Wnt5a and β-catenin signaling are both necessary for flow induced Runx2 upregulation.

<p>(A) Flow induced Runx2 expression was upregulated 2.2±0.2-fold in scrambled siRNA treated cells exposed to flow verses scrambled siRNA control cells. This fold increase was significantly different (p<0.05) than Wnt5a siRNA treated cells, in which the flow induced Runx2 expression was abrogated. (B) The fold change in Runx2 expression with flow was significantly different between untreated cells and cells with inhibited β-catenin signaling via endostatin treatment (p<0.01). Untreated cells exposed to oscillatory fluid flow had a 2.8±0.5-fold increase in Runx2 expression over control cells; while endostatin treated cells resulted in no difference between flowed and control cells. (C) Western blot analysis demonstrates that there is a significant decrease in β-catenin levels after a 24 hour incubation with endostatin. (Error bars: SEM (n≥6)).</p

Flow-induced β-catenin signaling may be mediated by cadherin signaling rather than canonical Wnt signaling.

<p>(A) Western blots were used to determine the level of phosphorylated β-catenin in cells exposed to oscillatory fluid flow versus controls. (B) Analysis of the western blot indicates that there is no significant difference between control and experimental cells in the level of phosphorylated β-catenin. (C) A western blot was also used to assay N-cadherin association with β-catenin as a function of mechanical stimulation. (D) Analysis of the western blot demonstrates that there is a significant 30% decrease in β-catenin/N-cadherin association with exposure to 35 minutes of oscillatory fluid flow (p<0.01). (Error bars: SEM (n≥4)).</p

A schematic diagram of potential signaling mechanisms involved in mechanically stimulated osteogenic differentiation.

<p>Oscillatory fluid flow, a potent mechanical signal within the microenvironment of bone has the potential to regulate non-canonical Wnt5a and β-catenin signaling pathways in MSCs, both of which are essential for fluid flow induced osteogenic lineage commitment via Runx2 upregulation. Furthermore, Wnt5a signals through Ror2 to activate RhoA, a small GTPase that is necessary and sufficient for osteogenic differentiation. Finally, flow induced β-catenin signaling appears to be mediated by alterations in N-cadherin/β-catenin association indicating that adherens junctions may be involved in the transduction of a mechanical signaling into a cell fate decision.</p

Wnt5a and Ror2 are necessary for flow-induced RhoA activation, but do not effect β-catenin translocation.

<p>(A) Western blots of nuclear β-catenin indicate that both scrambled and Wnt5a siRNA treated cells maintained their potential to initiate β-catenin signaling with flow. (B) Analysis of the western blots demonstrates that scrambled and Wnt5a siRNA treated cells had a 1.9±0.2-fold and 1.8±0.3-fold increase in nuclear β-catenin with flow, respectively. (C) β-catenin/TCF/LEF transcription of downstream genes was also maintained in both scramble and Wnt5a treated cells with a 1.8±0.4-fold and 2.2±0.3-fold increase in luciferase activity, respectively, indicating that Wnt5a is not necessary for mechanically induced β-catenin signaling. (D) Western blots were used to assay Rho activation in response to flow in scrambled, Wnt5a and Ror2 siRNA treated cells. (E) Analysis of the blots indicates that the 1.7±0.1-fold increase observed in scrambled treated cells is significantly greater than Wnt5a (p<0.01) and Ror2 (p<0.05) siRNA treated cells, both of which lost flow-induced RhoA activation. (Error bars: SEM (n≥4)).</p

Skeletally mature <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> mice exhibit less responsiveness to mechanical loading compared to control mice.

<p>(<b>A</b>) Representative images of non-loaded (left) and loaded (right) ulnae of <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> (top) and control (bottom) mice. Fluorochrome labels (Calcein-green and Alizarin Red-red) given on Days 5 and 12 after the onset of mechanical loading. (<b>B to D</b>) Relative mineralizing surface (rMS/BS, %, B), mineral apposition rate (rMAR, µm per day, C), and bone formation rate (rBFR/BS, µm³/µm² per year, D) of mechanically loaded mice. <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> mice exhibited a decrease of 32% in rMAR and 33% in rBFR/BS when compared to control mice. Data presented as mean ± SEM. * p<0.05.</p

<i>Kif3a</i> deletion in osteoblasts and osteocytes has no effect on tibial midshaft geometry.

<p>Imin and Imax are maximum and minimum second moment of inertia, respectively. pMOI is polar moment of inertia. Cortical bone geometry in 16 week old skeletally mature <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> and control mice was assessed using as microCT. Data presented as mean±SEM. N.S. is not significant (p>0.15).</p

<i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> were significantly less responsive to mechanical loading than control mice.

<p><i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> and control mice responded to mechanical loading with increased mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS), however, <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> were significantly less responsive to mechanical loading than control mice. Data presented as mean±SEM.</p>+++<p>p<0.001 for loaded vs. non-loaded values.</p>*<p>p<0.05 for <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> vs. control mice.</p

Generation and confirmation of bone-specific Kif3a conditional knockout mice.

<p>(<b>A</b>) A typical agarose gel resulting from PCR genotyping of genomic DNA from tail biopsies of transgenic mice. Bands indicate floxed (490 bp) and wild-type (360 bp) <i>Kif3a</i> and <i>Cre</i> recombinase (650 bp). 18S (870 bp) used as a positive control in the <i>Cre</i> PCR reactions. Floxed and recombined <i>Kif3a</i> allele present in <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> mice due to heterogeneous tissue in tail biopsies. (<b>B</b>) To assess <i>Cre</i> specificity, <i>Colα1(I) 2.3-Cre</i> mice were crossed with <i>Rosa26R</i> reporter mice. Effective <i>Cre</i> recombination was detected by LacZ staining in osteoblasts and osteocytes of <i>Colα1(I) 2.3-Cre;R26R</i> mice (right) but not in littermates lacking <i>Cre</i> (left). LacZ staining was not visible in muscle tissue of <i>Colα1(I) 2.3-Cre;R26R</i> mice. (b- bone, bm- bone marrow, m- muscle).</p

Axial ulnar loading leads to similar strain at the ulnar midshaft of <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> and control mice.

<p>(<b>A</b>) Image of strain gaging and axial ulnar loading experimental set-up. The right forearms of 16 week old skeletal mature mice were axially loaded for 120 cycles per day for 3 consecutive days with a 2 Hz sine wave using an electromagnetic loading system with feedback control. The left forearms were not loaded and used as non-loaded internal controls. (<b>B</b>) Strain in cortical bone at given mechanical loading levels. Open and closed circles indicate <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> (n = 35) and control (n = 27) mice, respectively. Data presented as mean ± SEM. * p<0.05.</p

Kif3a expression in osteoblasts and osteocytes is not critical for embryonic skeletal development.

<p>(<b>A,B</b>) Whole mount Alizarin Red (bone) and Alcian Blue (cartilage) staining of E18.5 <i>Colα1(I) 2.3-Cre;Kif3a^fl/f</i>^l (A) and control (B) embryos. The size and limb patterning of <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> mice was similar to that of the control mice. (<b>C,D</b>) Movat's pentachrome staining of cross-sections of the radial/ulnar growth plates (cartilage-blue) in E16.5 <i>Colα1(I) 2.3-Cre;Kif3a^fl/f</i>^l (C) and control (D) mice. (<b>E–J</b>) Cross-sections of E16.5 long bones stained with Picrosirius red (E,F-bright field; G,H-polarized light) to illuminate collagen and Safranin O (I,J) to demarcate cartilage. Both control and <i>Colα1(I) 2.3-Cre;Kif3a^fl/f</i>^l mice have similar patterns of osteogenic and chondrogenic differentiation. Scale bar: 100 µm.</p