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 (Ptcl, Ptc2), Smoothened (Smo), Glil, 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, Ptcl, Smo, Glil, 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 Ptcl 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 Glil was significantly decreased in OC samples, but there was no significant difference in expression of Gli3, Ptcl and Smo in OC cartilage compared to normal cartilage. The expression of Ptcl 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 PTHrP/Ihh feedback cycle exists in articular cartilage. The disparity between increased Ihh expression and decreased Glil expression in OC cartilage suggests a different primary transcription factor for Ihh or the presence of an elevated Ihh inhibitor in these tissues.

Altered gene expression in early osteochondrosis lesions

“…Runx2 is normally expressed in proliferating and hypertrophic chondrocytes in the growth plate, with highest expression in terminal hypertrophic chondrocytes. 5 Runx2 induces genes typical of terminal chondrocyte differentiation, including type X collagen, MMP-13, and Ihh, 5,29,30 thus it is not surprising that these genes are also up-regulated in OC lesions (as demonstrated here for MMP-13, and by Semevolos et al, 2005 11 for Ihh).…”

Mice with a Targeted Mutation of Patched2 Are Viable but Develop Alopecia and Epidermal Hyperplasia

“…In addition, PTCH2 splice variants were able to reconstitute a Dhh-dependent transcriptional response in cells lacking Ptc1 function (52). It was reported that Ptc2 and Ihh are highly expressed in equine osteochondrosis-affected cartilage and repair tissue, suggesting that Ihh-Ptc2 signaling might play a role in diseased adult cartilage (56).…”

Age‐related expression of molecular regulators of hypertrophy and maturation in articular cartilage

“…These riboprobes were previously determined to perform well on equine cartilage samples. 5,6 The probe was added to hybridization buffer containing 25% deionized formamide, 10% dextran sulfate, 300 mM NaCl, 10 mM TRIS, 1 mM EDTA, 1 Â Denhardt's solution (0.02% BSA, 0.02% Ficoll, and 0.02% polyvinylpyrrolidone), 0.5 mg yeast tRNA/mL, and 10 mM dithiothreitol. The probe (0.12 pmoles, specific activity 1.0-1.2 Â 10 7 cpm/pmol) in 40 mL of hybridization buffer was applied to a section on each slide, the slides coverslipped, and then incubated overnight in humid chambers at 438C.…”

“…In addition, alterations in the expression of PTHrP and Ihh have been shown to occur in osteochondrosis, a disease of articular cartilage development. 5,6 Previous studies have shown that the expression of regulatory molecules in articular cartilage may change during the postnatal period. [7][8][9][10][11][12][13][14][15] Qualitative assessment of PTHrP mRNA and protein expression in postnatal rats showed decreased expression in articular cartilage from youth to maturity.…”