Collagen transport and related pathways in Osteogenesis Imperfecta

Osteogenesis Imperfecta (OI) comprises a heterogeneous group of patients who share bone fragility and deformities as the main characteristics, albeit with different degrees of severity. Phenotypic variation also exists in other connective tissue aspects of the disease, complicating disease classification and disease course prediction. Although collagen type I defects are long established as the primary cause of the bone pathology, we are still far from comprehending the complete mechanism. In the last years, the advent of next generation sequencing has triggered the discovery of many new genetic causes for OI, helping to draw its molecular landscape. It has become clear that, in addition to collagen type I genes, OI can be caused by multiple proteins connected to different parts of collagen biosynthesis. The production of collagen entails a complex process, starting from the production of the collagen Iα1 and collagen Iα2 chains in the endoplasmic reticulum, during and after which procollagen is subjected to a plethora of posttranslational modifications by chaperones. After reaching the Golgi organelle, procollagen is destined to the extracellular matrix where it forms collagen fibrils. Recently discovered mutations in components of the retrograde transport of chaperones highlight its emerging role as critical contributor of OI development. This review offers an overview of collagen regulation in the context of recent gene discoveries, emphasizing the significance of transport disruptions in the OI mechanism. We aim to motivate exploration of skeletal fragility in OI from the perspective of these pathways to identify regulatory points which can hint to therapeutic targets

Exploration of the skeletal phenotype of the Col1a1 +/Mov13 mouse model for haploinsufficient osteogenesis imperfecta type 1

Introduction: Osteogenesis Imperfecta is a rare genetic connective tissue disorder, characterized by skeletal dysplasia and fragile bones. Currently only two mouse models have been reported for haploinsufficient (HI) mild Osteogenesis Imperfecta (OI); the Col1a1+/Mov13 (Mov13) and the Col1a1+/-365 mouse model. The Mov13 mice were created by random insertion of the Mouse Moloney leukemia virus in the first intron of the Col1a1 gene, preventing the initiation of transcription. Since the development of the Mov13 mice almost four decades ago and its basic phenotypic characterization in the 90s, there have not been many further studies. We aimed to extensively characterize the Mov13 mouse model in order to critically evaluate its possible use for preclinical studies of HI OI. Methods: Bone tissue from ten heterozygous Mov13 and ten wild-type littermates (WT) C57BL/6J mice (50% males per group) was analyzed at eight weeks of age with bone histomorphometry, micro computed tomography (microCT), 3-point bending, gene expression of different collagens, as well as serum markers of bone turnover Results: The Mov13 mouse presented a lower bone strength and impaired material properties based on our results of 3-point bending and microCT analysis respectively. In contrast, no significant differences were found for all histomorphometric parameters. In addition, no significant differences in Col1a1 bone expression were present, but there was a significant lower P1NP concentration, a bone formation marker, measured in serum. Furthermore, bone tissue of Mov13 mice presented significantly higher expression of collagens (Col1a2, Col5a1 and Col5a2), and bone metabolism markers (Bglap, Fgf23, Smad7, Edn1 and Eln) compared to WT. Finally, we measured a significantly lower Col1a1 expression in heart and skin tissue and also determined a higher expression of other collagens in the heart tissue. Conclusion: Although we did not detect a significant reduction in Col1a1 expression in the bone tissue, a change in bone structure and reduction in bone strength was noted. Regrettably, the variability of the bone phenotype and the appearance of severe lymphoma in adult Mov13 mice, does not favor their use for the testing of new long-term drug studies. As such, a new HI OI type 1 mouse model is urgently needed

Osteogenic transdifferentiation of primary human fibroblasts to osteoblast-like cells with human platelet lysate

Inherited bone disorders account for about 10% of documented Mendelian disorders and are associated with high financial burden. Their study requires osteoblasts which play a critical role in regulating the development and maintenance of bone tissue. However, bone tissue is not always available from patients. We developed a highly efficient platelet lysate-based approach to directly transdifferentiate skin-derived human fibroblasts to osteoblast-like cells. We extensively characterized our in vitro model by examining the expression of osteoblast-specific markers during the transdifferentiation process both at the mRNA and protein level. The transdifferentiated osteoblast-like cells showed significantly increased expression of a panel of osteogenic markers. Mineral deposition and ALP activity were also shown, confirming their osteogenic properties. RNA-seq analysis allowed the global study of changes in the transcriptome of the transdifferentiated cells. The transdifferentiated cells clustered separately from the primary fibroblasts with regard to the significantly upregulated genes indicating a distinct transcriptome profile; transdifferentiated osteoblasts also showed significant enrichment in gene expression related to skeletal development and bone mineralization. Our presented in vitro model may potentially contribute to the prospect of studying osteoblast-dependent disorders in patient-derived cells

Interaction between KDELR2 and HSP47 as a Key Determinant in Osteogenesis Imperfecta Caused by Bi-allelic Variants in KDELR2

Osteogenesis imperfecta (OI) is characterized primarily by susceptibility to fractures with or without bone deformation. OI is genetically heterogeneous: over 20 genetic causes are recognized. We identified bi-allelic pathogenic KDELR2 variants as a cause of OI in four families. KDELR2 encodes KDEL endoplasmic reticulum protein retention receptor 2, which recycles ER-resident proteins with a KDEL-like peptide from the cis-Golgi to the ER through COPI retrograde transport. Analysis of patient primary fibroblasts showed intracellular decrease of HSP47 and FKBP65 along with reduced procollagen type I in culture media. Electron microscopy identified an abnormal quality of secreted collagen fibrils with increased amount of HSP47 bound to monomeric and multimeric collagen molecules. Mapping the identified KDELR2 variants onto the crystal structure of G. gallus KDELR2 indicated that these lead to an inactive receptor resulting in impaired KDELR2-mediated Golgi-ER transport. Therefore, in KDELR2-deficient individuals, OI most likely occurs because of the inability of HSP47 to bind KDELR2 and dissociate from collagen type I. Instead, HSP47 remains bound to collagen molecules extracellularly, disrupting fiber formation. This highlights the importance of intracellular recycling of ER-resident molecular chaperones for collagen type I and bone metabolism and a crucial role of HSP47 in the KDELR2-associated pathogenic mechanism leading to OI