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The role of extracellular matrix phosphorylation on energy dissipation in bone

  1. Stacyann Bailey  Is a corresponding author
  2. Grazyna E Sroga
  3. Betty Hoac
  4. Orestis L Katsamenis
  5. Zehai Wang
  6. Nikolaos Bouropoulos
  7. Marc D McKee
  8. Esben S Sørensen
  9. Philipp J Thurner
  10. Deepak Vashishth  Is a corresponding author
  1. Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, United States
  2. Faculty of Dentistry, McGill University, Canada
  3. Faculty of Engineering and Physical Sciences, University of Southampton, United Kingdom
  4. Department of Mechanical, Aerospace, and Nuclear Engineering, Rensselaer Polytechnic Institute, United States
  5. Department of Material Science, University of Patras, Greece
  6. Department of Anatomy and Cell Biology, Faculty of Medicine, McGill University, Canada
  7. Department of Molecular Biology and Genetics, Aarhus University, Denmark
  8. Institute of Lightweight Design and Structural Biomechanics, Vienna University of Technology, Austria
Research Article
Cite this article as: eLife 2020;9:e58184 doi: 10.7554/eLife.58184
8 figures, 1 table and 5 additional files

Figures

Pre-immunoprecipation (Pre-IP) of mineral-bound OPN.

(a) and global phosphorylation (b) in protein extracts of long bones from WT, Hyp and Fgf23-/- mice. Post-immunoprecipation (Post-IP) indicates that despite similar levels of OPN (c), phosphorylation of OPN is reduced in these disease models (d).

Mean global protein phosphorylation.

(a) and change in phosphorylation (b) for WT and Opn KO groups. * indicates significance at p<0.05 and error bars represent standard deviation.

Mean global protein phosphorylation.

(a) and change in phosphorylation (b) after removal of phosphate groups (dephosphorylation) for WT and Opn KO groups. * indicates significance at p<0.05 and error bars represent standard deviation.

Mean fracture toughness (a) and change in fracture toughness (b) due to ex-vivo phosphorylation for WT and Opn KO groups.

* Indicates significance at p<0.05 and error bars represent standard deviation.

Figure 4—source data 1

Fracture toughness of phosphoryled WT and Opn KO mice.

https://cdn.elifesciences.org/articles/58184/elife-58184-fig4-data1-v3.xlsx
Mean fracture toughness (a) and change in fracture toughness (b) attributable to ex-vivo dephosphorylation for WT and Opn KO groups.

* Indicates significance at p<0.05 and error bars represent standard deviation.

Figure 5—source data 1

Fracture toughness of dephosphoryled WT and Opn KO mice.

https://cdn.elifesciences.org/articles/58184/elife-58184-fig5-data1-v3.xlsx
Figure 6 with 3 supplements
Energy dissipation of OPN networks during AFM-FS experiments.

Energies are normalized to dissipation levels in EDTA for OPN deposited on mica and pulled with a pristine AFM tip (pH 7.4) and to dissipation levels in H2O for OPN deposited on HA and pulled with a HA-functionalized tip. All values are significantly different except OPN between HA, pH 8.5 H2O vs. Na+. It should be noted that the relative differences are similar to what is seen for quantitative values, except for EDTA and H2O levels due to normalization. These values are provided in Supplementary files 1 and 2. * indicates significance at p<0.05 and error bars represent standard error (SE) of the mean.

Figure 6—source data 1

Energy dissipation of native (phosphorylated) and dephosphorylated OPN film on mica in EDTA and calcium solution.

https://cdn.elifesciences.org/articles/58184/elife-58184-fig6-data1-v3.xlsx
Figure 6—source data 2

Energy dissipation of native (phosphorylated) OPN film on HA under various pH and ionic conditions.

https://cdn.elifesciences.org/articles/58184/elife-58184-fig6-data2-v3.xlsx
Figure 6—figure supplement 1
Back‑scattered electron image of an AFM probe and HA surface.
Figure 6—figure supplement 2
Representative force spectroscopy curve of hydrated OPN.
Figure 6—figure supplement 3
Proposed model for the OPN-HA interaction in different ionic- and pH environments.
Schematic diagram showing differential effects of phosphorylation on conformation of protein systems.

In protein system (A), phosphorylation tends to increase inter- and intrafilament interactions, hence the interfilament distance is reduced. In protein system (B), phosphorylation tends to create interfilament repellant, hence increasing the protein system alignment and inter- filament distance.

Schematic of the relationship between global protein phosphorylation and fracture toughness of wild-type (a) and Opn KO (b) mice.

By continuing the increase in phosphorylation of WT bone, fracture toughness improves exponentially. There is no significant relationship between global phosphorylation and fracture toughness in Opn KO mice following ex-vivo phosphorylation and dephosphorylation.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (M. musculus)C57BL/6NCrlCharles RiverRRID:IMSR_CRL:27
Genetic reagent (M. musculus)B6.Cg-PhexHyp/JJackson LaboratoryCat#: 000528
RRID:IMSR_JAX:000528
Animals maintained in Dr M Mckee lab.
Genetic reagent (M. musculus)Fgf23-/-PMID:15579309Animals were a gift from Dr. B. Lanske
Genetic reagent (M. musculus)B6.129S6(Cg)-Spp1tm1Blh/JPMID:9661074Animals were a gift from Dr S. Rittling.
Genetic Reagent (B. taurus)Milk protein (Mammary gland)PMID:8320368Provided by Dr ES Sorensen
Chemical compound, drugSynthetic hydroxyapatiteAndriotis et al., 2010. Crystal Research and TechnologyProduced by Dr N. Bouropoulos
Commercial assay or kitpIMAGO-biotin HRP DetectionTymora AnalyticalCat# 900–100
Antibodyanti-OPN (goat polyclonal)R and D SystemsCat# AF808, RRID:AB_2194992(1:100,000 µL)
Antibodyanti-phosphoserine (rabbit polyclonal)Thermo Fisher ScientificCat# 61–8100, RRID:AB_2533940(1:2500 µL)

Additional files

Supplementary file 1

Adhesive properties of native (phosphorylated) and dephosphorylated OPN film on mica.

https://cdn.elifesciences.org/articles/58184/elife-58184-supp1-v3.docx
Supplementary file 2

Adhesive properties of native (phosphorylated) OPN film on HA.

https://cdn.elifesciences.org/articles/58184/elife-58184-supp2-v3.docx
Supplementary file 3

Mean maximum force of native (phosphorylated) OPN film on HA.

https://cdn.elifesciences.org/articles/58184/elife-58184-supp3-v3.docx
Supplementary file 4

Mean maximum force of native (phosphorylated) and dephosphorylated OPN film on mica.

https://cdn.elifesciences.org/articles/58184/elife-58184-supp4-v3.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/58184/elife-58184-transrepform-v3.docx

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