Figure 1.
Generation of a murine model of Cyclophilin B deficiency.
(A) Left, Diagram of knock-out allele containing β-geo reporter construct inserted into intron 1 of Ppib gene. Hemi-nested primers (arrows) for PCR-based genotyping selectively amplify the wild-type allele (255 bp) or the gene-trapped allele (364 bp). Right, Genotypes in offspring of matings between mice heterozygous for the gene-trapped allele are noted as wild-type (+/+), heterozygous (+/−) or homozygous (−/−). (B) Verification of Ppib expression by Real-time RT-PCR using total RNA isolated from primary cultures and tissues from newborns and 8-week mice, respectively. Values represent the average of two independent fibroblast and osteoblast cultures, and five independent dermal and femoral samples. (C) Lengths of femora and tibiae from 8 week-old wild-type (+/+), heterozygous (+/−) and homozygous (−/−) knock-out mice. Rhizomelia was not detected. (D) Growth curves of male and female mice from weaning (4 weeks) to 24 weeks of age.
Figure 2.
Absence of Ppib expression affects bone development.
(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 (n = 10/genotype).
Figure 3.
Whole bone structural and mechanical properties.
(A) Structural parameters of wild-type (+/+) and homozygous (−/−) femora at 8 weeks of age characterize reduced bone formation in CyPB-deficient mice (n = 9/genotype). Left, 3D reconstructions illustrate reduced trabecular and cortical bone volumes. Right, Trabecular parameters are decreased in homozygous (−/−) femora, including reduced bone volume (Tb BV/TV, p = 0.01), thickness (Tb Th, p = 0.04) and number (Tb N, p = 0.01). Cortical bone parameters of CyPB-deficient mice are also reduced, with reductions in cortical thickness (Ct Th, p = 0.003) and area (Ct Ar, p = 0.02). (B) Left, Representative load-displacement curve demonstrating differences between samples selected for median post-yield displacement. Right, CyPB-deficient femora are weaker in yield (Yd Load, p = 1.9×10−6), ultimate (Ult Load, p = 8.9×10−7) and failure loads (p = 2.1×10−5), with reduced stiffness (p = 0.001). Ppib−/− femora are also more brittle than wild-type femora, as demonstrated by decreased post-yield displacement (PYD, p = 0.001). Reduced toughness of Ppib−/− femora is evident by decreases in the elastic and plastic energy (E) values (p = 0.001 and 0.0003, respectively).
Figure 4.
(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 (+/−) and homozygous (−/−) fibroblasts (FB) and osteoblasts (OB). (C) Differential scanning calorimetry (DSC) analysis reveals no differences in thermal stability (Tm) of type I collagen secreted by fibroblast cultures.
Table 1.
Type I collagen post-translational hydroxylation.
Figure 5.
Cyclophilin B catalyzes folding of type I collagen.
(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.
Figure 6.
Post-translational modification of type I collagen from tissues.
(A) Total lysyl hydroxylation of type I collagen is dramatically reduced in dermal tissue from Ppib−/− mice (p<0.001 vs wild-type). (B) Post-translational hydroxylation and glycosylation of type I collagen lysyl residues in dermal tissue from Ppib−/− mice. (C) SDS-Urea PAGE analysis of pepsin extracts from dermal tissue demonstrates increased electrophoretic migration of Ppib−/− 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 (+/−) and homozygous (−/−) Ppib-null mice compared to wild-type (+/+) bone collagen. Both Ppib+/− and Ppib−/− bone collagen show increased Hyl compared to wild-type (p = 0.0002 and 0.005, respectively). (E) Analysis of post-translational lysine hydroxylation and glycosylation in Ppib−/− 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 α1(I) chains on SDS-Urea, indicated by arrow, and is consistent with post-translational overmodification. Arrowhead indicates a truncated form of α1(V) chains due to pepsin sensitivity.
Figure 7.
Altered post-translational modification of specific type I collagen lysine residues in the absence of Ppib.
Quantitation of type I collagen modifications at α1(I) and α2(I) K87 residues, which are important for crosslinking of type I collagen heterotypic fibrils in tissue. Results were obtained from collagen secreted in culture by primary osteoblasts, or from 3–5 independent tissue samples for each genotype. K, lysine; OH, hydroxylysine; G, galactosylhydroxylysine; GG, glucosylgalactosylhydroxylysine.
Table 2.
Post-translational modification of type I collagen lysine residues.
Figure 8.
Expression of ER resident collagen helical lysine modification enzymes.
(A–C) 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 β-actin in wild-type (+/+), heterozygous (+/−) and homozygous (−/−) Ppib-null cells and tissues.
Figure 9.
Pulse-chase analysis of type I collagen secretion.
There is a minimal delay in secretion of collagen by cyclophilin B-deficient fibroblasts and osteoblasts in culture.
Figure 10.
Dysregulation of collagen deposition and fibril assembly.
(A) Left, Deposition of type I collagen by osteoblasts into extracellular matrix in culture. Post-confluent cultures were pulsed for 24 hr, followed by serial extraction of incorporated collagens from the media (M), neutral salt (NS), acid soluble (AA, immaturely crosslinked) and pepsin soluble (P, maturely crosslinked) fractions of the matrix. Right, Matrix collagen to cell organics ratio from Raman micro-spectroscopy shows decreased collagen content in matrix deposited by homozygous Ppib-null (−/−) versus wild-type (+/+) osteoblasts in culture (p = 0.002). (B) Transmission electron micrographs of femoral and dermal collagen fibrils from 8 week-old wild-type (+/+) and CyPB-deficient mice (−/−). Diameters of 200 dermal fibrils were measured for each sample and plotted, right. (C) Quantitation of divalent crosslinks in murine humeri reveals increased HLNL (hydroxylysinonorleucine) crosslinks, which require helical lysine residues, but no change in DHLNL (dihydroxylysinonorleucine) crosslinks, which involve helical hydroxylysine residues. (D) Quantitation of trivalent crosslinks in murine humeri and femora. Total pyridinoline crosslinks are increased due to an increase in lysyl pyridinoline (LP), but not hydroxylysyl pyridinoline (HP) crosslinks in bone.