Impaired Osteoblastogenesis in a Murine Model of Dominant Osteogenesis Imperfecta: A New Target for Osteogenesis Imperfecta Pharmacological Therapy

Abstract

The molecular basis underlying the clinical phenotype in bone diseases is customarily associated with abnormal extracellular matrix structure and/or properties. More recently, cellular malfunction has been identified as a concomitant causative factor and increased attention has focused on stem cells differentiation. Classic osteogenesis imperfecta (OI) is a prototype for heritable bone dysplasias: it has dominant genetic transmission and is caused by mutations in the genes coding for collagen I, the most abundant protein in bone. Using the Brtl mouse, a well-characterized knockin model for moderately severe dominant OI, we demonstrated an impairment in the differentiation of bone marrow progenitor cells toward osteoblasts. In mutant mesenchymal stem cells (MSCs), the expression of early (Runx2 and Sp7) and late (Col1a1 and Ibsp) osteoblastic markers was significantly reduced with respect to wild type (WT). Conversely, mutant MSCs generated more colony-forming unit-adipocytes compared to WT, with more adipocytes per colony, and increased number and size of triglyceride drops per cell. Autophagy upregulation was also demonstrated in mutant adult MSCs differentiating toward osteogenic lineage as consequence of endoplasmic reticulum stress due to mutant collagen retention. Treatment of the Brtl mice with the proteasome inhibitor Bortezomib ameliorated both osteoblast differentiation in vitro and bone properties in vivo as demonstrated by colony-forming unit-osteoblasts assay and peripheral quantitative computed tomography analysis on long bones, respectively. This is the first report of impaired MSC differentiation to osteoblasts in OI, and it identifies a new potential target for the pharmacological treatment of the disorder.

Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta

“…42 We recently described ER stress in Brtl fibroblast and calvarial bone, as well as in MSCs differentiating toward the osteoblastic lineage. [30][31][32] In the latter case, we showed an increase in HSP47, a specific molecular chaperone known to bind folded type I procollagen molecules in the ER, similar to previous reports in some OI cases. 43 Thus, we evaluated the expression of HSP47 following silencing and found a significant reduction in F-Mut shRNA-transduced Brtl cells, compared with control fibroblasts, as might be expected based on about 40% total collagen suppression.…”

“…We also investigated the expression level of HSP47, a specific procollagen molecular chaperone, whose expression was shown to be increased in Brtl cells. 32 Interestingly, HSP47 evaluated by western blot demonstrated significant reduction (about 25%) in F-Mut-transduced Brtl cells compared with untransduced or LacZ-transduced Brtl fibroblasts (Figures 4c and d).…”

“…Our results resemble the data reported by Dawson et al using a ribozyme-based antisense approach against a Mut Col1a2 allele in patient fibroblasts. 33 We speculate 32 Many inherited skeletal dysplasias are associated with mutations in ECM components and their molecular basis has conventionally been attributed to the presence of an abnormal ECM and impairment of its structural integrity. It is now clear that many mutant proteins are retained intracellularly and accumulate in the ER, causing ER stress and triggering endoplasmic reticulum stress signaling, that, in turn, contributes to the skeletal outcomes.…”

“…In all, 60 mg of total proteins were separated by SDS-PAGE in denaturing conditions using 10% gels and electro-transferred to a PVDF membrane (Amersham, GE Healthcare, Chalfont St Giles, UK) for 2 h at 100 V. Incubation with primary antibodies against HSP47 and a-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and signal detection were performed as previously described. 32 …”

Genetic expression and functional characterization of the RUNX2 gene in human adult bone marrow mesenchymal stem cells

“…Owing to these prominent features and their ease of transplantation, MSCs have been used in treatments against various diseases including femoral head necrosis, spinal cord injury, immune-related diseases, and myocardial infarction (Chen et al, 2011). As MSCs are stably distributed in the adult body with pluripotent differentiation that is unlimited by cell replication, they have inspiring potential for clinical use within the existing ethical regulation (Gioia et al, 2012). However, as only limited numbers of MSCs exist in a normal adult's bone marrow, and extraction is difficult (Weng and Su, 2013), it is necessary to prepare large numbers of MSCs for clinical use.…”