Mechanism Of Intracellular Signaling Transduction Through β-catenin During Fracture Repair

BackgroundFracture healing is a complex repair process. In early phase of fracture repair, undifferentiated mesenchymal cells aggregate, proliferate, and differentiate in response to growth factors produced by the injured tissues at the site of injury. The process involves both intramembranous and endochondral ossification. Intramembranous ossification involves the formation of bone directly from osteoprogenitor cells and undifferentiated mesenchymal cells, resulting in bone formation in endosteum and periosteum. During endochondral ossification, mesenchymal cells differentiate into chondrocytes, producing cartilaginous matrix, which undergoes calcification and eventually replaced by bone. Primary formed bone experience remodelization until it restores original biomechanical character. When fracture healing is impaired, osteoblastic differentiation is inhibited, and that results in a delayed union, or non-union, usually requiring additional surgery for successful fracture healing. In some surgical operation, such as spinal fusion surgery or total joint replacement, a lack of bone ingrowth results in a failed outcome and the need for additional surgery. Adjunctive pharmacologic agents may useful for improving fracture healing to avoid unnecessary additional surgery in some cases. Although the histology and general mechanism of fracture healing are well established, understanding of molecular signaling level remains unclear.β-catenin signaling which activates Tcf-dependent transcription is a key regulator of embryonic bone development. Wnt ligands and antagonists of Wnt signaling bind to the transmembrane receptors including low-density lipoprotein receptor related protein-5/6 and Fz to regulate Wnt pathway. Activation of Wnt/β-catenin pathway produces a cytoplastic signaling cascade leading to the transcriptional regulation of gene expression. In the absence of appropriate Wnt ligands,β-catenin is phosphorylated, ubiquitinated and degraded by a multi-protein complex comprising glycogen synthase kinase-3β(GSK-3β), APC, and Axin. Appropriate Wnt signaling activates Wnt/β-catenin pathway andβ-catenin can translocate to the nucleus and mediate Tef/Tcf dependent gene transcription. During embryonic skeletogenesis, inhibition ofβ-catenin signaling can block osteoblast differentiation and shift pluripotential mesenchymal cells to develop a chondroblastic phenotype.β-catenin signaling also can regulate bone mass, and its activation results in increased bone density.Although the histology and general mechanism of fracture healing are well established, understanding of molecular signaling level remains unclear. Recently, it was found that Wnt proteins are expressed during fracture repair. Oral lithium treatment could be used to activateβ-catenin signaling and to increase the size of healing cutaneous wounds. It is not still unclear that whetherβ-catenin also plays a similar role in fracture repair. In this study, we aimed to investigate howβ-catenin signaling functions during fracture healing and that could be developed into a novel therapeutic approach to improve bone healing.Objectives1. To observe the changes ofβ-catenin during fracture repair and to investigate howβ-catenin function in mediating Tcf/Lef-dependent gene transcription as well as effect of downregulation ofβ-catenin on fracture repair.2. To explore mechanism of signaling transduction throughβ-catenin during fracture repair and to provide theorical bases for developing novel therapeutic approach.Methods1. In this study, model of tibia fracture was established using the modified Hiltunen's method. Mice were randomly divided into three groups: Group I-control group(n=2);Group II-fracture group(n=24) and Group III-Ad-Dkk1 group(n=6). At different time point (3, 7, 14, 21 days) after the fracture, mice were sacrificed and fracture callus was harvested for further analysis. At least three animals at each time point were studied.2.Using Horseradish peroxidase (HRP)-conjugated secondary antibody and the ECL chemoluminescence detection system, western blot analysis were employed for detectingβ-catenin in different time points during fracture repair. Relative expression levels were quantified by densitometry using AlphaEaseFC software (Alpha Innotech).3.β-galactosidase staining of histological sections were performed to observe relations betweenβ-catenin level andβ-galactosidase positive staining.4. Quantitative RT-PCR was used to detect Wnt ligmants and their receptors during fracture repair to understand whether signaling transduction throughβ-catenin during fracture healing was directly regulated by Wnts.5. Radiographic, histological analysis and histomorphometric measurements were used to observe effect of Dkk-1(an antagonist of canonical Wnt/β-catenin signaling that inhibits Wnt receptor activation) on fracture repair for further understanding function ofβ-catenin.Results1. Western analysis was employed to examineβ-catenin level from the healing callus at different time points in Tcf-reporter mice. It was found thatβ-catenin level is low in intact bone tissue, but is highly expressed during the entire period of fracture repair. Callus samples from animals treated with Ad-Dkk1 exhibited a greater reduction ofβ-catenin level, as compared to group II. There was statistical difference between the two groups (P<0.01). The results showed thatβ-catenin signaling was activated during facture healing.2. Usingβ-galactosidase staining, we examined Tcf-dependent transcription mediated byβ-catenin. Three days after the fracture, there was little positive staining. 1 week after the fracture, prechondrogenic mesenchymal-like cells and osteoblasts lining within the periosteum exhibited positive staining. Two weeks following the fracture, positive staining was detected in proliferating and pre-hypertrophic chondrocytes through the callus. Hypertrophic chondrocytes did not exhibit staining. Osteoblasts lining the periosteum also showed strong staining signal. As osteoblasts maturated into osteocytes 3 weeks after the fracture, there was a low level staining. The results demonstrated thatβ-catenin mediated Tcf-dependent transcription in proliferating chondroblasts and osteoblasts. 3. We examined the expression of Wnts and their receptors using RT-PCR in the fracture callus and found that some Wnt ligmands were expressed during fracture healing including Wnt-4,Wnt-5b. in addition, their receptors Fz-1 and Lrp-6 were also upregulated during fracture healing. It is possible that these Wnt ligmands are responsible for activatingβ-catenin signaling during fracture repair.4. Three weeks after the fracture, radiological examination showed complete healing in group II, while in Dkk-1 treated mice, fractures failed to heal. Using histological analysis and histomorphometric measurements, we found that except that trabecular separation (μm) is wider in Dkk-1 treated mice, there were substantial reduction of callus parameters including cartilage and bone volume as a percentage of total callus tissue volume, trabecular thickness (μm), trabecular number (per mm)(P<0.01). Our findings showed that Dkk-1(an antagonist of canonical Wnt/β-catenin signaling that inhibits Wnt receptor activation) inhibited bone and cartilage formation during fracture repair, and further confirmed thatβ-catenin played a key signaling role during fracture healing.ConclusionsOur results suggest that upregulation of many Wnt ligmands and their receptors during fracture healing can activate Wnt/β-catenin signaling pathway; High level expressedβ-catenin can mediate Tcf-dependent transcription and promote fracture healing during fracture repair;β-catenin signaling is activated mainly in proliferating chondrocytes, prehypertrophic chondrocytes and osteoblasts but not in hypertrophic chondrocytes; Dkk-1(an antagonist of canonical Wnt/β-catenin signaling that inhibits Wnt receptor activation) inhibited bone and cartilage formation during fracture repair, and further confirmed thatβ-catenin played a key signaling role during fracture healing.Our findings raise the possibility that therapy to activateβ-catenin could be used to enhance fracture repair, which could be developed into a novel therapeutic approach to improve bone healing.