Joint loading-driven bone formation and signaling pathways predicted from genome-wide expression profiles

Abstract

Joint loading is a recently developed loading modality that induces anabolic responses by lateral loads applied to a synovial joint such as an elbow and a knee. The present study extended this loading modality to an ankle and addressed a question: does ankle loading promote bone formation in the tibia? If so, what signaling pathways are involved in the anabolic responses? Using C57BL/6 female mice as a model system, lateral loads of 0.5 N were applied to the ankle at 5 Hz for 3 min/day for 3 consecutive days and load-driven bone formation was evaluated at three tibial cross-sections (the proximal, middle, and distal diaphysis). Furthermore, total RNA was isolated for 3 pairs of microarray experiments as well as quantitative real-time PCR analyses. The histomorphometric results revealed that in all cross-sections ankle loading elevated the cortical area and thickness as well as the calcein-labeled surface. Signaling pathway analysis from microarray-derived whole-genome mRNA expression profiles and quantitative real-time PCR predicted that molecules in phosphoinositide 3-kinase (PI3K), ECM-receptor interactions, TGFbeta signaling, and Wnt signaling were involved in the joint-loading driven responses. Since ankle loading stimulates bone formation throughout the tibia both in the endosteum and the periosteum, it may provide a non-pharmacological approach to effectively activate molecular signaling necessary for preventing bone loss.

FIG. 1

Experimental setup. (A) Mouse on a loading table for ankle loading. (B) Schematic diagram illustrating the piezoelectric mechanical loader. (C) Location of three cross-sections for bone morphometry. (D) Strain measurements with ankle and knee loading. The best-fit regression line is: y = 12.4 x + 3.7 with ankle loading (r² = 0.99), and y = 4.8 x + 1.2 with knee loading (r² = 0.99).

FIG. 2

Tibial cross-sections and measurements of cortical area and thickness (n = 18). The labels in (A-F) are: medial surface (med), lateral surface (lat), and posterior surface (post). White bar = 200 μm. The results in (G & H) are expressed as mean ± SEM, and the triple asterisk indicates p < 0.001. (A) Control proximal section. (B) Loaded proximal section. (C) Control middle section. (D) Loaded middle section. (E) Control distal section. (F) Loaded distal section. (G) Cross-sectional area (mm²). (H) Cortical thickness (mm).

FIG. 3

Percentage of the calcein-labeling surface to bone surface in the periosteum and the endosteum (n = 18). The results are expressed as mean ± SEM, and the asterisks show statistical significance at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). (A) Periosteal labeling (%). (B) Endosteal labeling (%). (C) Increase in labeling (% of control) in the periosteum and the endosteum.

FIG. 4

Increase in the morphometric parameters (n = 18). The results are expressed as mean ± SEM. The single asterisk indicates p < 0.05. (A) Increase in relative MS/BS (% of the control MS/BS value). (B) Increase in relative MAR (% of the control MAR value). (C) Increase in relative BFR/BS (% of the control BFR/BS value).

FIG. 5

List of 50 genes significantly upregulated or downregulated in microarray-derived data. (A) Mostly upregulated 25 genes with ankle loading and their fold change. (B) Mostly downregulated 25 genes with ankle loading and their fold change.

FIG. 6

Load-driven signaling pathways predicted with ankle loading at 1 h after the last loading. In the network diagrams the mRNA levels of the genes in the red boxes were altered by ankle loading. (A) PI3K pathway. (B) ECM-receptor interaction. (C) TGFβ signaling pathway. (D) Wnt signaling pathway.

FIG. 7

Alterations of mRNA levels with ankle loading. The harvest time (1 h, 1 week, or 2 weeks) is indicated from the last loading (day 3). (A) Elevated mRNA levels of c-fos, egr1, and atf3 at 1 h after the last loading for 7 mice. The amount of increases and decreases is color-coded in red and green, respectively. (B) Altered mRNA levels of col1α (type I collagen 1α), bmp2 (bone morphogenic protein 2), opn (osteopontin), ocn (osteocalcin), and bsp (bone sialoprotein) at three different time points after the last loading (7 mice at 1 h, 7 mice at 1 week, and 3 mice at 2 weeks). Upregulation and downregulation are colored coded in red and green, respectively.

FIG. 8

Summary of bone formation rate with joint loading. (A) Bone formation rate with ankle loading. Three cross-sections in the proximal, middle, and distal sites illustrate the rate of bone formation on the periosteal and endosteal surfaces. (B) Bone formation rate (BFR/BS) in the endosteum with ankle loading (n = 18) and knee loading (n = 8). The data with knee loading were taken from the studies published in (9, 20). All load-driven increases are statistically significant except for the proximal and middle sections with knee loading (p = 0.051 and 0.062, respectively).