Role of Cbl-PI3K Interaction during Skeletal Remodeling in a Murine Model of Bone Repair

<div><p>Mice in which Cbl is unable to bind PI3K (YF mice) display increased bone volume due to enhanced bone formation and repressed bone resorption during normal bone homeostasis. We investigated the effects of disrupted Cbl-PI3K interaction on fracture healing to determine whether this interaction has an effect on bone repair. Mid-diaphyseal femoral fractures induced in wild type (WT) and YF mice were temporally evaluated via micro-computed tomography scans, biomechanical testing, histological and histomorphometric analyses. Imaging analyses revealed no change in soft callus formation, increased bony callus formation, and delayed callus remodeling in YF mice compared to WT mice. Histomorphometric analyses showed significantly increased osteoblast surface per bone surface and osteoclast numbers in the calluses of YF fractured mice, as well as increased incorporation of dynamic bone labels. Furthermore, using laser capture micro-dissection of the fracture callus we found that cells lacking Cbl-PI3K interaction have higher expression of Osterix, TRAP, and Cathepsin K. We also found increased expression of genes involved in propagating PI3K signaling in cells isolated from the YF fracture callus, suggesting that the lack of Cbl-PI3K interaction perhaps results in enhanced PI3K signaling, leading to increased bone formation, but delayed remodeling in the healing femora.</p></div

Increased cartilaginous and bony matrix in remodeling fracture calluses of YF mice.

<p>For histological analysis, mid-sagittal sections of fractured femora were stained with Safranin O and fast green and quantitated by static histomorphometry. <b>A.</b> Representative sections at 7, 14, 21, and 28 days post-fracture. Bar charts show <b>B.</b> Total callus area <b>C.</b> Cartilaginous matrix area <b>D.</b> Bony matrix area. <b>E.</b> Ratio of cartilaginous matrix area over total callus area <b>F.</b> Ratio of bony matrix area over total callus area. n = 5–6, *p<0.05 vs. WT.</p

Lack of Cbl-PI3K interaction results in increased osteoblast surface, incorporation of dynamic bone labels, and up-regulation of master transcription factor Osterix in remodeling fracture calluses.

<p>Mid-saggital sections of fractured femora were stained with Safranin O and Fast Green, and counterstained with Hematoxylin. <b>A.</b> Representative sections at 7, 14, 21, and 28 days post-fracture showing bony regions of fracture calluses. <b>B.</b> Osteoblast surface over bone surface (ObS/BS) measured by static histomorphometry. n = 6, *p<0.05 vs. WT. <b>C.</b> Mice were injected with Alizarin Complexone (red) 4 days prior to sacrifice, and Calcein (green) 1 day prior to sacrifice. Mid-sagittal sections of fractured femora were used to image incorporation of dynamic bone labels in the center of the fracture calluses at 14 and 21 days post-fracture. Cells from sections of calluses at 7, 14, and 21 days post-fracture were harvested by LCM, and expression of <b>D.</b> Runx2, and <b>E.</b> Osterix were quantified by qRT-PCR n = 6, *p<0.05 vs. WT.</p

MicroCT measurements showed increased total callus and bony volume in mice lacking Cbl-PI3K interaction.

<p>A. Fractured femora were harvested from WT and YF mice at 14, 21 and 28 days post-fracture, and scanned by MicroCT. Bar charts show B. Total callus volume C. Bony callus volume D. Ratio of bony over total callus volume. n = 5–6, *p<0.05 vs. WT.</p

Histomorphometric parameters of bone resorption.

<p>Mid-sagittal sections of fractured femora were TRAP stained and counterstained with Alcian Blue and Hematoxylin. Three sections/mouse and 3 mice/genotype at 14 and 21, days post-fracture were analyzed. Osteoclast Surface, erosion surface and bone surface as determined by histomorphometry are shown. Values shown are mean ± SD from WT mice n = 3, YF mice n = 3</p><p>* p<0.05 was considered statistically significant as compared to respective controls using analysis of variance with post-hoc analysis (ANOVA) with Bonferroni post-hoc test.</p><p>Histomorphometric parameters of bone resorption.</p

Fractured femora lacking Cbl-PI3K interaction are delayed in restoring mechanical strength.

<p>Torsion testing was performed on fractured femora during remodeling of the hard callus at 21 and 35 days post-fracture. Bar graphs show <b>A.</b> Peak torque and <b>B.</b> Work. n = 11 *p<0.05 vs. WT.</p

Focal Adhesion Kinase is activated downstream of integrins and G_12/13 pathways.

<p>Aspirin treated, washed platelets were stimulated with 2MeSADP (100 nM) in presence or absence of reagents (as indicated) for 60 seconds under stirring conditions at 37°C (A). The lysates were then subjected to western blotting analysis and probed with anti- phospho- FAK (Y-397) and total FAK antibodies as lane loading control. The data are representative of mean ± S.E.M (n = 3). The data was analyzed by ANOVA and * P≤0.05 was considered significant (B). Aspirin-treated washed platelets were stimulated with AYPGKF (500 µM) in presence or absence of YM254890 (150 nM) (C), The lysates were then subjected to western blotting analysis and probed with anti- phospho- FAK (Y-397) and total FAK antibodies as lane loading control.</p

Role of c-Src and Syk downstream of integrin αIIbβ3 in thromboxane generation in platelets.

<p>Aspirin–treated, washed platelets were stimulated with 2MeSADP (100 nM) at various time points (A) or with varying concentrations of 2MeSADP (B) for one minute under stirring conditions at 37°C. The lysates were then subjected to western blotting analysis and probed with anti-phospho-(Y416) and total c-Src antibodies as lane loading control. Washed murine (WT or c-Src KO) platelets, without aspirin-treatment, were stimulated with 2MeSADP (100 nM) for 3.5 minutes and TXB₂ levels were analyzed (C) as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016586#pone-0016586-g001" target="_blank">Figure 1</a>. The data are represented as the % Fold increase over the control. Aspirin-treated washed platelets were stimulated with 2MeSADP (100 nM) for (30–120 seconds) or convulxin (100 ng/ml) for 30 seconds under stirring conditions at 37°C (D). The lysates were then subjected to western blotting analysis and probed with anti- phospho- Syk(Y 525/526) and total Syk antibodies as lane loading control. The data are representative of at least 3 separate experiments.</p

LCM Primer Sequences.

<p>LCM Primer Sequences.</p

Model depicting the regulation of TXA₂ generation by G_12/13 pathways and integrins through FAK.

<p>Integrin clustering leads to FAK activation (1). Fibrinogen receptor antagonist SC57101 prevents integrin clustering hence inhibits FAK activation (2). FAK can be activated in an integrin-independent manner by G_12/13 pathways (3). FAK is activated downstream of Rho kinase and SFKs upon stimulation of G_12/13 pathways (4). FAK can be inhibited by TAE-226 (5). Common effector molecule downstream of integrins and G_12/13 pathways contributing to thromboxane generation are unknown (6).</p