Showing papers by "Joan C. Marini published in 2014"

Osteogenesis imperfecta due to mutations in non-collagenous genes: lessons in the biology of bone formation.

Abstract: Purpose of Review Osteogenesis imperfecta (OI), or “brittle bone disease”, has mainly been considered a bone disorder caused by collagen mutations. Within the last decade, however, a surge of genetic discoveries has created a new paradigm for OI as a collagen-related disorder, where autosomal dominant type I collagen defects cause most cases, while rare, mostly recessive forms are due to defects in genes whose protein products interact with collagen protein. This review is both timely and relevant in outlining the genesis, development and future of this paradigm shift in the understanding of OI.

Abnormal Type I Collagen Post-translational Modification and Crosslinking in a Cyclophilin B KO Mouse Model of Recessive Osteogenesis Imperfecta

Abstract: Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib−/− mice that recapitulate the OI phenotype Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots Only residual (2–11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity Ppib−/− fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87 Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios The altered crosslink pattern was associated with decreased collagen deposition into matrix in culture, altered fibril structure in tissue, and reduced bone strength These studies demonstrate novel consequences of the indirect regulatory effect of CyPB on collagen hydroxylation, impacting collagen glycosylation, crosslinking and fibrillogenesis, which contribute to maintaining bone mechanical properties

A novel IFITM5 mutation in severe atypical osteogenesis imperfecta type VI impairs osteoblast production of pigment epithelium-derived factor.

Abstract: Osteogenesis imperfecta (OI) types V and VI are caused, respectively, by a unique dominant mutation in IFITM5, encoding BRIL, a transmembrane ifitm-like protein most strongly expressed in the skeletal system, and recessive null mutations in SERPINF1, encoding pigment epithelium-derived factor (PEDF) We identified a 25-year-old woman with severe OI whose dermal fibroblasts and cultured osteoblasts displayed minimal secretion of PEDF, but whose serum PEDF level was in the normal range SERPINF1 sequences were normal despite bone histomorphometry consistent with type VI OI and elevated childhood serum alkaline phosphatase We performed exome sequencing on the proband, both parents, and an unaffected sibling IFITM5 emerged as the candidate gene from bioinformatics analysis, and was corroborated by membership in a murine bone co-expression network module containing all currently known OI genes The de novo IFITM5 mutation was confirmed in one allele of the proband, resulting in a pS40L substitution in the intracellular domain of BRIL but was absent in unaffected family members IFITM5 expression was normal in proband fibroblasts and osteoblasts, and BRIL protein level was similar to control in differentiated proband osteoblasts on Western blot and in permeabilized mutant osteoblasts by microscopy In contrast, SERPINF1 expression was decreased in proband osteoblasts; PEDF was barely detectable in conditioned media of proband cells Expression and secretion of type I collagen was similarly decreased in proband osteoblasts; the expression pattern of several osteoblast markers largely overlapped reported values from cells with a primary PEDF defect In contrast, osteoblasts from a typical case of type V OI, with an activating mutation at the 5'-terminus of BRIL, have increased SERPINF1 expression and PEDF secretion during osteoblast differentiation Together, these data suggest that BRIL and PEDF have a relationship that connects the genes for types V and VI OI and their roles in bone mineralization

Adult Brtl/+ mouse model of osteogenesis imperfecta demonstrates anabolic response to sclerostin antibody treatment with increased bone mass and strength

Abstract: Treatments to reduce fracture rates in adults with osteogenesis imperfecta are limited. Sclerostin antibody, developed for treating osteoporosis, has not been explored in adults with OI. This study demonstrates that treatment of adult OI mice respond favorably to sclerostin antibody therapy despite retention of the OI-causing defect. Osteogenesis imperfecta (OI) is a heritable collagen-related bone dysplasia, characterized by brittle bones with increased fracture risk. Although OI fracture risk is greatest before puberty, adults with OI remain at risk of fracture. Antiresorptive bisphosphonates are commonly used to treat adult OI, but have shown mixed efficacy. New treatments which consistently improve bone mass throughout the skeleton may improve patient outcomes. Neutralizing antibodies to sclerostin (Scl-Ab) are a novel anabolic therapy that have shown efficacy in preclinical studies by stimulating bone formation via the canonical wnt signaling pathway. The purpose of this study was to evaluate Scl-Ab in an adult 6 month old Brtl/+ model of OI that harbors a typical heterozygous OI-causing Gly > Cys substitution on Col1a1. Six-month-old WT and Brtl/+ mice were treated with Scl-Ab (25 mg/kg, 2×/week) or Veh for 5 weeks. OCN and TRACP5b serum assays, dynamic histomorphometry, microCT and mechanical testing were performed. Adult Brtl/+ mice demonstrated a strong anabolic response to Scl-Ab with increased serum osteocalcin and bone formation rate. This anabolic response led to improved trabecular and cortical bone mass in the femur. Mechanical testing revealed Scl-Ab increased Brtl/+ femoral stiffness and strength. Scl-Ab was successfully anabolic in an adult Brtl/+ model of OI.

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

Abstract: Gene silencing approaches have the potential to become a powerful curative tool for a variety of monogenic diseases caused by gain-of-function mutations. Classical osteogenesis imperfecta (OI), a dominantly inherited bone dysplasia, is characterized in its more severe forms by synthesis of structurally abnormal type I collagen, which exerts a negative effect on extracellular matrix. Specific suppression of the mutant (Mut) allele would convert severe OI forms to the mild type caused by a quantitative defect in normal collagen. Here, we describe the in vitro and ex vivo investigation of a small interfering RNA (siRNA) approach to allele-specific gene silencing using Mut Col1a1 from the Brtl mouse, a well-characterized model for classical human OI. A human embryonic kidney cell line, which expresses the firefly luciferase gene, combined with either wild-type or Mut Brtl Col1a1 exon 23 sequences, was used for the first screening. The siRNAs selected based on their specificity and the corresponding short hairpin RNAs (shRNAs) subcloned in a lentiviral vector were evaluated ex vivo in Brtl fibroblasts for their effect on collagen transcripts and protein. A preferential reduction of the Mut allele of up to 52% was associated with about 40% decrease of the Mut protein, with no alteration of cell proliferation. Interestingly, a downregulation of HSP47, a specific collagen chaperone known to be upregulated in some OI cases, was detected. Our data support further testing of shRNAs and their delivery by lentivirus as a strategy to specifically suppress the Mut allele in mesenchymal stem cells of OI patients for autologous transplantation.