15 research outputs found
Abnormal Type I Collagen Post-translational Modification and Crosslinking in a Cyclophilin B KO Mouse Model of Recessive Osteogenesis Imperfecta
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 Ppibβ/β 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 (2β11%) 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. Ppibβ/β 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
Inhibition of Collagen Fibrillogenesis by Cells Expressing Soluble Extracellular Domains of DDR1 and DDR2
Absence of <i>Ppib</i> expression affects bone development.
<p>(A) Staining of newborn skeletons with Alizarin red (bone) and Alcian blue (cartilage) reveals undermineralization of calvaria and ribs. Homozygous mice have smaller size of whole skeleton and long bones, and a deformed rib cage. (B) X-rays of 8 week-old mice. (C) DXA analysis of 8 week-old mice (nβ=β10/genotype).</p
Synthesis of type I collagen.
<p>(A) Western blots of cell lysates with antibodies to collagen 3-hydroxylation complex components. Lysates are derived from two independent cultures for each genotype. (B) SDS-Urea PAGE analysis of steady-state labeled type I collagen from wild-type (+/+), heterozygous (+/β) and homozygous (β/β) fibroblasts (FB) and osteoblasts (OB). (C) Differential scanning calorimetry (DSC) analysis reveals no differences in thermal stability (T<sub>m</sub>) of type I collagen secreted by fibroblast cultures.</p
Whole bone structural and mechanical properties.
<p>(A) Structural parameters of wild-type (+/+) and homozygous (β/β) femora at 8 weeks of age characterize reduced bone formation in CyPB-deficient mice (nβ=β9/genotype). <i>Left</i>, 3D reconstructions illustrate reduced trabecular and cortical bone volumes. <i>Right</i>, Trabecular parameters are decreased in homozygous (β/β) femora, including reduced bone volume (Tb BV/TV, pβ=β0.01), thickness (Tb Th, pβ=β0.04) and number (Tb N, pβ=β0.01). Cortical bone parameters of CyPB-deficient mice are also reduced, with reductions in cortical thickness (Ct Th, pβ=β0.003) and area (Ct Ar, pβ=β0.02). (B) <i>Left</i>, Representative load-displacement curve demonstrating differences between samples selected for median post-yield displacement. <i>Right</i>, CyPB-deficient femora are weaker in yield (Yd Load, pβ=β1.9Γ10<sup>β6</sup>), ultimate (Ult Load, pβ=β8.9Γ10<sup>β7</sup>) and failure loads (pβ=β2.1Γ10<sup>β5</sup>), with reduced stiffness (pβ=β0.001). <i>Ppib</i><sup>β/β</sup> femora are also more brittle than wild-type femora, as demonstrated by decreased post-yield displacement (PYD, pβ=β0.001). Reduced toughness of <i>Ppib</i><sup>β/β</sup> femora is evident by decreases in the elastic and plastic energy (E) values (pβ=β0.001 and 0.0003, respectively).</p
Post-translational modification of type I collagen lysine residues.
<p>*p<0.05 between +/+ and β/β;</p>#<p>p<0.05 between +/β and β/β;</p><p>no significant difference between +/+ and +/β in bone.</p
Post-translational modification of type I collagen from tissues.
<p>(A) Total lysyl hydroxylation of type I collagen is dramatically reduced in dermal tissue from <i>Ppib</i><sup>β/β</sup> mice (p<0.001 vs wild-type). (B) Post-translational hydroxylation and glycosylation of type I collagen lysyl residues in dermal tissue from <i>Ppib</i><sup>β/β</sup> mice. (C) SDS-Urea PAGE analysis of pepsin extracts from dermal tissue demonstrates increased electrophoretic migration of <i>Ppib</i><sup>β/β</sup> type I collagen alpha chains compared to wild-type. Arrows indicate faster migrating alpha chains. (D) Total lysyl hydroxylation of type I collagen extracted from bone tissue of heterozygous (+/β) and homozygous (β/β) <i>Ppib</i>-null mice compared to wild-type (+/+) bone collagen. Both <i>Ppib</i><sup>+/β</sup> and <i>Ppib</i><sup>β/β</sup> bone collagen show increased Hyl compared to wild-type (pβ=β0.0002 and 0.005, respectively). (E) Analysis of post-translational lysine hydroxylation and glycosylation in <i>Ppib</i><sup>β/β</sup> bone-derived type I collagen demonstrates increased galactosyl-hydroxylysine (G-HYL) content compared to wild-type (p<0.001). (F) Type I collagen extracted from bone tissue displays backstreaking of Ξ±1(I) chains on SDS-Urea, indicated by arrow, and is consistent with post-translational overmodification. Arrowhead indicates a truncated form of Ξ±1(V) chains due to pepsin sensitivity.</p
Cyclophilin B catalyzes folding of type I collagen.
<p>(A) Assay for intracellular folding of type I collagen in fibroblast cultures. (B) Assay for intracellular folding of type I collagen in calvarial osteoblast cultures. Data represents the average from two independent cell lines for each genotype.</p
Expression of ER resident collagen helical lysine modification enzymes.
<p>(AβC) Real-time RT-PCR of total RNA from two independent cell cultures of newborn fibroblasts (FB) and calvarial osteoblasts (OB), and 5 independent skin and femoral samples for each genotype at 8 weeks of age. (D) Western blots of cell lysates probing for lysyl hydroxylase 1 (LH1/PLOD1), lysyl hydroxylase 3 (LH3/PLOD3), glycosyltransferase 25 domain containing 1 (GLT25D1), and Ξ²-actin in wild-type (+/+), heterozygous (+/β) and homozygous (β/β) <i>Ppib</i>-null cells and tissues.</p