Abbi Smith, Íris Halldórsdóttir, Eiríkur Steingrímsson and Sara Sigurbjörnsdóttir
Introduction: Osteoarthritis is an incurable debilitating joint disease characterized by degenerative articular cartilage. The hedgehog signaling pathway is aberrantly activated in osteoarthritic cartilage. Smoothened (SMO) is a gatekeeper of downstream hedgehog pathway activation and binds a cholesterol molecule to potentiate downstream signaling. A GWAS study identified a missense mutation of SMO leading to an arginine substitution for cysteine at residue 173 (R173C) correlated with an increased risk of developing hip osteoarthritis.
Methods: smo-/- zebrafish are embryonic lethal at 5 days post fertilization (dpf) and exhibit severe morphological defects which make them a useful tool for phenotypic recovery experiments. Fertilized zebrafish eggs were microinjected with mRNA transcripts encoding human SMO with wildtype (WT), R173C mutation and other mutations in the cholesterol binding pocket (D95N and Y130F) known to abolish cholesterol binding. The morphological phenotype was assessed at 2 dpf to determine the amount of phenotypic recovery. Immunofluorescent and histochemical staining is in progress to assess cartilage development.
Results: Human SMOWT and SMOR173C mRNAs microinjected into smo-/- zebrafish eggs were capable of rescuing a normal morphological phenotype, however the degree of complete rescue was higher in the SMOWT mRNA injected cohort. SMOD95N and SMOY130F mRNA both failed to rescue a normal morphological phenotype.
Conclusions: Human SMO mRNA is functional in zebrafish and can successfully activate hedgehog signaling. The R173C mutation is a viable mutation and can sufficiently activate hedgehog signaling to rescue a normal morphological phenotype. However, more experiments are necessary to elucidate its role in osteoarthritis development.
