FASEB J. 2016 Aug;30(8):2837-48. doi: 10.1096/fj.201500027R. Epub 2016 Apr 28.
Pigment epithelium-derived factor restoration increases bone mass and improves bone plasticity in a model of osteogenesis imperfecta type VI via Wnt3a blockade.
Belinsky GS1, Sreekumar B1, Andrejecsk JW2, Saltzman WM2, Gong J1, Herzog RI1, Lin S3, Horsley V3, Carpenter TO4, Chung C5.
Department of Medicine, Yale University School of Medicine, New Haven, Connecticut, USA; Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut, USA chuhan.chung@yale.edu.
Null mutations in for pigment epithelium-derived factor (PEDF), the protein product of the SERPINF1 gene, are the cause of osteogenesis imperfecta (OI) type VI. The PEDF-knockout (KO) mouse captures crucial elements of the human disease, including diminished bone mineralization and propensity to fracture. Our group and others have demonstrated that PEDF directs human mesenchymal stem cell (hMSC) commitment to the osteoblast lineage and modulates Wnt/β-catenin signaling, a major regulator of bone development; however, the ability of PEDF to restore bone mass in a mouse model of OI type VI has not been determined. In this study, PEDF delivery increased trabecular bone volume/total volume by 52% in 6-mo-old PEDF-KO mice but not in wild-type mice. In young (19-d-old) PEDF-KO mice, PEDF restoration increased bone volume fraction by 35% and enhanced biomechanical parameters of bone plasticity. A Wnt-green fluorescent protein reporter demonstrated dynamic changes in Wnt/β-catenin signaling characterized by early activation and marked suppression during terminal differentiation of hMSCs. Continuous Wnt3a exposure impeded mineralization of hMSCs, whereas the combination of Wnt3a and PEDF potentiated mineralization. Interrogation of the PEDF sequence identified a conserved motif found in other Wnt modulators, such as the dickkopf proteins. Mutation of a single amino acid on a 34-mer PEDF peptide increased mineralization of hMSC cultures compared with the native peptide sequence. These results indicate that PEDF counters Wnt signaling to allow for osteoblast differentiation and provides a mechanistic insight into how the PEDF null state results in OI type VI.-Belinsky, G. S., Sreekumar, B., Andrejecsk, J. W., Saltzman, W. M., Gong, J., Herzog, R. I., Lin, S., Horsley, V., Carpenter, T. O., Chung, C. Pigment epithelium-derived factor restoration increases bone mass and improves bone plasticity in a model of osteogenesis imperfecta type VI via Wnt3a blockade.
PEDF increases trabecular bone volume/total volume (BV/TV), the bone volume fraction, in 6-mo-old mouse femurs. A) μCT and Toluidine blue images of adult mouse femurs after treatment with vehicle or PEDF. WT or PEDF-KO as indicated. B) BV/TV and trabecular thickness parameters for adult PEDF-KO mice and WT littermates. Values are expressed as means ± sd; n = 4 mice/group. Original magnification, ×4.
PEDF increases bone mass in young (19-d-old) mice. A) Representative µCT images of femurs from vehicle- and PEDF-treated mice where PEDF restoration enhanced trabecular bone volume. B) Quantification of trabecular bone volumes after PEDF. C) Increased cortical BV/TV and cortical thickness after PEDF treatment. Values are expressed as means ± sd; n = 9 mice/group.
PEDF enhances the plasticity of femurs in PEDF-KO mice. A) Femur length was not different between groups. B) PEDF increased bone stiffness, the slope of the load vs. displacement. C) Maximum load was not different in PEDF-treated mice. D) Displacement at fracture (FX) increased after PEDF treatment. E) Displacement at yield. F) Preyield work. G) Displacement of femurs upon maximum load. H) Postyield deflection, the deformation of bone after the yield point, which is a measure of bone plasticity, increased after PEDF treatment. I) Postyield work. Values are expressed as means ± sd; n = 3 vehicle-treated mice, n = 5 PEDF-treated mice. *P < 0.05 by Student’s t test.
Endogenous PEDF secretion by hMSCs increases with osteogenesis and is suppressed by Wnt3a. A) hMSC transduced with a Wnt-GFP reporter grown in osteogenic or control medium starting at d 0. Fields imaged at indicated days (original magnification, ×10); GFP+ cells counted; n = 5–20 fields per time point. B) Temporal quantitation of endogenously secreted PEDF over 21 d in osteogenic medium, with Coomassie blue–stained gel to show equal loading of conditioned medium. C) Suppression of PEDF secretion in hMSCs at d 21 with continuous Wnt3a exposure. DIC, differential interference contrast. Values are expressed as means ± sd. *P < 0.05 by Student’s t test; **P < 0.0001 compared with control.
Unopposed Wnt3a results in impaired mineralization that is rescued by PEDF and PEDF peptides. A) Alizarin red S staining of 21-d-old hMSC cultures with continuous Wnt3a (50 ng/ml) exposure vs. controls (ctl). B) PEDF amino acid sequence homology of putative Wnt modulating motif (in boxes). Left box contains lysine that was changed to alanine, k→a. C) Alizarin red S staining of 21-d-old hMSC cultures with continuous Wnt3a 50 ng/ml alone or in combination with PEDF 34-mer 100 nM, or k→a 34-mer. For late groups, PEDF 34-mer was added only during the last 8 d of differentiation protocol. D) Quantitation of Alizarin red S staining in panel C. PEDF 300 ng/ml. Values are expressed as means ± sd; n = 3–4 wells/group. *P < 0.05, **P < 0.01 by Student’s t test compared with Wnt3a group; 1-way ANOVA, P = 0.0022.
PEDF peptides inhibit Wnt/β-catenin signaling. A) Western blots on active (nonphosphorylated) β-catenin and total β-catenin after continuous 50 ng/ml Wnt3a treatment or continuous Wnt3a + 100 nM PEDF 34-mer treatment of hMSCs at day 21 of osteogenic differentiation. Duplicate wells done for each treatment. B) Short-term exposure to PEDF peptides or DKK1 antagonizes Wnt signaling in hMSCs. Cells were incubated in 200 nM peptides or 50 ng/ml DKK1 for 6 h, then protein extracts were made and Western blots were performed as indicated. C) Quantitation of bands in panel A. D) Quantitation of bands in panel B. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Values are expressed as means ± sd; 2 biologic replicates per group.
PEDF protein restoration in PEDF-KO mice inhibits Wnt signaling. A) Bone proteins were extracted and the indicated immunoblots were performed. B) Quantitation of bands from 2 separate blots; n = 6 bands per treatment. Each lane is a separate tibia + femur. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Values are expressed as means ± sd. *P < 0.05, **P < 0.001 by Student’s t test.
Schematic diagram of PEDF's effect on blocking Wnt3a, allowing for terminal differentiation of osteoblasts. In the absence of PEDF, unopposed Wnt3a exposure impedes terminal osteoblast differentiation and appropriate mineralization. PEDF potentiates osteoblast precursors to differentiate into mature osteoblasts. MSC, mesenchymal stem cell.