Research ArticleCELL BIOLOGY

Dysregulation of ectonucleotidase-mediated extracellular adenosine during postmenopausal bone loss

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Science Advances  21 Aug 2019:
Vol. 5, no. 8, eaax1387
DOI: 10.1126/sciadv.aax1387
  • Fig. 1 Deficient CD73 and CD39 expressions and extracellular adenosine concentration in BM of OVX animals.

    (A to K) Characterization of healthy (sham) and OVX animals 4 weeks after ovariectomy. (A) Immunofluorescence staining of CD73 (green) and (B) CD39 (red) in vertebrae of OVX animals. Nuclear staining (blue). Scale bars, 100 μm. Inset shows magnified image of bone surface. Yellow arrowheads indicate cells positive for CD73 or CD39 on bone surface. Scale bars, 50 μm. (C) Flow cytometric analysis of CD73 and CD39 membrane expression of hematopoietic cells from mouse BM cells 4 weeks after ovariectomy (OVX) and healthy controls. (D) Percentage and median fluorescence intensity of hematopoietic cells expressing CD73. (E) Percentage and median fluorescence intensity of hematopoietic cells expressing CD39. (F) Flow cytometric analysis of CD73 and CD39 membrane expression of nonhematopoietic cells from mouse BM cells 4 weeks after ovariectomy and healthy controls. (G) Percentage and median fluorescence intensity of nonhematopoietic cells expressing CD73. (H) Percentage and median fluorescence intensity of nonhematopoietic cells expressing CD39. (I) CD73 gene expression and (J) CD39 gene expression of cells from bone chips. (K) Extracellular adenosine concentration in BM plasma of sham and OVX animals. n = 5. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2 Regulation of CD73 and CD39 cell membrane expressions and extracellular adenosine levels by ERs in osteoprogenitor cells.

    (A) Flow cytometric analyses and (B) quantification of CD73 and CD39 in osteoprogenitors in the absence or presence of E2 (100 nM) for 3 days. (C to E) Single (ESR1 or ESR2) or dual (ESR1 and ESR2) ER knockdown (KD) by siRNA in primary mouse osteoprogenitors and analyzed after 3 days. (C) Flow cytometric analyses of CD73 and CD39 after single knockdown (ESR1 or ESR2) and dual knockdown (ESR1 and ESR2). (D) Percentage of double-positive (CD73/CD39) cells in single knockdown and dual knockdown cells. (E) In vitro adenosine levels normalized by cell number in single knockdown and dual knockdown cells. Control (scrambled) siRNA concentration for single knockdown and dual knockdown are 5 and 10 nM, respectively. n = 5. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 Regulation of CD73 and CD39 cell membrane expression and extracellular adenosine levels by ERs in osteoclasts.

    (A) Flow cytometric analyses and (B) quantification of CD73 and CD39 in primary mouse mononuclear cells undergoing osteoclast differentiation in the absence or presence of E2 (100 nM) for 3 days. (C to E) Single (ESR1 or ESR2) or dual (ESR1 and ESR2) ER knockdown by siRNA during macrophage differentiation for 3 days and subsequent osteoclast differentiation for 6 days. (C) Flow cytometric analyses of CD73 and CD39 after single knockdown (ESR1 or ESR2) and dual knockdown (ESR1 and ESR2). (D) Percentage of double-positive (CD73/CD39) cells in single knockdown and dual knockdown cells. (E) In vitro adenosine levels normalized by cell number in single knockdown and dual knockdown cells. Control (scrambled) siRNA concentration for single knockdown and dual knockdown are 5 and 10 nM, respectively. n = 4. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 4 Adenosine A2BR signaling promote osteogenic and inhibit osteoclast differentiation in vitro.

    (A and B) In vitro knockdown of adenosine A2BR using siRNA in primary mouse osteoprogenitor cells isolated from the BM for 2 days, followed by adenosine treatment (ADO; 30 μg/ml) for 7 or 14 days. Gene expression of (A) osteoblast-specific marker and (B) Opn. (C to F) In vitro knockdown of adenosine A2BR by siRNA in mouse mononuclear cells isolated from BM undergoing macrophage differentiation for 3 days, followed by osteoclast differentiation along with treatment of small-molecule adenosine (30 μg/ml) for 6 days. Gene expressions of (C) osteoclast transcription factor Nfatc1, (D) ACP5, and (E) CTSK. (F) TRAP staining. Second group from the left is no siRNA control, while the third group from the left involves control (scrambled) siRNA. Scale bar, 200 μm. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 Adenosine A2BR agonist BAY 60-6583 attenuates bone loss in OVX animals.

    (A to L) Administration of BAY 60-6583 and vehicle for 8 weeks in OVX animals (4 weeks after ovariectomy). Groups are compared to healthy control with no surgery and no treatment (CTL). (A) TRAP staining (purple). Scale bar, 50 μm. (B) Quantification of TRAP-positive cells on bone surface. n = 4. (C) Double-fluorescence bone labeling by calcein (green) and alizarin complexone (red). Scale bar, 100 μm. (D) Quantification of mineral apposition rate (MAR) from bone labeling images. (E) Quantification of bone formation rate (BFR/BS) from bone labeling images. n = 5. (F) Reconstructed microCT images of L4 vertebra. Scale bar, 500 μm. (G to J) Quantification of microCT images. (G) BMD. (H) BV/TV. (I) Tb.N. (J) Tb.Sp. n = 5. Mechanical measurement for (K) maximum load and (L) stiffness of tibia. n = 12. OV, ovariectomy, vehicle [dimethyl sulfoxide (DMSO)]; OB, ovariectomy, BAY 60-6583 (1 mg/kg). *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaax1387/DC1

    Fig. S1. Measurement of estradiol (E2) and microCT imaging in OVX mice.

    Fig. S2. ER knockdown in osteoprogenitors and immunofluorescent staining of ectonucleotidase expression.

    Fig. S3. ER knockdown in osteoprogenitors and flow cytometric analyses of ectonucleotidase expression.

    Fig. S4. ER knockdown in osteoclasts and immunofluorescent staining of ectonucleotidase expression.

    Fig. S5. ER knockdown in osteoclasts and flow cytometric analyses of ectonucleotidase expression.

    Fig. S6. ER knockdown of BM cells undergoing macrophage differentiation and flow cytometric analyses of ectonucleotidase expression.

    Fig. S7. siRNA knockdown of A2BR and reverse transcriptase quantitative PCR.

    Fig. S8. Immunofluorescent staining of adenosine A2BR expression in vertebra of animals.

    Fig. S9. H&E staining and microCT of BAY 60-6583–treated mice.

    Fig. S10. Immunofluorescent staining of CD73 and CD39 in vertebra of BAY 60-6583–treated animals.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Measurement of estradiol (E2) and microCT imaging in OVX mice.
    • Fig. S2. ER knockdown in osteoprogenitors and immunofluorescent staining of ectonucleotidase expression.
    • Fig. S3. ER knockdown in osteoprogenitors and flow cytometric analyses of ectonucleotidase expression.
    • Fig. S4. ER knockdown in osteoclasts and immunofluorescent staining of ectonucleotidase expression.
    • Fig. S5. ER knockdown in osteoclasts and flow cytometric analyses of ectonucleotidase expression.
    • Fig. S6. ER knockdown of BM cells undergoing macrophage differentiation and flow cytometric analyses of ectonucleotidase expression.
    • Fig. S7. siRNA knockdown of A2BR and reverse transcriptase quantitative PCR.
    • Fig. S8. Immunofluorescent staining of adenosine A2BR expression in vertebra of animals.
    • Fig. S9. H&E staining and microCT of BAY 60-6583–treated mice.
    • Fig. S10. Immunofluorescent staining of CD73 and CD39 in vertebra of BAY 60-6583–treated animals.

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