Tumour necrosis factor-α promotes BMHSC differentiation by increasing P2X7 receptor in oestrogen-deficient osteoporosis

Abstract

The exact mechanism of tumour necrosis factor α (TNF-α) promoting osteoclast differentiation is not completely clear. A variety of P2 purine receptor subtypes have been confirmed to be widely involved in bone metabolism. Thus, the purpose of this study was to explore whether P2 receptor is involved in the differentiation of osteoclasts. Mouse bone marrow haematopoietic stem cells (BMHSCs) were co-cultured with TNF-α to explore the effect of TNF-α on osteoclast differentiation and bone resorption capacity in vitro, and changes in the P2 receptor were detected at the same time. The P2 receptor was silenced and overexpressed to explore the effect on differentiation of BMHSCs into osteoclasts. In an in vivo experiment, the animal model of PMOP was established in ovariectomized mice, and anti-TNF-α intervention was used to detect the ability of BMHCs to differentiate into osteoclasts as well as the expression of the P2 receptor. It was confirmed in vitro that TNF-α at a concentration of 20 ng/mL up-regulated the P2X7 receptor of BMHSCs through the PI3k/Akt signalling pathway, promoted BMHSCs to differentiate into a large number of osteoclasts and enhanced bone resorption. In vivo experiments showed that more P2X7 receptor positive osteoclasts were produced in postmenopausal osteoporotic mice. Anti-TNF-α could significantly delay the progression of PMOP by inhibiting the production of osteoclasts. Overall, our results revealed a novel function of the P2X7 receptor and suggested that suppressing the P2X7 receptor may be an effective strategy to delay bone formation in oestrogen deficiency-induced osteoporosis.

Keywords: P2X7; osteoclast; osteoporosis; tumour necrosis factor-α.

Figure 1

The results of in vitro experiments confirmed that TNF‐α at 20 ng/mL significantly up‐regulated the expression of P2X7 receptor protein of BMHSCs and promoted their differentiation into more osteoclasts. A, BMHSCs were stimulated by different concentrations of TNF‐α to differentiate into osteoclasts, and then TRAP staining was used for statistical comparison (n = 5, scale bars were 100 μm). B, BMHSCs were stimulated by a 20 ng/mL concentration of TNF‐α to differentiate into osteoclasts, and then stained with TRAP for statistical comparison (n = 5, scale bars were 100 μm). C, The mRNA of P2 receptors in BMHSCs were detected by qRT‐PCR after stimulation by different concentrations of TNF‐α for different times (n = 5). D, P2X7 receptor protein of BMHSCs was detected by Western blot after stimulation by different concentrations of TNF‐α for different times (n = 5). *P < .05, **P < .01, ***P < .001

Figure 2

The results of an in vitro experiment showed that TNF‐α was involved in the differentiation of BMHSCs into osteoclasts by up‐regulating the P2X7 receptor. A, The silencing effect of transfected shRNA‐P2X7 was detected by Western blot (n = 5). B, The specific proteins related to osteoclast differentiation expressed by BMHSCs transfected with shRNA‐P2X7 were detected by Western blot, including MMP‐9, Cathepsin K, TRAP, and C‐Src (n = 5). C, The differentiation of osteoclasts from BMHSCs transfected with shRNA‐P2X7 was detected by TRAP staining and statistically analysed (n = 5, scale bars were 100 μm). D, After BMHSCs differentiated into osteoclasts, they were stained with cyclopeptide and immunofluorescence, and the number of osteoclasts and P2X7 receptors were counted (P2X7‐Green) (n = 5, scale bars were 100 μm). E, The experiment of osteoclast bone resorption was carried out with bovine bone slices and a biomimetic bone coated petri dish (two different methods). And the bone resorption areas of the two materials were analysed quantitatively (n = 5, scale bars were 100 μm). *P < .05, **P < .01, ***P < .001

Figure 3

TNF‐α up‐regulates the expression of P2X7 through the PI3k/Akt signalling pathway, thus promoting osteoclast differentiation induced by RANKL. A, The transient phosphorylation of the signalling pathway proteins in BMHSCs stimulated by TNF‐α was detected by Western blot (n = 5). B, The quantitative data of the phosphorylation change of instantaneous signal protein in (A) were presented (n = 5). C, BMHSCs were stimulated by Ly294002, an inhibitor of the PI3k/Akt signal pathway, and the differentiated osteoclasts were stained with TRAP (n = 5, scale bars were 100 μm). D, Changes of PI3k/Akt protein phosphorylation and NFATc1 protein expression in BMHSCs stimulated by Ly294002 were detected by Western blot and analysed statistically (n = 5). E, Changes of P2X7 receptor mRNA and protein expression in BMHSCs stimulated by inhibitor Ly294002/activator HY101625 were detected by Western blot and analysed statistically (n = 5). F, The change of number of BMHSCs under the stimulation of different reagents was detected by CCK8 (n = 5). *P < .05, **P < .01, ***P < .001

Figure 4

With increased serum TNF‐α in ovariectomized mice, the number of the osteoclasts increased, while bone trabeculae, bone mineral density and bone content decreased, compared to the SHAM mice. At the same time, the expression of P2X7 in BMHSCs was up‐regulated. Anti‐TNF‐α can effectively inhibit the effect of TNF‐α factor on BMHSCs. A, Randomly selected specimens were scanned by Micro‐CT and reconstructed. The image shows three groups of typical images (n = 4, scale bars were 50 μm). B, Three groups of specimens were stained with HE (scale bars were 200 μm), TRAP (scale bars were 200 μm) and P2X7 protein fluorescence staining (IHC, arrows show a large number of P2X7 protein positive osteoclasts in the OVX group, scale bars were 100 μm). The image shows three groups of typical images (n = 4). C, Quantitative data analysis of Micro‐CT scan reconstruction (Tb.N, BMD, BV/TV), HE staining (HE Tb.N‐area) and TRAP staining (TRAP area) was calculated statistically, and the content of TNF‐α in serum (TNF‐α level) of three groups of mice was detected by ELISA (n = 4). D, The P2X7 receptor mRNA was detected by qRT‐PCR (relative fold changes) and protein expression in bone tissue was detected by Western blot (n = 4). *P < .05, **P < .01, ***P < .001