Enhanced Chondrogenesis and Wnt Signaling in PTH-Treated Fractures

Abstract

Studies have shown that systemic PTH treatment enhanced the rate of bone repair in rodent models. However, the mechanisms through which PTH affects bone repair have not been elucidated. In these studies we show that PTH primarily enhanced the earliest stages of endochondral bone repair by increasing chondrocyte recruitment and rate of differentiation. In coordination with these cellular events, we observed an increased level of canonical Wnt-signaling in PTH-treated bones at multiple time-points across the timecourse of fracture repair, supporting the conclusion that PTH responses are at least in part mediated through Wnt signaling.Introduction: Since FDA approval of PTH ; Forteo] as a treatment for osteoporosis, there has been interest in its use in other musculoskeletal conditions. Fracture repair is one area in which PTH may have a significant clinical impact. Multiple animal studies have shown that systemic PTH treatment of healing fractures increased both callus volume and return of mechanical competence in models of fracture healing. Whereas the potential for PTH has been established, the mechanism(s) by which PTH produces these effects remain elusive. Materials and Methods: Closed femoral fractures were generated in 8-wk-old male C57Bl/6 mice followed by daily systemic injections of either saline (control) or 30 g/kg PTH(1-34) for 14 days after fracture. Bones were harvested at days 2, 3, 5, 7, 10, 14, 21, and 28 after fracture and analyzed at the tissue level by radiography and histomorphometry and at the molecular and biochemical levels level by RNase protection assay (RPA), real-time PCR, and Western blot analysis. Results: Quantitative CT analysis showed that PTH treatment induced a larger callus cross-sectional area, length, and total volume compared with controls. Molecular analysis of the expression of extracellular matrix genes associated with chondrogenesis and osteogenesis showed that PTH treated fractures displayed a 3-fold greater increase in chondrogenesis relative to osteogenesis over the course of the repair process. In addition, chondrocyte hypertrophy occurred earlier in the PTH-treated callus tissues. Analysis of the expression of potential mediators of PTH actions showed that PTH treatment significantly induced the expression of Wnts 4, 5a, 5b, and 10b and increased levels of unphosphorylated, nuclear localized ␤-catenin protein, a central feature of canonical Wnt signaling. Conclusions: These results showed that the PTH-mediated enhancement of fracture repair is primarily associated with an amplification of chondrocyte recruitment and maturation in the early fracture callus. Associated with these cellular effects, we observed an increase in canonical Wnt signaling supporting the conclusion that PTH effects on bone repair are mediated at least in part through the activation of Wnt-signaling pathways.

Loss of Gi G-Protein-Coupled Receptor Signaling in Osteoblasts Accelerates Bone Fracture Healing

“…(34) In addition, systemic administration of PTH can enhance bone repair in animal models by increasing callus bone volume, stimulating bone formation, and restoring mechanical competence. (7,8) A similar effect has been reported in several small studies with recombinant PTH in humans to speed fracture healing. (27,35) Although these prior results indicated that PTH could activate the bone repair process, the fact that PTHR1 and other GPCRs can each activate G s , G i , and G q GPCR signaling pathways (36) poses a challenge to identifying how the G s or G i pathways specifically regulate fracture repair.…”

“…We focused our assessments on days 10 and 28 because the peak of osteogenesis occurs between 10 to 14 days after fracture and because cartilage replacement with bone in the callus to bridge the fracture gap is usually completed by 28 days. (8,23) mCT imaging demonstrated that the bone fracture gap was bridged by bony tissue by day 28 ( Fig. 2A, Supporting Fig.…”

“…(8) In addition, activation of G s signaling in osteoblasts can increase bone formation by favoring the commitment of mesenchymal progenitors into the osteoblast lineage. (41) These findings indicate that this process is favored via G s activation in the Rs1-expressing mice but not loss of G i activity.…”

“…Systemic administration of PTH can enhance endochondral and intramembranous bone repair by increasing bone callus volume, stimulating the bone formation rate, and restoring mechanical competence in animal models of normal and impaired fracture repair. (7,8) The G s and G i pathways may have independent functions in osteoblasts despite their common convergence on the cAMP second messenger. (9,10) There has been growing interest in molecular crosstalk between different GPCRs as a way to increase the dynamic complexity of cell signaling (11,12) ; however, this crosstalk also makes it difficult to assess how individual pathways control cellular function.…”

Intermittent Administration of Human Parathyroid Hormone before Osteosynthesis Stimulates Cancellous Bone Union in Ovariectomized Rats

“…Cancellous bone repair of the proximal tibia is caused by endochondral ossification (Tsuchie et al 2011), and when cartilage tissues combine after wiring, it seems that bone union is accelerated more in the PTH treated group. Recently, some reports stated that the effect of accelerated bone union by intermittent PTH administration was related to the accelerated formation of cartilage tissue (Nakazawa et al 2005;Kakar et al 2007;Kaback et al 2008). However, Sox9 expression, which is involved in the differentiation of cartilage tissue, was not accelerated at all time points.…”