Abstract
The ubiquitin/proteasome system controls the stability of Runx2 and JunB, proteins essential for differentiation of mesenchymal progenitor/stem cells (MPCs) to osteoblasts. Local administration of proteasome inhibitor enhances bone fracture healing by accelerating endochondral ossification. However, if a short-term administration of proteasome inhibitor enhances fracture repair and potential mechanisms involved have yet to be exploited. We hypothesize that injury activates the ubiquitin/proteasome system in callus, leading to elevated protein ubiquitination and degradation, decreased MPCs, and impaired fracture healing, which can be prevented by a short-term of proteasome inhibition. We used a tibial fracture model in Nestin-GFP reporter mice, in which a subgroup of MPCs are labeled by Nestin-GFP, to test our hypothesis. We found increased expression of ubiquitin E3 ligases and ubiquitinated proteins in callus tissues at the early phase of fracture repair. Proteasome inhibitor Bortezomib, given soon after fracture, enhanced fracture repair, which is accompanied by increased callus Nestin-GFP+ cells and their proliferation, and the expression of osteoblast-associated genes and Runx2 and JunB proteins. Thus, early treatment of fractures with Bortezomib could enhance the fracture repair by increasing the number and proliferation of MPCs.
Highlights
Successful fracture repair restores broken bones to their original structural geometry and biomechanical integrity
To determine if E3-mediated ubiquitination and degradation of these proteins play a role in fracture repair, we examined the expression levels of Nedd4 sub-class of E3s, Wwp1, Itch, Smurf1 and Smurf2, in callus samples at different time points after fracture by qPCR
The rationale why Btz was administered at day 1, 3, and 5 include 1) we hypothesize that Btz will increase mesenchymal progenitor/stem cells (MPCs) at the early phase of fracture repair and day 1–5 is the time when MPCs are rapidly expanded [27, 46]; 2) a major mechanism of action of Btz is to block the degradation of Ub-proteins while we found increased Ub-proteins at the early phase of fracture repair (Fig 1B); and 3) our pharmacology study reported recently that blood Btz levels return to un-detectable levels at 12 hours [47] and that the inhibitory effect of Btz on Ubprotein degradation is abolished at 24 hours following a single Btz administration
Summary
Successful fracture repair restores broken bones to their original structural geometry and biomechanical integrity. The fracture repair process is comprised of four phases: inflammatory response; recruitment of mesenchymal progenitor/stem cells (MPCs) and their differentiation into osteoblasts (OBs); new bone formation; and remodeling of the new bone from woven to lamellar bone. Fracture repair is rapid and efficient, about 5–10% of patients with fractures, mainly aged subjects, develop delayed union or nonunion, resulting in unsuccessful fracture.
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