Ablation of Enpp6 results in transient bone hypomineralization

C.F. was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) via an Institute Strategic Programme Grant Funding (BB/J004316/1). S.D. was supported through a BBSRC EASTBIO Doctoral Training Partnership studentship award (1803936) and N.M.M. was supported by a Wellcome Trust New Investigator Award (100981/Z/13/Z). S.D. wrote the manuscript. S.D., K.S., S-N.H., and L.A.S. carried out experimental work. W.P.C., R.W. and N.M.M. provided reagents and materials. A.J.S., F.N. and C.F. contributed to conceptualization of the study and experimental design. All authors reviewed and edited the manuscript and approved the final version. All authors state that they have no conflicts of interest.Biomineralization is a fundamental process key to the development of the skeleton. The phosphatase orphan phosphatase 1 (PHOSPHO1), which likely functions within extracellular matrix vesicles, has emerged as a critical regulator of biomineralization. The biochemical pathways which generate intravesicular PHOSPHO1 substrates are however currently unknown. We hypothesized that the enzyme ectonucleotide pyrophosphatase/phosphodiesterase (ENPP6) is an upstream source of PHOSPHO1 substrate. To test this, we characterized skeletal phenotypes of mice homozygous for a targeted deletion of Enpp6 (Enpp6‒/‒). Micro-computed tomography of the trabecular compartment revealed transient hypomineralization in Enpp6‒/‒ tibiae (p 0.01) and osteoid surface (p < 0.05) which recovered by 12 weeks but was not accompanied by changes in osteoblast or osteoclast number. This study is the first to characterize the skeletal phenotype of Enpp6‒/‒ mice, revealing transient hypomineralization in young animals compared to wild-type controls. These data suggest that ENPP6 is important for bone mineralization and may function upstream of PHOSPHO1 as a novel means of generating its substrates inside matrix vesicles.Publisher PDFPeer reviewe

An Investigation of the Mineral in Ductile and Brittle Cortical Mouse Bone

Bone is a strong and tough material composed of apatite mineral, organic matter, and water. Changes in composition and organization of these building blocks affect bone's mechanical integrity. Skeletal disorders often affect bone's mineral phase, either by variations in the collagen or directly altering mineralization. The aim of the current study was to explore the differences in the mineral of brittle and ductile cortical bone at the mineral (nm) and tissue (µm) levels using two mouse phenotypes. Osteogenesis imperfecta model, oim(-/-) , mice have a defect in the collagen, which leads to brittle bone; PHOSPHO1 mutants, Phospho1(-/-) , have ductile bone resulting from altered mineralization. Oim(-/-) and Phospho1(-/-) were compared with their respective wild-type controls. Femora were defatted and ground to powder to measure average mineral crystal size using X-ray diffraction (XRD) and to monitor the bulk mineral to matrix ratio via thermogravimetric analysis (TGA). XRD scans were run after TGA for phase identification to assess the fractions of hydroxyapatite and β-tricalcium phosphate. Tibiae were embedded to measure elastic properties with nanoindentation and the extent of mineralization with backscattered electron microscopy (BSE SEM). Results revealed that although both pathology models had extremely different whole-bone mechanics, they both had smaller apatite crystals, lower bulk mineral to matrix ratio, and showed more thermal conversion to β-tricalcium phosphate than their wild types, indicating deviations from stoichiometric hydroxyapatite in the original mineral. In contrast, the degree of mineralization of bone matrix was different for each strain: brittle oim(-/-) were hypermineralized, whereas ductile Phospho1(-/-) were hypomineralized. Despite differences in the mineralization, nanoscale alterations in the mineral were associated with reduced tissue elastic moduli in both pathologies. Results indicated that alterations from normal crystal size, composition, and structure are correlated with reduced mechanical integrity of bone

Follistatin-like 3 (FSTL3) mediated silencing of transforming growth factor (TGF ) signaling is essential for testicular aging and regulating testis size

Follistatin-like 3 (FSTL3) is a glycoprotein that binds and inhibits the action of TGFβ ligands such as activin. The roles played by FSTL3 and activin signaling in organ development and homeostasis are not fully understood. The authors show mice deficient in FSTL3 develop markedly enlarged testes that are also delayed in their age-related regression. These FSTL3 knockout mice exhibit increased Sertoli cell numbers, allowing for increased spermatogenesis but otherwise showing normal testicular function. The data show that FSTL3 deletion leads to increased AKT signaling and SIRT1 expression in the testis. This demonstrates a cross-talk between TGFβ ligand and AKT signaling and leads to a potential mechanism for increased cellular survival and antiaging. The findings identify crucial roles for FSTL3 in limiting testis organ size and promoting age-related testicular regression

Reference point indentation is not indicative of whole mouse bone measures of stress intensity fracture toughness

AbstractBone fragility is a concern for aged and diseased bone. Measuring bone toughness and understanding fracture properties of the bone are critical for predicting fracture risk associated with age and disease and for preclinical testing of therapies. A reference point indentation technique (BioDent) has recently been developed to determine bone's resistance to fracture in a minimally invasive way by measuring the indentation distance increase (IDI) between the first and last indentations over cyclic indentations in the same position. In this study, we investigate the relationship between fracture toughness KC and reference point indentation parameters (i.e. IDI, total indentation distance (TID) and creep indentation distance (CID)) in bones from 38 mice from six types (C57Bl/6, Balb, oim/oim, oim/+, Phospho1−/− and Phospho1 wild type counterpart). These mice bone are models of healthy and diseased bone spanning a range of fracture toughness from very brittle (oim/oim) to ductile (Phospho1−/−). Left femora were dissected, notched and tested in 3-point bending until complete failure. Contralateral femora were dissected and indented in 10 sites of their anterior and posterior shaft surface over 10 indentation cycles. IDI, TID and CID were measured. Results from this study suggest that reference point indentation parameters are not indicative of stress intensity fracture toughness in mouse bone. In particular, the IDI values at the anterior mid-diaphysis across mouse types overlapped, making it difficult to discern differences between mouse types, despite having extreme differences in stress intensity based toughness measures. When more locations of indentation were considered, the normalised IDIs could distinguish between mouse types. Future studies should investigate the relationship of the reference point indentation parameters for mouse bone in other material properties of the bone tissue in order to determine their use for measuring bone quality

Comparative analysis of β-actin expression is highly variable across a broad range of tissues.

<p>A. Representative images of the tissue samples in which actin expression was assessed. From left to right: Muscle (Gastrocnemius), heart, bone (femur), calvaria, spleen and fat (gonadal). Scale bar = 1 cm. B. LICOR image of QWB demonstrating considerable variability of β-actin expression (green) in muscle, heart, bone, calvaria, spleen and fat extracts. Total protein stain gel image (red) is overlaid on QWB as a control. C. Total protein measurements for different molecular weight ranges demonstrates the accuracy of protein loading across the different tissue samples. D. Stacked bar graph demonstrating the comparative variability of β-actin (green bars) and total protein measurements (red bars) for each tissue examined.</p

Linear range and sensitivity of total protein stain is greater than the conventional loading controls β-actin or β-tubulin.

<p>(A) Representative LICOR image for a protein dilution series of whole brain homogenate 1, 5, 10, 20, 30, 40 µg demonstrating the working range of β-actin and β-tubulin when using QWB. (B) Quantification of protein dilution series showing the linear ranges of β-actin (black circle) and β-tubulin (open triangle). Note that tubulin expression appears to saturate at less than 10 ug of brain homogenate. (C) In order to pinpoint the saturation level a tubulin specific protein dilution series over a smaller range (0.5, 1, 2, 4, 6, 8, 10,12 and 14 µg) establishing the saturation point of β-tubulin when using QWB. (D) Quantification of β-tubulin linear range. (E) Total protein stain of dilution series 2, 10, 20, 40, 80 µg made using the bovine serum albumin standard (2 µg/µl) from the Pierce BCA kit (see methods). BSA molecular weight is 66.5 kDa. Imaging of this dilution series demonstrates imaging of a broad concentration range without saturation at a single protein mass. (F) Graphical representation of quantification from BSA dilution series in panel E. This demonstrates wide linear detection and high correlation (0.998) validating the use of total protein measurements as a viable method for detecting protein load using the LICOR system. (G) Total protein stain of whole brain homogenate dilution series 1, 5, 10, 20, 30, 40 µg demonstrates the broad concentration range detectable without saturation. (H) Correlation between the total protein stain quantification (red line) and the BCA OD (blue line) for the protein dilution series demonstrates wide linear detection and high correlation (0.996 & 0.979 respectively) validating the use of total protein measurements as a viable “loading control” for QWB using the LICOR system.</p

Examination of publicly available mass screening data reveals alterations in expression levels of commonly used “loading control” proteins.

<p>Note: Alterations listed in table are in units published in the original manuscript or accompanying supplementary material.</p

Actin & NF-L levels are not stable throughout different regions of the mouse sciatic nerve.

<p>Differential expression in proximal versus distal sciatic nerve preparations. A. Photograph depicting the lower half of a Bl6 mouse with sciatic nerve exposed on the right leg. B. Higher magnification photograph shows sciatic nerve and subsequent branches (anatomical nomenclature taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072457#pone.0072457-Silva1" target="_blank">[26]</a>). Scale bar: A = 1 cm, B = 0.5 cm. C. Representative LICOR overlay image of β-actin QWB (green) and total protein stained gel (red) in proximal and distal portions of sciatic nerve from the same mouse. D. Representative LICOR overlay image of NF-L QWB (green) and total protein stained gel (red) in proximal and distal portions of sciatic nerve from the same mouse. E. Stacked bar graph demonstrating the comparative expression of β-actin (green bars) and total protein stain (red bars) expression in proximal and distal sections of sciatic nerve. F. Comparative expression of NF-L (green bars) and total protein stain (red bars) expression in proximal and distal sections of sciatic nerve.</p