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Procollagen C-propeptide Processing Impacts Bone Matrix

ABSTRACT: Classical Osteogenesis imperfecta (OI), characterized by short stature, fragile bones and low bone mass, is predominantly caused by genetic defects in the structure or synthesis of type I collagen. Unexpectedly, there is a rare high bone mass form of OI (HBM OI) with extremely high bone mineralization by quantitative backscattered electron imaging (qBEI) and a moderately severe skeletal phenotype. HBM OI is caused by dominant missense mutations at residues comprising the type I procollagen C-propeptide cleavage site, preventing processing of the C-propeptide. To elucidate the role of C-propeptide processing in bone mineralization and development, we generated heterozygous HBM OI mice, with substitutions in the Col1a1 C-propeptide cleavage site. Femoral extracts from HBM OI mice contain increased monomeric proα1(I) C-propeptide and pC-collagen, resulting in a “barbed-wire” appearance to bone collagen fibrils. The decrease in type I procollagen cleavage has a profound impact on bone, leading to diminished bone strength and integrity despite normal collagen helical primary sequence. HBM OI femora have thin cortices with decreased BV/TV leading to decreased stiffness, yield and fracture load, which does not improve with age. Their femora are also extremely brittle, with increased mineral content and HP crosslinks. Similar to the high to normal DXA bone density of HBM OI patients, µCT bone density is normal in femora. However, murine HBM OI cortical bone is hypermineralized in excess of other OI mouse models, with thinner, more disorganized mineral particles. Increased expression of ossification genes is detected in both murine and human HBM OI osteoblasts, with enhanced mineral deposition in culture. There is decreased expression of genes involved with extracellular matrix organization, correlating with changes in actin and vinculin staining when osteoblasts are grown on mutant matrix. Murine HBM OI bone mineralization is increased throughout life and increases with age, raising concerns for long-term management of HBM OI patients.

ORGANISM(S): Mus musculus

PROVIDER: GSE279055 | GEO | 2025/10/08

REPOSITORIES: GEO

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Project description:Osteogenesis Imperfecta (OI) is a genetic, rare disease characterized by bone fragility, with a wide range in the severity of clinical manifestations. The majority of the cases are due to mutations in COL1A1 or COL1A2 genes which code for the type I collagen molecule. Mesenchymal stem cells (MSCs), as the progenitors of the osteoblasts, the main type I collagen secreting cell type in the bone, have been proposed and tested as an innovative therapy for OI with promising but transient outcomes. In this study, we performed a phase I clinical trial based on reiterative infusions of histocompatible MSCs in two pediatric patients affected by severe and moderate OI. The aim of this study was to assess the safety and effectiveness of this cell therapy in non-immunosuppressed OI patients. The host response to MSCs was also evaluated by collecting the sera from OI patients before, along and after the cell therapy. The results of this clinical trial are twofold: the sequential administration of MSCs is safe and improve the bone parameters and quality of life of OI patients. Moreover, MSCs therapy elicits a pro-osteogenic paracrine response in patients, especially noticeable in that affected by severe OI. These results indicate the feasibility and potential of reiterative MSCs infusion for OI and highlight the paracrine response shown by OI patients as a consequence of MSCs treatment. Study design and patients: The Mesenchymal Stem Cell Therapy for the Treatment of Osteogenesis Imperfecta (TERCELOI) study (www.clinical trials.gov: # NCT02172885, EudraCT number: 2012-002553-38) is an independent multi-center Phase I clinical trial to evaluate the feasibility, safety and potential efficacy of infused sibling HLA-matched MSCs in non-immunosuppressed children with OI. PBMCs isolation and serum collection: Venous blood samples were collected before the cell therapy (basal serum), along the cell therapy (1 week, 1 month and 4 months after each MSCs infusion) and during the follow-up period (1 and 2 years after the fifth and last MSCs infusion). MLR assay: PBMCs isolated from patients were resuspended in PBS+FBS (5%) and stained with carboxyfluoroscein succinate-ester (CFSE) (Molecular Probes, USA). Bone mineral density: Bone mineral density (BMD) at the lumbar spine (LS) from L1 to L4, was measured by dual energy x-ray absorptiometry (DXA) on a whole-body scanner within a pediatric platform (Hologic QDR densitometer). Raw measurements were converted to Z-scores for analysis using reference standards for age and pubertal status. OI-MSCs isolation and characterization and culture: OI-MSCs were derived from discarded and donated bone fragments of OI pediatric patients undergoing corrective surgery. All patients suffered Type III-IV OI, with mutations in either COL1A1 or COL1A2 genes. The donation was approved by the Basque Clinical Research Ethics Committee. Briefly, bone chips were mechanically flushed with PBS and then cut into small pieces to extrude cells without the use of enzymes. The cut bone pieces were let undisturbed during 14 days until OI-MSCs migrated to cell culture plate. OI-MSCs were assessed for the expression of CD105, CD90 and CD73 and the absence of CD14, CD34, CD45, CD19 and HLA-DR. MSCs were also tested for their potential to differentiate to osteoblasts and adipocytes using specific cell culture media. OI-MSCs were cultured under standard growth medium (DMEM low glucose with glutamax (Gibco), penicillin/streptomycin (Gibco) and fetal bovine serum at 10% (Sigma-Aldrich, USA). Osteogenesis induction medium (OIM) was composed of standard medium plus ascorbic acid 0.2 mM, beta-glycerophosphate 10 mM, and dexamethasone 10 nM (all from Sigma-Aldrich). When specified, FBS was replaced by P01 and P02 serum samples at 2.5%. RNAseq and Q-PCR validation: OI-MSCs from 10 OI pediatric patients were seeded in 96-well plates (1 000 cells/well) and the following day cultured in the presence of OIM. Next, total RNA was isolated with the AllPrep kit (QIAGEN, USA). cDNA library (TrueSeq stranded Total cDNA library, Illumina, USA) and sequencing at HiSeq 4000 (PE100nt, 50 million reads/sample).

Project description:Osteogenesis imperfecta (OI) is a group of diseases caused by defects in type I collagen processing which result in skeletal fragility. While these disorders have traditionally been regarded as defects in osteoblast function, the role of matrix-embedded osteocytes, descendants of osteoblasts, in OI pathogenesis remains unknown. The homozygous human SP7 (c.946C > T, R316C) mutation results in a recessive form of osteogenesis imperfecta characterized by short stature, fragility fractures, low bone mineral density, and osteocyte dendrite defects. To better understand how the osteogenesis imperfecta-causingSP7 R316Cmutation affects the function of this transcription factor in different osteoblast lineage cells in bone, we generatedSp7 R342Cknock-in mice. Homozygous mutantSp7 R342C/R342Cmice demonstrate increased cortical porosity and reduced cortical bone mineral density, findings consistent with phenotypes observed in patients with this mutation. Sp7 R342Cmice show osteocyte dendrite defects, increased osteocyte apoptosis, and intracortical bone remodeling characterized by ectopic intracortical osteoclasts and elevated Tnfsf11 expression by osteocytes. Remarkably, these overt defects in osteocyte function contrast to preserved osteoblast function, suggesting that this Sp7 point mutation selectively interferes with the function of this transcription factor in osteocytes but not osteoblasts. Osteocyte morphology changes in Sp7 R342C/R342Cmice were not restored by inhibiting osteoclast formation, indicating that dendrite defects lie upstream of high intra-cortical osteoclast activity in this model. Moreover, transcriptomic profiling reveals that the expression of a core set osteocyte-enriched genes is highly dysregulated by the R342C mutation. Thus, this model supports a model in which osteocyte dysfunction can drive osteogenesis imperfecta pathogenesis, and provides a valuable resource to test novel therapeutic approaches and to understand the osteocyte-specific role of SP7 in bone homeostasis and remodeling.

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Procollagen C-propeptide Processing Impacts Bone Matrix

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