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.
Highlights
Recessive Osteogenesis imperfecta (OI) cases have been reported more recently and have been shown to be caused by mutations in the cartilage-associated protein (CRTAP), prolyl 3-hydroxylase 1 or leprecan (LEPRE1) gene and cyclophilin B (CypB) (4 –9)
These abnormalities are shown to be due to the loss of 3-hydroxyproline from key sites in collagen extracted from tissues, as well as due to the loss of the functions of the P3H11⁄7CRTAP1⁄7CypB complex during active collagen biosynthesis
Prolyl 3-hydroxylase 1 has been shown to be crucial for proper bone formation as human mutations result in a severe recessive bone disorder that resembles osteogenesis imperfecta [5,6, 10]
Summary
P3H1 Null Mice—P3H1 null mice were purchased from Deltagen (San Mateo, CA). Directed knockouts were created in which exons 1–3 (nucleotides 15– 817) of the mouse P3H1 or leprecan 1 gene (NCBI Reference sequence number: NM_019782.2) were deleted. X-ray Scans and Bone Mineral Density—X-rays were performed on adult mice using a Faxitron cabinet instrument (model #43855B) made by Hewlett Packard. Bone mineral measurements were performed by dual energy x-ray absorptiometry using the PIXImus instrument (Lunar Corp., Madison, WI). Electron Microscopy Analysis of Tendon and Skin—Freshly obtained tissues were fixed in cacodylate-buffered 1.5% glutaraldehye/1.5% paraformaldehyde containing 0.05% tannic acid (w/v), rinsed, exposed to 1% osmium tetroxide, dehydrated in a grade series of ethanol to 100%. The tomography tilt series was acquired from a 350 nm thick section at a magnification of 29,000ϫ using an FEI Tecnai G2 transmission electron microscope operated at 200 KV. Solution was centrifuged to remove insoluble material, NaCl was added to a final concentration of 0.7 M to precipitate collagen, and solution was incubated. Pellet was resolubilized in 0.1 M acetic acid, analyzed on SDS-PAGE gels, and lyophilized for further digestion and analysis
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