Proprotein convertase PC5/6 (Pcsk5) is expressed in mouse bone and Pcsk5 epiblast-specific conditional knockout mice have a bone phenotype displaying small size, delayed ossification and additional thoracic segments and ribs. Some features are attributed to growth and developmental factor 11 (GDF11) a known substrate for PC5/6 while the delayed mineralization has yet to be explained. Osteopontin (OPN) is a bone matrix protein with roles in mineralization, cell adhesion and migration, and contains the consensus sequence, K/R-Xn-K/R↓ (n=0,2,4 or 6 variable amino acids) for cleavage by PC5/6. This study investigated OPN as a substrate for PC5/6. In situ hybridization of normal mouse bone using Pcsk5, Opn/Spp1 and Dmp1 anti-sense and control sense cRNA probes, and quantitative RT-PCR on primary murine bone cells and bone cell lines showed expression of Pcsk5 in bone-forming cells (similar to Opn and Dmp1). Long-bone extracts from Pcsk5 conditional-knockout E18.5 embryos revealed an OPN fragment at ~15 kDa in normal bone that was not present in PC5/6-deficient bone, as determined by OPN immunoblotting. Pcsk5 conditional-knockout E18.5 embryos were also analyzed by micro-computed tomography, confirming the delayed skeletal mineralization. In addition, enzyme-substrate assays using recombinant human PC5/6 and OPN, as well as co-transfections of V5-tagged OPN/Opn and Pcsk5 into Cos-1 cells, showed that PC5/6 efficiently cleaved human OPN (~65 kDa) into ~55 kDa and 17 kDa fragments, consistent with the co-transfection results. Co-transfections with mouse Opn cDNA did not show any direct cleavage by PC5/6. In conclusion, we demonstrate that Pcsk5 is expressed in bone-forming cells, and that OPN is a novel substrate for PC5/6. Cleavage of OPN by PC5/6 could modify the function of OPN in bone, and/or modify downstream enzymatic processing of OPN leading to the bone phenotype. Animal studies were approved by the institutional animal care committee. Funded by CIHR.
17 May 2014 - 20 May 2014