Abstract
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.
Highlights
The E3 ubiquitin ligase Cbl is a multi-domain adaptor protein [1], which regulates bone resorption by interacting with several molecules in osteoclasts[2,3,4,5,6]
In mice, a single point mutation that disrupts the interaction between Cbl and the p85 subunit of Phosphatidylinositol-3 Kinase (PI3K) is capable of uncoupling bone resorption and bone
The key observations of this study were that the absence of Cbl-mediated sequestration of PI3K resulted in a net increase in the total callus size, and the amount of newly formed bone in response to fracture, without any apparent effect on soft callus formation (Figs 2 and 3)
Summary
The E3 ubiquitin ligase Cbl is a multi-domain adaptor protein [1], which regulates bone resorption by interacting with several molecules in osteoclasts[2,3,4,5,6]. Cbl was shown to control ubiquitylation of signaling molecules and regulate proliferation, differentiation, and survival of human mesenchymal-derived osteoblasts [7]. Cbl’s role in fine tuning signaling pathways in bone turnover appears to be important in both normal and pathological conditions. Phosphatidylinositol-3 Kinase (PI3K), a lipid kinase, plays a central role in regulating cell proliferation, survival, and migration [9]. PI3K signaling plays an important role in skeletal homeostasis [11, 12]. A reduction in alkaline phosphatase (ALP) activity and osteocalcin mRNA expression was reported in p85α-/- deficient mesenchymal stem cells suggesting a role for PI3K signaling in osteoblast differentiation [19]. Regulation of PI3K plays a significant role during osteogenesis
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