Endocrine Abstracts

Ossification defects have been described in patients with peroxisomal disorders, autosomal-recessive diseases due to the impairment of peroxisome biogenesis. However, the functions of peroxisomes in skeletal tissues are unknown. In the present study, we used a knockout (KO) mouse model, defective in the PEX11β gene, to investigate the molecular pathogenesis of the ossification defect. Alizarin Red/Alzian Blue stainings, flat-panel volume-CT (fpvCT), and immunofluorescence (IF) preparations of paraffin sections were performed to show differences in ossification. Primary cultures of calvaria osteoblasts with subsequent subcellular fractionation and western blot analyses were used to study the molecular pathogenesis of the ossification defect. Skeleton stainings showed impairment in ossification of distal bone elements of the limbs, sacral and coccygeal vertebrae as well as parts of the skull and the sternum in KO mice. FpvCT analyses revealed a substantially lower total bone volume and a decreased whole-body bone mineral density in PEX11β KO animals. The ossification impairment in KO mice was corroborated by analyses of Alizarin red S preparations and IF stainings for osteopontin on paraffin sections. Subcellular fractionation and western blot analyses revealed a significant increase of peroxisomal catalase in KO osteoblasts. Additionally, the levels of other antioxidant enzymes (SOD1, SOD2 and hemoxygenase-1) were upregulated as well. Most interestingly, a higher level of p-GSK-3β was present and less β-catenin was shifted into the nucleus in KO osteoblasts. Furthermore, these changes were accompanied with the increasing PPARγ expression and the alteration of Runx2 localization in KO osteoblasts, showing a more prominent abundance in the cytoplasm and a reduction in the nuclear area. Taken together, our data suggest that peroxisomes might play a vital role in osteoblast ROS metabolism and ossification. PPARγ activation and Runx2 inactivation through less active Wnt signaling could contribute to impaired ossification due to peroxisomal deficiency.