Showing papers by "Joan C. Marini published in 2012"

Absence of FKBP10 in recessive type XI osteogenesis imperfecta leads to diminished collagen cross-linking and reduced collagen deposition in extracellular matrix.

Abstract: Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% over-modification. Normal chain incorporation, helix folding, and collagen T(m) support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix.

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.

Cardiopulmonary dysfunction in the Osteogenesis imperfecta mouse model Aga2 and human patients are caused by bone-independent mechanisms

Abstract: Osteogenesis imperfecta (OI) is an inherited connective tissue disorder with skeletal dysplasia of varying severity, predominantly caused by mutations in the collagen I genes (COL1A1/COL1A2). Extraskeletal findings such as cardiac and pulmonary complications are generally considered to be significant secondary features. Aga2, a murine model for human OI, was systemically analyzed in the German Mouse Clinic by means of in vivo and in vitro examinations of the cardiopulmonary system, to identify novel mechanisms accounting for perinatal lethality. Pulmonary and, especially, cardiac fibroblast of perinatal lethal Aga2/+ animals display a strong down-regulation of Col1a1 transcripts in vivo and in vitro, resulting in a loss of extracellular matrix integrity. In addition, dysregulated gene expression of Nppa, different types of collagen and Agt in heart and lung tissue support a bone-independent vicious cycle of heart dysfunction, including hypertrophy, loss of myocardial matrix integrity, pulmonary hypertension, pneumonia and hypoxia leading to death in Aga2. These murine findings are corroborated by a pediatric OI cohort study, displaying significant progressive decline in pulmonary function and restrictive pulmonary disease independent of scoliosis. Most participants show mild cardiac valvular regurgitation, independent of pulmonary and skeletal findings. Data obtained from human OI patients and the mouse model Aga2 provide novel evidence for primary effects of type I collagen mutations on the heart and lung. The findings will have potential benefits of anticipatory clinical exams and early intervention in OI patients.

A novel mutation in LEPRE1 that eliminates only the KDEL ER- retrieval sequence causes non-lethal osteogenesis imperfecta.

Abstract: Prolyl 3-hydroxylase 1 (P3H1), encoded by the LEPRE1 gene, forms a molecular complex with cartilage-associated protein (CRTAP) and cyclophilin B (encoded by PPIB) in the endoplasmic reticulum (ER). This complex is responsible for one step in collagen post-translational modification, the prolyl 3-hydroxylation of specific proline residues, specifically α1(I) Pro986. P3H1 provides the enzymatic activity of the complex and has a Lys-Asp-Glu-Leu (KDEL) ER-retrieval sequence at the carboxyl terminus. Loss of function mutations in LEPRE1 lead to the Pro986 residue remaining unmodified and lead to slow folding and excessive helical post-translational modification of type I collagen, which is seen in both dominant and recessive osteogenesis imperfecta (OI). Here, we present the case of siblings with non-lethal OI due to novel compound heterozygous mutations in LEPRE1 (c.484delG and c.2155dupC). The results of RNA analysis and real-time PCR suggest that mRNA with c.2155dupC escapes from nonsense-mediated RNA decay. Without the KDEL ER- retrieval sequence, the product of the c.2155dupC variant cannot be retained in the ER. This is the first report of a mutation in LEPRE1 that eliminates only the KDEL ER-retrieval sequence, whereas other functional domains remain intact. Our study shows, for the first time, that the KDEL ER- retrieval sequence is essential for P3H1 functionality and that a defect in KDEL is sufficient for disease onset.

Deficiency of CRTAP in non-lethal recessive osteogenesis imperfecta reduces collagen deposition into matrix

Abstract: Osteogenesis imperfecta (OI) is a heterogeneous heritable connective tissue disorder characterized by bone fragility and deformity. The majority of OI cases have dominant inheritance (Sillence types I to IV OI) and result from mutations in the COL1A1 or COL1A2 genes, encoding the proα1(I) and proα2(I) chains of type I collagen, the major structural protein of bone (1, 2). Biochemically, collagen structural defects delay helical folding, exposing the chains to post-translational prolyl 4-hydroxylation and lysyl hydroxylation for a longer time, resulting in ‘over-modification’ and delayed electrophoretic migration of collagen chains. In the last 5 years, a few recessive forms of OI have been shown to be caused by defects in the genes encoding the components of the collagen prolyl 3-hydroxylation complex (3, 4): cartilage-associated protein (CRTAP) (type VII OI, OMIM #610682) (5, 6), LEPRE1 (7–9) (type VIII OI, OMIM #610915), and PPIB (10–12) (type IX OI, OMIM #259440). Recently, additional disease loci responsible for recessive OI have been identified: FKBP10 (13), SERPINH1 (14), SP7/OX (15), and SERPINF1 (16). While both FKBP10 and SERPINH1 code for collagen chaperones resident in the ER, products of the latter two genes instead are not directly involved in collagen production or secretion but are key factors in osteoblasts differentiation and activity. Patients with defects in the components of the ER-resident 3-hydroxylation complex have moderate to severe/lethal OI, with white sclerae, small to normal head circumference and structurally normal collagen. Loss-of-function mutations in CRTAP and LEPRE1 result in rhizomelia, decreased to absent 3-hydroxylation of α1(I)Pro986, and collagen helical overmodification indicative of delayed folding. Of the three components of the 3-hydroxylation complex, CRTAP is known to be secreted into the extracellular matrix (17, 18). Normally, about 10% of CRTAP is secreted, while most is retained in the ER in a complex with prolyl 3-hydroxylase 1 (P3H1). Sixteen CRTAP mutant alleles, occurring in 15 index probands, have been reported (5–7, 18–20). Most null cases are lethal in the perinatal period or within the first year of life. Five non-lethal cases have been described. We present here a 7-year-old Egyptian boy whose severe OI is caused by homozygosity for a frameshift mutation in CRTAP exon 1. His dermal fibroblast type I collagen has typical post-translational modification defects for type VII OI. We report here the novel finding that the collagen content of matrix deposited by patient cells in culture is severely decreased. This data is supported by an in vitro collagen matrix-chase assay. These investigations describe matrix deficiency and disorganization associated with CRTAP deficiency which may reflect the absence of the crucial functions of CRTAP in extracellular matrix.

Replenishing Cartilage from Endogenous Stem Cells

Abstract: A feature of osteoarthritis is the erosion of cartilage. Investigators have recently described the identification of a small molecule that induces mesenchymal stem cells to differentiate and to secrete cartilage.