Expression patterns of hedgehog signaling peptides in naturally acquired equine osteochondrosis

Abstract

Hypertrophic differentiation and endochondral ossification of growth cartilage are regulated by a complex array of signaling peptides, including parathyroid hormone-related protein (PTH-rP), Indian hedgehog (Ihh), and bone morphogenetic proteins (BMPs). This study investigated the expression of Ihh, Patched1 and 2 (Ptc1, Ptc2), Smoothened (Smo), Gli1, and Gli3, in naturally acquired articular osteochondrosis, using an equine model. Cartilage was harvested from osteochondrosis (OC) affected femoropatellar or scapulohumeral joints from immature horses and normal control horses of similar age. Ihh, Ptc1, Smo, Gli1, and Gli3 mRNA expression levels were evaluated by real-time quantitative PCR. Spatial tissue expression was determined by in situ hybridization for Ihh and Smo and immunohistochemistry for Ptc1 and Ptc2. The expression of Ihh was significantly increased in OC cartilage compared to normal control cartilage and was localized mainly to the deep layer of articular cartilage, just above the calcified zone, with some mild expression also present in the middle cartilage layer. The expression of Gli1 was significantly decreased in OC samples, but there was no significant difference in expression of Gli3, Ptc1 and Smo in OC cartilage compared to normal cartilage. The expression of Ptc1 protein was present at the junction of deep and calcified layers, while Ptc2 protein was expressed throughout the middle, deep, and calcified cartilage layers. Spatial expression of Smo was variable between animals and confined mainly to the middle and deep layers when present. Half of the OC samples displayed areas of moderate to strong Smo expression compared to mild or minimal expression in normal controls. The increased Ihh expression in OC suggests a role of Ihh in diseased cartilage, although it is not known if a PTH-rP/Ihh feedback cycle exists in articular cartilage. The disparity between increased Ihh expression and decreased Gli1 expression in OC cartilage suggests a different primary transcription factor for Ihh or the presence of an elevated Ihh inhibitor in these tissues.