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. 2016 Dec;31(12):2215-2226.
doi: 10.1002/jbmr.2900. Epub 2016 Sep 7.

Activation of Wnt Signaling by Mechanical Loading Is Impaired in the Bone of Old Mice

Nilsson Holguin  1 , Michael D Brodt  1 , Matthew J Silva  1   2
Affiliations

Activation of Wnt Signaling by Mechanical Loading Is Impaired in the Bone of Old Mice

Nilsson Holguin et al. J Bone Miner Res. 2016 Dec.

Abstract

Aging diminishes bone formation engendered by mechanical loads, but the mechanism for this impairment remains unclear. Because Wnt signaling is required for optimal loading-induced bone formation, we hypothesized that aging impairs the load-induced activation of Wnt signaling. We analyzed dynamic histomorphometry of 5-month-old, 12-month-old, and 22-month-old C57Bl/6JN mice subjected to multiple days of tibial compression and corroborated an age-related decline in the periosteal loading response on day 5. Similarly, 1 day of loading increased periosteal and endocortical bone formation in young-adult (5-month-old) mice, but old (22-month-old) mice were unresponsive. These findings corroborated mRNA expression of genes related to bone formation and the Wnt pathway in tibias after loading. Multiple bouts (3 to 5 days) of loading upregulated bone formation-related genes, e.g., Osx and Col1a1, but older mice were significantly less responsive. Expression of Wnt negative regulators, Sost and Dkk1, was suppressed with a single day of loading in all mice, but suppression was sustained only in young-adult mice. Moreover, multiple days of loading repeatedly suppressed Sost and Dkk1 in young-adult, but not in old tibias. The age-dependent response to loading was further assessed by osteocyte staining for Sclerostin and LacZ in tibia of TOPGAL mice. After 1 day of loading, fewer osteocytes were Sclerostin-positive and, corroboratively, more osteocytes were LacZ-positive (Wnt active) in both 5-month-old and 12-month-old mice. However, although these changes were sustained after multiple days of loading in 5-month-old mice, they were not sustained in 12-month-old mice. Last, Wnt1 and Wnt7b were the most load-responsive of the 19 Wnt ligands. However, 4 hours after a single bout of loading, although their expression was upregulated threefold to 10-fold in young-adult mice, it was not altered in old mice. In conclusion, the reduced bone formation response of aged mice to loading may be due to failure to sustain Wnt activity with repeated loading. © 2016 American Society for Bone and Mineral Research.

Keywords: AGING; ANABOLIC; OSTEOBLAST; OSTEOCYTE; WNT/β-CATENIN SIGNALING.

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Conflict of interest statement

Disclosures

All authors state that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1
(A) An example of a mid-diaphyseal section from a single-bout loaded tibia imaged by fluorescence microscopy. For dynamic histomorphometry, the entire cross-section was analyzed. The dashed square (20× magnification) highlights the posterolateral region of peak compressive strain where we determined the percentage of sclerostin-positive (Fig. 3) and Wnt-positive (Fig. 4) osteocytes. The image is from a 5-month-old loaded tibia. The solid square (40×) illustrates a higher magnification of the region. (B) Periosteal and (C) endocortical single-labeled surface (based on calcein, day 5) of 5-month-old, 12-month-old, and 22-month-old tibias loaded for 5 days and control tibias. (D) Periosteal and endocortical mineralizing surface (based on calcein day 3, alizarin day 8) of 5-month-old, 12-month-old, and 22-month-old tibias loaded for 1 day and control tibias. Data are represented as mean ±SD. *Loaded versus Control; Bars = 5-month-old versus 12-month-old versus 22-month-old of loaded tibias; p <0.05. Ec = endocortical; Ct = cortical; Ps = periosteal; BS = bone surface; Ps.sLS = periosteal single-labeled surface; Ps.MS = periosteal mineralizing surface; Ec.sLS = endocortical single-labeled surface; Ec.MS = endocortical mineralizing surface.
Fig. 2
Fig. 2
Gene expression of osteoblast differentiation marker (A) Osx and bone formation (B) Col1a1 in 5-month-old, 12-month-old, and 22-month-old tibias loaded daily for up to 5 days and tibias harvested 4 hours after each day. Gene expression of (C) Osx and (D) Col1a1 in 5-month-old, 12-month-old, and 22-month-old tibias loaded once and tibias harvested at 4 hours, 24 hours, and 72 hours. Data are represented as mean ± SD. *Loaded versus Control; Bars = 5-month-old versus 12-month-old versus 22-month-old; p <0.05.
Fig. 3
Fig. 3
Gene expression of negative regulators of Wnt signaling (A) Sost and (B) Dkk1 in 5-month-old, 12-month-old, and 22-month-old tibias loaded daily for up to 5 days and tibias harvested 4 hours after each day. Gene expression of (C) Sost and (D) Dkk1 in tibias loaded once and tibias harvested at 4 hours, 24 hours, and 72 hours. Immunohistochemistry staining for Sclerostin (brown) in 5-month-old (E, F) and 12-month-old (G, H) mice after 1 day (E, G) or 5 days of loading (F, H). (I) Control tibia of a 5-month-old animal that also exemplifies the staining of a 12-month-old control tibia. (J) Percentage of sclerostin-positive osteocytes of 5-month-old and 12-month-old mice subjected to 1 day or 5 days of tibial compression (−650 με or −2200 με). Data are represented as mean ± SD. Scale bar = 125 μm; *Loaded versus Control; p <0.05.
Fig. 4
Fig. 4
LacZ expression (arrow) of Wnt signaling in 5-month-old (A, B) and 12-month-old (C, D) mice after 1 day (A, C) or 5 days of loading (B, D). (E) Control tibia of a 5-month-old animal that also exemplifies the staining of a 12-month-old control tibia. (F) Percentage of Wnt-positive osteocytes of 5-month-old and 12-month-old mice subjected to 1 day or 5 days of tibial compression (−650 με or −2200 με). Data are represented as mean ± SD. Scale bar = 60 μm; *Loaded versus Control; p <0.05.
Fig. 5
Fig. 5
Gene expression of 19 Wnt ligands in 5-month-old, 12-month-old, and 22-month-old tibias loaded for (A) 1 day, (B) 3 days, or (C) 5 days and tibias harvested 4 hours after the last day of loading. Gene expression of (D) Wnt1 and (E) Wnt7b in 5-month-old, 12-month-old, and 22-month-old tibias harvested 4, 24, or 72 hours after a single day of loading. Data are represented as mean ± SD. *Loaded versus Control; p <0.05.
Fig. 6
Fig. 6
(A) Compilation of the number of osteoblast (OB; black bar), Wnt ligand (gray), and Wnt-related (Wnt-Rel; white) genes regulated by 1, 3, or 5 days of loading in 5-month-old, 12-month-old, and 22-month-old tibias, where the solid bars denote upregulation and the patterned bars denote downregulation. Overall, aging reduced the response of bone formation–related genes to multiple days of loading. (B) Venn diagram depicting the number of significant correlations between gene pairs as a function of age. Of the 780 possible gene-pairs analyzed, there were 216, 190, and 133 significant correlations at 5 months, 12 months, and 22 months, respectively; 53 of these were common to all ages (See Supplemental Methods and Results for details of correlation analyses.). OB = osteoblast.
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