Abstract
Summary Statement
Wnt/β-catenin gene expression is altered in early osteochondrosis, particularly in chondrocytes surrounding cartilage canals, and may be associated with disease initiation and/or pathogenesis.
Introduction
Osteochondrosis (OC) is a disease of articular cartilage development involving abnormal endochondral ossification along the osteochondral junction. Associated etiological factors of OC have included rapid growth rate, biomechanical trauma, abnormal collagen turnover, aberrant paracrine signaling, and altered blood supply involving cartilage canals. Wnt signaling regulates chondrocyte differentiation/maturation during pre-/post-natal cartilage development. Gene expression profiling of leukocytes has revealed aberrant expression of Wnt/β-catenin pathway in early OC. The objective of this study was to elucidate the expression of molecules associated with Wnt/β-catenin signaling in early OC using an equine model. Our hypothesis was that there would be increased expression of Wnt signaling molecules in chondrocytes adjacent to cartilage canals and the osteochondral junction in early OC lesions compared to normal controls.
Patients & Methods
Osteochondral samples were obtained (IACUC-approved) from femoropatellar joints of 15 euthanised immature horses (1–6 months old). Disease status was determined based on histology of osteochondral junctions (7 early OC, 8 normal controls). Osteochondral sections were frozen in OCT for laser capture microdissection (LCM) or fixed in 4% paraformaldehyde and paraffin-embedded for immunohistochemistry. Chondrocytes surrounding cartilage canals and osteochondral junctions were captured using LCM. RNA isolation and reverse transcription were performed. Equine-specific β-catenin, Wnt-4, Wnt-5b, Wnt-11, Dickkopf-1(Dkk-1), Lrp-4 and -6, Axin1, Wnt inhibitory factor(WIF)-1, secreted Frizzled-related protein-1, -3, and -5(Sfrp), retinoic acid receptor-gamma(RARG), RAR-inducible serine carboxypeptidase(SC-PEP) and 18S mRNA expression was evaluated by two-step real-time qPCR. Spatial protein expression was determined by immunohistochemistry using rabbit polyclonal (β-catenin, Wnt-11) or mouse monoclonal (Wnt-4, Dkk1) primary antibodies (confirmed by Western blot). Statistical analysis of early OC vs. normal controls was performed using Wilcoxon rank sum test (p <0.05).
Results
Chondrocytes adjacent to cartilage canals had significantly increased gene expression of β-catenin (p=0.026), Wnt-5b (p=0.04), Lrp6 (p=0.026), WIF-1 (p=0.026), Dkk-1 (p=0.015), Axin1 (0.041), and SC-PEP (p=0.026), and decreased expression of Wnt-11 (p=0.04), in OC vs. normal controls. OC chondrocytes along osteochondral junctions had significantly increased gene expression of β-catenin (p=0.004) and SC-PEP (p=0.026), with a trend for increased Wnt-4 (p= 0.06) and Wnt-5b (p=0.06) compared to normal controls. Immunostaining for β-catenin was moderate in deep cartilage layers, including osteochondral junction chondrocytes. Wnt-4 immunostaining was moderate along the osteochondral junction and minimal along cartilage canals. Strong Wnt-11 protein expression was apparent in superficial cartilage layers and vascular cells lining cartilage canals and osteochondral junction. Mild to moderate Dkk1 immunostaining was found along osteochondral junction.
Discussion/Conclusion
Wnt/β-catenin signaling regulates cartilage differentiation during development and is important in endochondral ossification. Increased gene expression of β-catenin in OC chondrocytes may affect chondrocyte hypertrophy or induce cartilage degeneration, depending on the stage of cartilage development, as β-catenin has been shown to play a dual role in cartilage growth and degeneration. In cells surrounding cartilage canals, increased gene expression of Lrp6, a co-receptor for Wnt proteins, provides further evidence of upregulation of canonical signaling in OC. However, increased Wnt inhibitor gene expression in OC chondrocytes, including Dkk1, WIF-1, and Axin1, may be an attempt to control activation of the canonical pathway.
