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. 2010 May 28;285(22):17253-62.
doi: 10.1074/jbc.M110.102228. Epub 2010 Apr 2.

Prolyl 3-hydroxylase 1 null mice display abnormalities in fibrillar collagen-rich tissues such as tendons, skin, and bones

Janice A Vranka  1 , Elena Pokidysheva, Lauren Hayashi, Keith Zientek, Kazunori Mizuno, Yoshihiro Ishikawa, Kerry Maddox, Sara Tufa, Douglas R Keene, Robert Klein, Hans Peter Bächinger
Affiliations

Prolyl 3-hydroxylase 1 null mice display abnormalities in fibrillar collagen-rich tissues such as tendons, skin, and bones

Janice A Vranka et al. J Biol Chem. .

Erratum in

  • J Biol Chem. 2010 Jun 25;285(26):20421

Abstract

Osteogenesis imperfecta (OI) is a skeletal disorder primarily caused by mutations in the type I collagen genes. However, recent investigations have revealed that mutations in the genes encoding for cartilage-associated protein (CRTAP) or prolyl 3-hydroxylase 1 (P3H1) can cause a severe, recessive form of OI. These reports show minimal 3-hydroxylation of key proline residues in type I collagen as a result of CRTAP or P3H1 deficiency and demonstrate the importance of P3H1 and CRTAP to bone structure and development. P3H1 and CRTAP have previously been shown to form a stable complex with cyclophilin B, and P3H1 was shown to catalyze the 3-hydroxylation of specific proline residues in procollagen I in vitro. Here we describe a mouse model in which the P3H1 gene has been inactivated. Our data demonstrate abnormalities in collagen fibril ultrastructure in tendons from P3H1 null mice by electron microscopy. Differences are also seen in skin architecture, as well as in developing limbs by histology. Additionally bone mass and strength were significantly lower in the P3H1 mice as compared with wild-type littermates. Altogether these investigations demonstrate disturbances of collagen fiber architecture in tissues rich in fibrillar collagen, including bone, tendon, and skin. This model system presents a good opportunity to study the underlying mechanisms of recessive OI and to better understand its effects in humans.

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Figures

FIGURE 1.
FIGURE 1.
Reduced body size and x-ray analysis of mice. P3H1 knock-out mice typically appear smaller in size (A). Wild-types (B, black curve), heterozygotes (B, red curve), and homozygotes (B, blue curve) weights are plotted as a function of age with standard deviations shown. Null mice are considerably smaller already at 3 weeks of age and stay smaller through the lifetime. Full body x-rays (C) show pronounced curvature of the vertebrae and thinner skull of knock-out mice as compared with age- and sex-matched wild-type mice. The difference is obvious at 3 month age but progresses further in life as seen in the x-rays of 12 month old mice (C, right side). Hind limb x-rays (D) of P3H1 null mice show decreased bone mineral density as well as a foreshortening of the proximal bone relative to the distal bone in comparison with wild-type hind limb.
FIGURE 2.
FIGURE 2.
Tendon fibrils of P3H1 null mice compared with wt mice. Electron microscope images of tail tendon fibrils from adult wild-type (A and C) and adult P3H1 null mice (B and D). Cross-sections of the collagen fibrils (A and B) show a more heterogeneous distribution of fibril diameters as well as some disturbances in the overall shape of some of the fibrils in the P3H1 null mice as compared with the wild-type fibrils. Longitudinal sections of the tendon collagen fibrils (C and D) show an alternating pattern of very small fibrils interspersed with very large fibrils as well as abnormal branching at the ends of some of the P3H1 null fibrils as compared with the wild-type fibrils. (Scale bar in D represents 500 nm and is the same for images A–D.) Tendon fibril size versus number of fibrils measured was graphed (E) and demonstrates a shift in the size distribution from 100 nm to 350 nm in the wild-type to primarily 0–100 nm in the null (n = 1000 for wt and n = 1000 for P3H1 null). F, a projection through a 350 nm thick section of longitudinally sectioned P3H1 null tendon from which the tomogram (supplemental Fig. S1) was collected. Arrows point to regions in the tendon in which thicker fibrils branch into thinner fibrils and where axial twists are evident.
FIGURE 3.
FIGURE 3.
Skeleton preparations of E18 mice. Skeletons of E18 mice were stained with alizarin red and Alcian blue to detect differences in mineralized bone and cartilage, respectively (A and B). The primary difference detected at this developmental stage is the lack of ossification in the parietal bone in the skull of null mice (B) as compared with heterozygous mice (A) indicating a delay in ossification in the P3H1 null mice. (Note: bone mineral density in heterozygous mice was indistinguishable from that of wild-type mice (see Table 2)).
FIGURE 4.
FIGURE 4.
Histochemistry of hind limbs. Newborn (P0) hind limbs were cryosectioned and stained with Masson trichrome stain to detect collagen fibrils (blue) in skin of wild-type (A) and P3H1 null mice (B). Collagen fibril staining in the skin of newborn null mice was less intense and less dense suggesting an overall decrease in the amount of collagen in the dermis. Sections of P1 fore limbs were also stained with hematoxylin and eosin to look at the overall organization of the chondrocytes in wild-type (C) and null (D) radial cartilage. The hypertropic zone in the P3H1 null limb (D) appears to be severely affected compared with the same region in the wild-type limb (C). Newborn hind limb sections were also stained with Von Kossa stain to detect mineralized bone (E and F). P3H1 null femurs (F) have less mineralized bone in the trabeculae with more spaces throughout as compared with wild-type femurs (scale bar = 120 μm).
FIGURE 5.
FIGURE 5.
Skin of P3H1 null mice. Electron microscope images of adult wild-type (A and C) and P3H1 null mouse skin (B and D) were analyzed and show abnormalities in the reticular dermis of the null skin collagen fibrils where many irregular, fused collagen fibrils are found (arrows in B), as well as more spaces between fibril bundles (D) as compared with wild-type skin (scale bar = 2 μm). Cryosections of adult skin were also stained with Masson trichrome stain to detect collagen fibrils in wild-type (E) and null (F) dermis (scale bar = 120 μm). P3H1 null skin was overall thinner and slightly less dense than the wild-type skin.
FIGURE 6.
FIGURE 6.
3-hydroxyproline in wt and P3H1 null mice. SDS-PAGE of extracted and pepsin digested wild-type and P3H1 null collagen I from tail tendon (A) shows a mobility shift in P3H1 null collagen as compared with wild-type indicating overmodified collagen molecules. Mass spectrometry data (B) of the trypsin fragment containing the single 3-hydroxyproline site in the alpha 1 chain of collagen I show the presence of 3-hydroxyproline at proline 986 in the wild type, but it is not present at the same location in the P3H1 null mouse collagen. O, 4(R)-hydroxyproline; Z, 3(S)-hydroxyproline in the indicated sequences.
FIGURE 7.
FIGURE 7.
Thermal stability of type I collagen. Temperature scan of type I collagen extracted from mutant and wild-type tail tendons. The circular dichroism signal was monitored at 221 nm, and the rate of heating was 10 °C/h. The curves for wild-type (solid line) and mutant (dashed line) type I collagen are shown.
FIGURE 8.
FIGURE 8.
Secretion of type I collagen. Collagen secretion rate assay was performed using wild-type and P3H1 null primary mouse skin fibroblasts. Cell numbers were equalized prior to labeling, and results demonstrate a delay in collagen secretion in the null cells by at least 20 min and a lesser total amount of labeled collagen in P3H1 null fibroblasts. The secretion rate of type I collagen is represented graphically and shows maximal secretion at 60 min for the wild-type collagen (solid line with filled square) and 120 min for the null mouse collagen (dotted line with open circle).
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References

    1. Sillence D. O., Senn A., Danks D. M. (1979) J. Med. Genet. 16, 101–116 - PMC - PubMed
    1. Rauch F., Glorieux F. H. (2004) Lancet 363, 1377–1385 - PubMed
    1. Royce P. M., Steinmann B. (eds) (2002) Connective Tissue and Its Heritable Disorders: Molecular, Genetic, and Medical Aspects, pp. 385–430, Wiley-Liss, New York
    1. Morello R., Bertin T. K., Chen Y., Hicks J., Tonachini L., Monticone M., Castagnola P., Rauch F., Glorieux F. H., Vranka J., Bächinger H. P., Pace J. M., Schwarze U., Byers P. H., Weis M., Fernandes R. J., Eyre D. R., Yao Z., Boyce B. F., Lee B. (2006) Cell 127, 291–304 - PubMed
    1. Baldridge D., Schwarze U., Morello R., Lennington J., Bertin T. K., Pace J. M., Pepin M. G., Weis M., Eyre D. R., Walsh J., Lambert D., Green A., Robinson H., Michelson M., Houge G., Lindman C., Martin J., Ward J., Lemyre E., Mitchell J. J., Krakow D., Rimoin D. L., Cohn D. H., Byers P. H., Lee B. (2008) Hum. Mutat. 29, 1435–1442 - PMC - PubMed

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