Expression of phosphorylated Pyk2 in FAK+/+ and FAK−/− osteoblasts.

<p>Expression and localization of phosphorylated Pyk2 at Tyr-402 (red) in FAK+/+ (A) and FAK−/− (B) osteoblasts. DAPI nuclei stain in blue. Magnification = 60×. Scale bar represents 25 µm.</p

Trabecular Microarchitecture in the Distal Femur of Adult WT and FAK−/− Mice.

<p>Mean ± SD; <i>n</i>, sample number; BV/TV, bone volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular spacing; Conn.D, connectivity density; SMI, structure model index; ^a<i>p</i><0.05 versus gender-matched WT group.</p

Relative bone formation parameters in WT and FAK−/− male and female mice.

<p>Conditional deletion of FAK did not affect relative mineralizing surface (rMS/BS), relative mineral apposition rate (rMAR) or relative bone formation rate (rBFR/BS), which is a product of rMS/BS and rMAR. Data are presented as box and whisker plots where the median, Q2, Q3 and whiskers, representing the 5% and 95% confidence intervals, are depicted.</p

Transverse cross-sections at ulnar midshaft in nonloaded (left) and loaded (right) forearms in WT (top) and FAK−/− (bottom) mice given calcein (green) and alizarin (red) fluorochrome bone labels at 4 and 11 days, respectively, after the first day of loading.

<p>In response to applied mechanical loading, most new bone is formed on the medial and lateral surfaces where bending strains are highest. Note the appearance of double bone labels (green and red) on the medial and lateral surfaces of the ulna, as well as on the rostral surface, in the loaded ulna (top, right) compared to the nonloaded internal control (top, left) where very little bone formation is observed. The loaded ulna (bottom, right) in FAK−/− mice exhibit bone formation on the medial and lateral ulnar surfaces, but much less new bone formation, in terms of percent mineralizing surface, is observed relative to the nonloaded ulna (bottom, left). Sections are representative of the response observed for WT and FAK−/− mice. Magnification = 10×.</p

Structural Properties of the Femur in WT and FAK−/− Adult Mice.

<p>Mean ± SD; <i>n</i>, sample number; I_MAX, maximum second moment of area; I_MIN, minimum second moment of area; Ct.Ar, cortical area; Ct.Th, cortical thickness; ^a<i>p</i><0.05 versus gender-matched WT group.</p

Focal adhesion formation and actin cytoskeleton in wild-type and FAK−/− osteoblasts.

<p>(A–C) FAK+/+ osteoblasts form focal adhesions (red) as shown by vinculin staining and display prominent actin fiber formation (green) as shown by phalloidin staining. (D–F) FAK−/− osteoblasts also exhibit actin fiber and focal adhesion formation. Panels C and F show merged images. DAPI nuclei stain in blue. Magnification = 60×. Scale bar represents 25 µm.</p

Rodent ulnar loading model.

<p>The right forearm in mice is subjected to cycle axial compression across the olecranon and flexed carpus while under isoflurane anesthesia. Due to the natural curvature of the ulna-radius complex in the medial direction, compressive loading creates a bending moment about the craniocaudal axis creating compressive and tensile bending strains on the medial and lateral surfaces, respectively.</p

Western blot analysis of focal adhesion kinase expression in FAK−/− osteoblasts transiently transfected with the wild-type focal adhesion kinase (WT FAK) construct.

<p>Actin was used as a loading control. Expression in FAK−/−, FAK+/+, and MC3T3 osteoblast-like cells is shown as controls. WT FAK protein expression is similar to FAK expression in FAK+/+ and MC3T3 cells.</p

PGE₂ release in response to oscillatory fluid flow.

<p>(A) Fold-change in PGE₂ release in FAK+/+ and FAK−/− osteoblasts following 2 hours of oscillatory flow and a 4 hour post-flow incubation. FAK−/− osteoblasts exhibited a significantly lower fold-change in PGE₂ release in response to fluid flow compared to FAK+/+ osteoblasts. (B) Fold-change in PGE2 release following fluid flow in mutant osteoblasts transiently re-expressing wild-type focal adhesion kinase and phosphospecific site mutations. Note that differences in absolute fold-changes in PGE2 release following flow in Panel A and Panel B represent variations in parallel assays.</p