ATP, adenosine 5′-diphosphate (ADP), or uridine 5′-triphosphate (UTP) failed to elicit a response. The potentiation of β-catenin transcriptional activity elicited by BzATP was also inhibited by two distinct P2X7 antagonists: A 438079 and A 740003. Furthermore, responses to Wnt3a in calvarial cells isolated from P2rx7 knockout mice were significantly less than in cells from wild-type controls. In MC3T3-E1 cells, BzATP increased inhibitory phosphorylation of glycogen synthase kinase 3β (GSK3β), a process that was blocked by A 438079 and diminished by inhibition of protein kinase C. Thus, P2X7 signaling may potentiate the canonical Wnt pathway through GSK3β inhibition. Taken together, we show that P2X7 activation prolongs and potentiates Wnt/β-catenin signaling. Consequently, cross-talk between P2X7 and Wnt/β-catenin pathways may modulate osteoblast activity in response to mechanical loading.

Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations

“…It is well-known that bone adapts to mechanical loading by increasing its mass. In this regard, activation of P2X7 receptors by extracellular ATP in the millimolar range has been proposed to stimulate anabolic responses in bone (Ke et al, 2003 ; Li et al, 2005 ; Panupinthu et al, 2008 ; Grol et al, 2013 , 2016 ). Therefore, we propose that the second, P2X7-mediated threshold in calcium responses to ATP provides an anabolic signal following robust mechanical stimulation as well as during the early stages of wound healing following trauma.…”

“…Importantly, MC3T3-E1 cells endogenously express multiple subtypes of P2X and P2Y receptors (Gartland et al, 2012 ; Grol et al, 2013 ; Xing et al, 2014 ). Functionally, extracellular nucleotides acting through P2 receptors on MC3T3-E1 cells have been reported to stimulate: cell proliferation (Shimegi, 1996 ); prostaglandin release (Genetos et al, 2005 ); cell metabolism (Grol et al, 2012 ); Ca 2+ -NFATc1 signaling (Grol et al, 2013 ); and the Wnt/β-catenin signaling pathway (Grol et al, 2016 ). Thus, the MC3T3-E1 cell line is an excellent system for examining interactions among endogenously expressed P2 receptors in a physiologically relevant cell type.…”

The potential of P2X7 receptors as a therapeutic target, including inflammation and tumour progression

“…Different polymorphic variants of the P2X7R are associated with high or reduced periprosthetic osteolysis in the long-term complication of total hip arthroplasty due to osteoarthritis [ 192 ]. Cross-talk between P2X7R and Wnt/β-catenin pathways may modulate osteoblast activity in response to mechanical loading [ 193 ]. Treatment with AZD9056, a selective P2X7R antagonist, produced pain-relieving and anti-inflammatory effects in rats with osteoarthritis [ 194 ].…”

Highly‐expressed P2X7 receptor promotes growth and metastasis of human HOS/MNNG osteosarcoma cells via PI3K/Akt/GSK3β/β‐catenin and mTOR/HIF1α/VEGF signaling

“…Moreover, PI3K/Akt/mTOR signaling regulates tumor cell growth and angiogenesis by inducing expression of hypoxia inducible factor (HIF) 1α and VEGF . S. Jeffrey's group recently reported that P2X7 receptor activation upregulated the canonical Wnt/β‐catenin pathway via increased phosphorylation and inhibition of glycogen synthase kinase 3β (GSK3β) in osteoblast‐like cells . Inactivation of GSK3β evokes Wnt/β‐catenin/T‐cell factor (TCF) signaling and mediates Snail expression, which is responsible for epithelial to mesenchymal transition (EMT) .…”

“…23,24 S. Jeffrey's group recently reported that P2X7 receptor activation upregulated the canonical Wnt/β-catenin pathway via increased phosphorylation and inhibition of glycogen synthase kinase 3β (GSK3β) in osteoblast-like cells. 25,26 Inactivation of GSK3β evokes Wnt/β-catenin/T-cell factor (TCF) signaling and mediates Snail expression, which is responsible for epithelial to mesenchymal transition (EMT). 27,28 EMT is characterized by loss of the epithelial marker E-cadherin and increased expression of mesenchymal markers, such as Fibronectin and Vimentin.…”