Our funded grant, R01DE030716, ?Rspondin-Lgr Axis in Bone Regeneration?, is focused on examining the role of Rspo2-Lgr6 signaling in calvarial regeneration. While there are some similarities in healing processes that exist between long bones and bones of the skull, there are also important differences. As an example, the calvaria heals exclusively through an intramembranous process while long bones heal through both intramembranous and endochondral bone formation. An additional important difference that exists between calvarial and long bone healing is the source and type of progenitor cell that contributes to bone healing. In long bones, a critical aspect of the bone healing process begins with the expansion of mesenchymal progenitors from the marrow and periosteum, which occurs immediately after injury. These cells then become bone forming osteoblasts and chondrocytes. However, mechanisms that control long-bone skeletal progenitor/stem cell activation, expansion, and differentiation in response to injury are poorly described. This supplement will study the role of the R-spondin (ligand) ? Lgr (receptor) signaling axis in regulating long bone progenitors and bone regeneration. R-spondins (roof plate specific spondin) are a family of four secreted matricellular proteins (Rspo1-4) that bind to Leucine-rich repeat-containing G-protein coupled receptors 4/5/6 (Lgrs). Rspo-Lgr interaction potentiate canonical Wnt pathway by preventing the turnover of Wnt Frizzled receptors, and hence determines canonical Wnt signaling levels. While canonical Wnt signaling is known to play an important role in bone regeneration, very little research has explored positive modulators of Wnt signaling. In particular, the requirement of Rspo-Lgr in the context of long-bone fracture healing has never been examined, due to the lack of appropriate models. Our primary goal is to define the requirement of Rspo2/3 and Lgr6 in mesenchymal progenitors in response to femoral fracture. We have defined two specific aims to address this goal.
In Aim1, we will use single and compound Rspo2 and Rspo3 floxed mice crossed with an alphaSMACreERT2 mouse to disrupt the Rspo2/3 genes in long-bone mesenchymal progenitors at the time of fracture. We will assess in vivo proliferation, Wnt signaling, and osteogenesis of progenitors, and analyze healing by using CT, histology, and mechanical testing.
In Aim 2, Lgr6 knockout mice will be investigated for their bone healing properties using parameters similarly to Aim 1. Completion of this project will identify the requirement of Rspo2/3-Lgr6 interaction in long-bone fracture healing and will provide new mechanistic insights into the action of cWnt signaling in bone healing that can be directly compared to similar experiments conducted with calvarial healing in the parent grant.
Canonical Wnt signaling is used therapeutically to enhance bone formation, but the related mechanisms utilized by osteoprogenitors remain poorly understood. This project aims to identify and characterize the role(s) novel ligand-receptor combination of Rspondins and Lgrs, which modulate cWnt signaling. Compromised, non-healing fractures pose a significant public health problem; understanding the molecular drivers of osteogenesis will help in developing new approaches to reduce the incidence of delayed union and nonunion in fracture healing of long bones.
