Silencing NTPDase3 activity rehabilitates the osteogenic commitment of post-menopausal stem cell bone progenitors

Abstract

Background Endogenously released adenine and uracil nucleotides favour the osteogenic commitment of bone marrow-derived mesenchymal stromal cells (BM-MSCs) through the activation of ATP-sensitive P2X7 and UDP-sensitive P2Y6 receptors. Yet, these nucleotides have their osteogenic potential compromised in post-menopausal (Pm) women due to overexpression of nucleotide metabolizing enzymes, namely NTPDase3. This prompted us to investigate whether NTPDase3 gene silencing or inhibition of its enzymatic activity could rehabilitate the osteogenic potential of Pm BM-MSCs. Methods MSCs were harvested from the bone marrow of Pm women (69 ± 2 years old) and younger female controls (22 ± 4 years old). The cells were allowed to grow for 35 days in an osteogenic-inducing medium in either the absence or the presence of NTPDase3 inhibitors (PSB 06126 and hN3-B3s antibody); pre-treatment with a lentiviral short hairpin RNA (Lenti-shRNA) was used to silence the NTPDase3 gene expression. Immunofluorescence confocal microscopy was used to monitor protein cell densities. The osteogenic commitment of BM-MSCs was assessed by increases in the alkaline phosphatase (ALP) activity. The amount of the osteogenic transcription factor Osterix and the alizarin red-stained bone nodule formation. ATP was measured with the luciferin-luciferase bioluminescence assay. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC Results The extracellular catabolism of ATP and UDP was faster in BM-MSCs from Pm women compared to younger females. The immunoreactivity against NTPDase3 increased 5.6-fold in BM-MSCs from Pm women vs. younger females. Selective inhibition or transient NTPDase3 gene silencing increased the extracellular accumulation of adenine and uracil nucleotides in cultured Pm BM-MSCs. Downregulation of NTPDase3 expression or activity rehabilitated the osteogenic commitment of Pm BM-MSCs measured as increases in ALP activity, Osterix protein cellular content and bone nodule formation; blockage of P2X7 and P2Y6 purinoceptors prevented this effect. Conclusions Data suggest that NTPDase3 overexpression in BM-MSCs may be a clinical surrogate of the osteogenic differentiation impairment in Pm women. Thus, besides P2X7 and P2Y6 receptors activation, targeting NTPDase3 may represent a novel therapeutic strategy to increase bone mass and reduce the osteoporotic risk of fractures in Pm women.

Mechanical stimulation-induced purinome priming fosters osteogenic differentiation and osteointegration of mesenchymal stem cells from the bone marrow of post-menopausal women

“…Bone marrow samples were obtained from the neck of the femur of thirteen Pm women (70 ± 3 years old) undergoing total hip arthroplasty to resolve non-inflammatory degenerative osteoarthrosis. Handling of bone marrow samples and culture of adherent cells was performed until near confluence for 10–15 days, as previously described [ 8 – 10 , 18 ]. In brief, bone marrow samples were placed immediately in fresh-frozen α-minimal essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B (standard culture medium) and transported to the laboratory on the day or following day of surgery.…”

“…Cells proliferation and differentiation were assessed at culture days 7, 14 and 21; bone nodules formation were evaluated at culture day 35. Protein was collected from the cultures at day 21 for Western blot analysis of osterix (OSX) and osteopontin (OPN), as previously described [ 9 , 10 ]. Intra- and extracellular ATP amounts were evaluated using the quinacrine staining assay and the luciferin-luciferase ATP bioluminescence assay, respectively, at culture days 7 and 21.…”

“…Although the bone fracture risk in post-menopausal (Pm) women might result from excessive bone resorption, our research group demonstrated that osteoprogenitor bone marrow-derived MSCs (BM-MSCs) retain their proliferative potential throughout life, but their ability to differentiate into bone-forming osteoblasts is severely compromised [ 8 – 10 ]. Among many other local factors, adenine and uracil nucleotides, namely ATP and UDP, are paramount to regulate the osteogenic commitment of BM-MSCs, via the activation of ionotropic P2X7 and metabotropic P2Y 6 purinoceptors, respectively (for a review, see [ 11 ]).…”

“…Among many other local factors, adenine and uracil nucleotides, namely ATP and UDP, are paramount to regulate the osteogenic commitment of BM-MSCs, via the activation of ionotropic P2X7 and metabotropic P2Y 6 purinoceptors, respectively (for a review, see [ 11 ]). Interestingly, we found that the osteogenic activity of these two purinoceptors is diminished in BM-MSCs from Pm women compared to younger females and aged-matched men ([ 8 – 10 ]; reviewed in [ 11 ]). Experimental data using Pm BM-MSCs undergoing osteogenic differentiation in culture showed that rehabilitation of the osteogenic commitment of these cells may be achived through activation of P2X7 and P2Y 6 purinoceptors using enzymatically stable ATP and UDP analogues, but not when the cells were treated with the native compounds.…”

“…Experimental data using Pm BM-MSCs undergoing osteogenic differentiation in culture showed that rehabilitation of the osteogenic commitment of these cells may be achived through activation of P2X7 and P2Y 6 purinoceptors using enzymatically stable ATP and UDP analogues, but not when the cells were treated with the native compounds. These findings, together with the discovery that Pm MSCs overexpress the nucleotide hydrolysing enzyme, NTPDase3 [ 10 ], explain the failure of endogenous adenine and uracil nucleotides to reach high enough extracellular concentrations to activate membrane-bound purinoceptors, which is needed to trigger the osteogenic differentiation of these cells [ 12 , 13 ].…”