Bone Abstracts

Osteogenesis imperfecta (OI) type VI is caused by recessive null mutations in SERPINF1, encoding pigment epithelium-derived factor (PEDF), an anti-angiogenic secretory glycoprotein. Dominant mutations in IFITM5, encoding BRIL (Bone Restricted Ifitm-Like), a transmembrane protein upregulated in osteoblasts during mineralization, cause either type V OI (c.-14C>T, addition of 5 amino acids on BRIL) or atypical type VI OI (c.119C>T, p.Ser40Leu substitution in BRIL intracellular domain). Atypical type VI OI patients have extremely severe bone dysplasia, histological features of type VI OI bone, but no characteristics of type V OI. We reported that type V OI IFITM5 mutations cause an increased SERPINF1 expression and PEDF secretion in proband osteoblasts, while patient osteoblasts with the Ser40Leu mutation display decreased SERPINF1 expression and PEDF secretion. Currently, little is known about how point mutations in BRIL impact SERPINF1 expression. Interestingly, overexpression of BRIL (Ser40Leu) in mesenchymal stem cells did not impact on PEDF secretion, which was significantly decreased in the osteoblasts from the atypical OI type VI patient. To further investigate the underlying mechanism by which BRIL regulates PEDF secretion and thereby osteoblast matrix mineralization, we generated a cellular system that carries the IFITM5 (c.119C>T) mutation, using the CRISPR/Cas9-mediated gene editing system. We designed a donor DNA for homology-directed repair-mediated gene modification and obtained several clones that harbor the heterozygous IFITM5 (c.119C>T) mutation. In CRISPR/Cas9-edited clones, significantly decreased SERPINF1 expression and PEFD secretion recapitulated our previous observation with cells from the atypical OI type VI proband. Moreover, the IFITM5 (c.119C>T) mutation resulted in an decreased expression of PPARγ, also seen in osteoblasts from the serpinf1-null mice. These results demonstrate that BRIL-mediated induction of PEDF activates PPARγ-mediated cellular events. This study also demonstrated that CRISPR/Cas9-mediated gene editing can be a useful tool to simulate cellular status and to study molecular mechanisms of OI-causing mutations.