Molecular Consequences of the SERPINH1/HSP47 Mutation in the Dachshund Natural Model of Osteogenesis Imperfecta

Abstract

Osteogenesis imperfecta (OI) is a heritable connective tissue disease characterized by bone fragility and increased risk of fractures. Up to now, mutations in at least 18 genes have been associated with dominant and recessive forms of OI that affect the production or post-translational processing of procollagen or alter bone homeostasis. Among those, SERPINH1 encoding heat shock protein 47 (HSP47), a chaperone exclusive for collagen folding in the ER, was identified to cause a severe form of OI in dachshunds (L326P) as well as in humans (one single case with a L78P mutation). To elucidate the disease mechanism underlying OI in the dog model, we applied a range of biochemical assays to mutant and control skin fibroblasts as well as on bone samples. These experiments revealed that type I collagen synthesized by mutant cells had decreased electrophoretic mobility. Procollagen was retained intracellularly with concomitant dilation of ER cisternae and activation of the ER stress response markers GRP78 and phospho-eIF2α, thus suggesting a defect in procollagen processing. In line with the migration shift detected on SDS-PAGE of cell culture collagen, extracts of bone collagen from the OI dog showed a similar mobility shift, and on tandem mass spectrometry, the chains were post-translationally overmodified. The bone collagen had a higher content of pyridinoline than control dog bone. We conclude that the SERPINH1 mutation in this naturally occurring model of OI impairs how HSP47 acts as a chaperone in the ER. This results in abnormal post-translational modification and cross-linking of the bone collagen.

Keywords: SERPINH1; bone; collagen; connective tissue; cross-links; endoplasmic reticulum stress (ER stress); extracellular matrix; heat shock protein 47; osteogenesis.

FIGURE 1.

Mutant HSP47 is expressed as a stable protein and localizes to the ER. A and B, Western blot of HSP47 in primary cultured fibroblasts of mutant (OI) and a control dog (Contr. 1). The mutant protein level is reduced to half of the control level. Proteasome inhibition by treatment with MG-132 (50 μm, 16 h) does not influence the amount of either mutant or wild-type protein. Error bars indicate ± S.D. C, immunofluorescent staining of HSP47 in primary cultured fibroblasts of mutant and control dog. The protein is similarly detectable in both mutant and control fibroblasts and co-localizes with the endoplasmic reticulum marker GRP78. Cell nuclei stained with DAPI are shown in blue.

FIGURE 2.

Overmodification of type I collagen and intracellular retention of types I, III, and V collagen in HSP47 mutant fibroblasts. A and B, steady-state analysis of pepsinized (A) and unpepsinized (B) procollagen by SDS-PAGE in primary cultured fibroblasts of HSP47 mutant (OI) and a control dog (Contr. 1). The proα1(I) and proα2(I) (in panel B) as well as the α1(I) and α2(I) (in panel A) bands from mutant fibroblasts show decreased electrophoretic mobility when compared with the control, indicating overmodification of the triple helical and possibly the telopeptide region. Proα1(I) and proα2(I) are retained in the cell layer. Also, type III and type V collagen are retained in the cell layer as evident in the pepsinized collagen analysis (A).

FIGURE 3.

Delayed secretion of type I and type V collagen. A, pulse-chase analysis of pepsinized collagen in primary fibroblasts showing a delay in secretion of type I and type V collagen from the cell layer (C) into the medium (M) in the OI dog (OI) when compared with the control dog (Contr. 1). B, quantification of the pulse-chase experiment for type I collagen.

FIGURE 4.

Type I collagen accumulates in HSP47 mutant fibroblasts. A and B, co-immunofluorescent staining of primary skin fibroblasts for Col I (green) and the ER marker GRP78 (A) or the Golgi marker GM130 (B), both shown in red. The two control cell lines (Contr. 1 and 2) show weak collagen I staining overlapping with the Golgi marker. In HSP47 mutant fibroblasts (OI), collagen I staining is much stronger and more spread out overlapping with both the ER marker and the Golgi marker.

FIGURE 5.

Dilation of endoplasmic reticulum cisternae in HSP47 mutant fibroblasts. Transmission electron microscopy of primary fibroblasts from HSP47 mutant dog (OI) shows increased abundance of enlarged endoplasmic reticulum cisternae (red arrows) when compared with control dog fibroblasts.

FIGURE 6.

ER stress markers are up-regulated in HSP47 mutant fibroblasts. A–D, Western blot analysis (A and C) and quantification (B and D) of three independent experiments of GRP78 and phospho-eIF2α (P-eIF2α). The amount of both ER stress marker proteins is increased in untreated (-) HSP47 mutant fibroblasts (OI) when compared with the level in control cells (Contr. 2). Induction of ER stress by treating cells with thapsigargin (Thap.) leads to an increase in control cells, whereas remaining on a high level in HSP47 mutant cells. GRP78 is detected as a double band, probably due to post-translational modification. Error bars indicate ± S.D.

FIGURE 7.

Decreased electrophoretic mobility of type I collagen chains from HSP47 mutant bone and skin. SDS-PAGE (6%) of type I collagen extracted by heat denaturation or pepsin from HSP47 mutant (OI) bone and heat denaturation of skin shows decreased electrophoretic mobility of the α and β chains when compared with the 9-year-old control dog (Control).

FIGURE 8.

Overmodification of lysine residues at cross-linking sites in HSP47 mutant bone collagen revealed by tandem mass spectrometry. A, peptides from the four molecular sites of cross-linking in collagen type I prepared by bacterial collagenase digestion of decalcified bone were identified by LC-MS. Their post-translational profiles are compared between HSP47 mutant and control (9-year-old dog) bone. N-telo, N-telopeptide; C-telo, C-telopeptide. B and C, spectra from the two helical sites (Lys-87 and Lys-930) (B) and two telopeptide sites (C) show that Lys-87 is mostly glcgalHyl, Lys-930 is mostly Hyl, and both telopeptides are essentially all Hyl from mutant bone, whereas from control bone Lys-87 is mostly galHyl, Lys-930 is highly underhydroxylated, and both telopeptides are about 50% hydroxylated.

FIGURE 9.

Proposed pathomechanism of OI induced by the HSP47 mutation in the dog. Symbols used are: green spheres, HSP47; yellow triangles, hydroxylysyl residues; gray circles, glucosyl-galactosyl residues. Decreased binding of HSP47 to the collagen triple helix delays collagen folding overall and allows for local micro-unfolding of the triple helix after its formation, thus leading to an increased exposure to lysyl-hydroxylases and glucosyl-galactosyltransferases and to increased post-translational modification of the triple helix and the telopeptides involving the cross linking sites (87 and 930, N-telopeptide and C-telopeptide). A portion of overmodified collagen is transported from the rough ER (rER) into the Golgi, and afterward into the extracellular space, whereas a portion of it accumulates in the rough ER, inducing ER stress. The secretion of overmodified collagen molecules to the extracellular space leads to abnormal collagen cross-linking in bone, i.e. an increase of HP and a decrease of pyrrole cross-links (gray vertical bar, pyrrole; dark gray vertical bar, HP; light gray vertical bar, LP). Both induction of ER stress and altered collagen cross-linking may contribute to the pathomechanism of OI.