The molecular chaperone Hsp47 is essential forcartilage and endochondral bone formation

Yusaku Masago1,2, Akihiro Hosoya3, Kunito Kawasaki1,2, Shogo Kawano4, Akira Nasu5,6, Junya Toguchida5,Katsumasa Fujita7, Hiroaki Nakamura3, Gen Kondoh8 and Kazuhiro Nagata1,2,*1Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan2Laboratory of Molecular and Cellular Biology, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo, Kita-ku Kyoto, 603-8555, Japan3Department of Oral Histology, Matsumoto Dental University, Shiojiri, 399-0781, Japan4Department of Frontier Biosciences, Osaka University, Osaka, 565-0871, Japan5Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan6Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto, 606-8507, Japan7Department of Applied Physics, Osaka University, Osaka, 565-0871, Japan8Laboratory of Animal Experiments for Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan

*Author for correspondence (nagata@cc.kyoto-su.ac.jp)

Accepted 12 August 2011Journal of Cell Science 125, 1118–1128� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.089748

SummaryHeat shock protein 47 kDa (Hsp47) is considered as a molecular chaperone essential for the correct folding of type I and type IVprocollagen in the ER. However, the function of Hsp47 for other types of procollagen and its importance for chondrogenesis have neverbeen elucidated. To examine the function of Hsp47 in cartilage formation and endochondral ossification, we conditionally inactivatedthe Hsp47 gene in chondrocytes using Hsp47 floxed mice and mice carrying a chondrocyte-specific Col2a1–Cre transgene. Hsp47

conditional null mutant mice died just before or shortly after birth, and exhibited severe generalized chondrodysplasia and bonedeformities with lower levels of type II and type XI collagen. Second-harmonic generation (SHG) analysis and electron microscopyrevealed the accumulation of misaligned type I collagen molecules in the intervertebral discs and a substantial decrease in type II

collagen fibers, respectively. Whole-mount skeletal staining showed no calcified region in the vertebral bodies of sacral vertebrae, andrevealed that the endochondral bones were severely twisted and shortened. These results demonstrate that Hsp47 is indispensable forwell-organized cartilage and normal endochondral bone formation.

Key words: Collagen, Hsp47, Cartilage, Intervertebral disc, Endochondral bone

IntroductionCartilage and bone are primarily composed of type II and type I

collagen bundles, respectively, with covalently bound collagen

fibers (Eyre et al., 1986; Wu et al., 1987; Wu et al., 1992; Knott and

Bailey, 1998; Chung et al., 2008). Collagen fibers form fibrillar

networks in cartilage and interact with various extracellular matrix

(ECM) components, including aggrecan and hyaluronan (Yaeger

et al., 1997; Cortes et al., 2009; Matsumoto et al., 2009). Bones in

mammals, except for the cranial, clavicle and some facial bones,

are formed by endochondral ossification, a dynamic process in

which cartilage is newly synthesized by chondrocytes, followed by

the formation of hypertrophic cartilage after the differentiation of

chondrocytes into hypertrophic chondrocytes, and finally bone

formation by ossification of the hypertrophic cartilage region

(Stickens et al., 2004; Mackie et al., 2008).

As many as 28 types of mammalian collagen have been

reported and classified on the basis of their supramolecular

structures: fibrillar collagens including types I, II, III, V, XI,

XXIV and XXVII; fibril-associated collagens with interrupted

triple helices (FACIT) including types IX, XII, XIV, XVI, XIX,

XX, XXI, XXII and XXVI; collagen with transmembrane

domains, types XIII, XVII, XXIII and XXV; network-forming

collagen composed of types VIII and X; basement membrane

collagen of type IV; and other types of collagen (Van der Rest

and Garrone, 1991; Prockop and Kivirikko, 1995; Hubert et al.,

2009). Collagen is composed of three a-chains, each of which

contains characteristic Gly-x-y triplet repeats in their amino acid

sequence. The three a-chains assemble at the C-terminus in the

endoplasmic reticulum (ER) and form a triple helix structure

from the C-terminus to the N-terminus (Bachinger et al., 1980;

Engel and Prockop, 1991; Bulleid et al., 1997). The majority of

the proline residues at the Y position of the Gly-x-y triplet are

hydroxylated by prolyl 4-hydroxylase (P4H), which stabilizes the

triple helices of procollagen. Mutations in fibrillar collagen genes

have been reported to cause osteodysplasias such as osteogenesis

imperfecta (OI), which is mainly the result of mutations in the

COL1A1 and COL1A2 genes. In addition, mutations in cartilage-

associated protein (CRTAP), 65 kDa FK506-binding protein

(FKBP65 also known as FKBP10; a member of the collagen

chaperone complex) and prolyl-3-hydroxylase-1 (P3H1, encoded

by the LEPRE1 gene) result in OI (Baldridge et al., 2008; Vranka

et al., 2004; Alanay et al., 2010). Mutations in collagen genes,

result in either quantitative deficiencies or sequence alterations

that lead to structural defects of the triple helix, altered secretion

and abnormal fibril formation and/or assembly (Rauch and

Glorieux, 2004; Marini et al., 2007).

Cartilage collagen fibrils are composed of type II collagen and

other quantitatively minor collagens, including types IX and XI

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(Eyre, 2002). Type II collagen is a homotrimer of a1(II) chains

encoded by a single gene, COL2A1. Defects in COL2A1 have

been identified in a phenotypic continuum of chondrodysplasias

that includes achondrogenesis type II, hypochondrogenesis,

spondyloepiphyseal dysplasia congenita, Kniest dysplasia and

Stickler syndrome (Spranger, 1988; Cole, 1994; Chan et al.,

1995). In addition, mutations in COL9A1 or COL11A2 have also

been linked to Stickler syndrome (Kuivaniemi et al., 1997).

During protein biosynthesis, a number of molecular

chaperones are involved in ensuring the correct folding of

protein in the cell (Hendrick and Hartl, 1993; Frydman et al.,

1994; Hartl and Hayer-Hartl, 2002). In the case of procollagen

biosynthesis in the ER, these molecular chaperones include BiP

(Grp78), Grp94, PDI, P4H and Hsp47 (Chessler and Byers, 1993;

Ferreira et al., 1994; Lamande et al., 1995; Wilson et al., 1998;

Walmsley et al., 1999; Nagata, 2003). Hsp47 was first identified

as a collagen binding protein that resides in the ER and functions

as a collagen-specific molecular chaperone (Nagata et al., 1986).

Hsp47 transiently associates with procollagen in the ER and

dissociates before reaching the cis-Golgi (Satoh et al., 1996).

Recent studies have revealed that the SERPINH1 gene, which

encodes human HSP47, is one of the genes responsible for

recessive OI (Christiansen et al., 2010). Our previous studies

showed that disruption of murine Hsp47 caused early embryonic

lethality at embryonic day (E) 11.5 with impaired basement

membrane formation (Nagai et al., 2000). Type I collagen

secreted from Hsp47 null cells formed abnormal fibrillar

structures with thin and branched fibrils (Ishida et al., 2006).

Thus, Hsp47 was established as essential for development due to

its role as a collagen-specific molecular chaperone in the ER, at

least for type I and type IV collagen maturation.

Here we report that chondrocyte-specific disruption of Hsp47

caused severe chondrodysplasia and defective endochondral bone

formation. Conditional null mutant mice, which died just before

or after birth, exhibited a marked decrease in type II and type XI

collagen accumulation in the cartilage. Thus, Hsp47 has an

essential role as a molecular chaperone for type II collagen, a

major component of cartilage.

ResultsGeneration of floxed Hsp47 mice

Previous results revealed that gene disruption of murine Hsp47

caused embryonic lethality at E11.5 (Nagai et al., 2000). To

examine the role of Hsp47 in the molecular maturation of

collagens at later stages of mouse development, a Cre-loxP

system was used to disrupt the Hsp47 gene in a tissue-specific

manner (Gu et al., 1994). To accomplish this, a transgenic mouse

harboring a floxed Hsp47 gene in which the exon-6-containing

region was flanked by loxP sites was established (Fig. 1A). Exon

6 is the final exon and encodes approximately 24% of the amino

acid sequence of Hsp47, including a region essential for binding

to collagen (T. Homma et al., personal communication) and a

poly(A) addition signal. Correct targeting by homologous

recombination and germline transmission was confirmed by

Southern blotting (Fig. 1B), PCR and genomic sequencing

around the loxP site (data not shown). Heterozygous and

homozygous mice were viable and fertile without noticeable

phenotypic differences between littermates on a mixed genetic

Fig. 1. Targeting strategy for conditional inactivation

of the Hsp47 gene. (A) Strategy to generate a conditional

allele for Hsp47. Structure of the genomic Hsp47 locus,

targeting vector and the homologous recombined allele.

Exons are depicted as black numbered boxes, and intronic

sequences are shown as solid lines. The PGK-neo cassette

is depicted as a white box. Forward and reverse primers

used for PCR genotyping for floxed alleles are shown as

arrows. Neo, neomycin; DTA, diphtheria toxin A.

(B) Southern blot analysis of fetal genomic DNA.

Genomic DNA isolated from the skin of wild-type,

Hsp47flox/wt and Hsp47flox/flox mice were digested with

BamHI and then hybridized with the 39 prove. The wild-

type and floxed alleles were detected as 9.4-kb and 2.9-kb

fragments, respectively. (C) Genotyping of wild-type,

Hsp47flox/wt and Hsp47flox/flox mice from tail DNA by

genomic PCR shows germline transmission.

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background (129SvEvTaq/C57BL6). Mice were used forexperiments after backcrossing with C57BL/6 mice for at least

two generations. Crossing the mice harboring the Hsp47 flox allelewith mice harboring Cre recombinase in many cell types, includingfibroblasts and osteoblasts (Col1a1–Cre), resulted in embryonic

lethality of Hsp47flox homozygous mice harboring Col1a1–Cre

(data not shown), similar to Hsp47 null mice (Hsp47–/–) (Nagaiet al., 2000). These results indicated that Hsp47 floxed mice couldbe used for tissue-specific disruption of Hsp47.

Phenotypic changes induced by chondrocyte-specificdisruption of Hsp47

To examine the role of Hsp47 during chondrogenesis, a conditionalknockout of Hsp47 in chondrocytes was created by crossing Hsp47

floxed mice with Col2a1–Cre transgenic mice expressing Crerecombinase in chondrocytes (Ovchinnikov et al., 2000), resulting inheterozygous and homozygous Hsp47flox mice carrying the Col2a1–

Cre transgene. The heterozygous Hsp47flox mice carrying Col2a1–

Cre showed no phenotypic abnormalities, similar to Hsp47

heterozygous knockout (KO) mice (Nagai et al., 2000). However,Hsp47 conditional knockout mice (Hsp47 cKO: Hsp47flox/flox;

Col2a1–Cre) exhibited marked phenotypic changes. Approximately8% of the Hsp47 cKO mice were embryonic lethal at approximatelyE18.5 because of severe amnionic bleeding (n537). Conversely, 92%

of Hsp47 cKO mice died either just before or within 2 hours afterbirth. This could have been because of their cleft palate and breathingdifficulties, resulting from their abnormal rib cage, narrowed airway

and abnormal bronchial cartilage (see supplementary material Fig.S1L, arrow). Hsp47 cKO neonates exhibited severely twisted anddeformed limbs, some of which bled in the joint region (Fig. 2A,arrowheads). The body weights of cKO mice were ,17% less than

control (Hsp47flox/flox) littermates.

To examine skeletal abnormalities in cKO mice, micro-

computed tomography (m-CT) examination was performed onneonatal mice. The maxilla, mandible, basilar bone, rib, humerus,femur and all the vertebral bodies were visible in newborncontrol mice by m-CT scanning (Fig. 2B, left). By contrast, only

the maxilla, mandible and part of the cervical spine were visiblein Hsp47 cKO mice by m-CT scanning (Fig. 2B, right),suggesting the impairment of normal bone formation.

The effects of Hsp47 gene disruption on chondrogenesisand bone formation

Skeletal staining of E15.0 and E18.5 Hsp47 cKO mouse embryosrevealed severe systemic chondrodysplasia and deformities of the

long bones (Fig. 3B,D). The vertebral bodies of the thoracic,lumbar, sacral and caudal vertebrae of cKO mice were notossified, but ectopic ossification were observed in cervicalvertebrae (Fig. 3F, arrow; supplementary material Fig. S1). And

the sacral vertebral arches of cKO mice failed to fuse (Fig. 3H,arrowhead), whereas the counterparts in control mice wereconnected by blue-stained cartilaginous bridges (Fig. 3G). The

rib cartilage, humerus, radius, ulna, femur, tibia and fibula wereconsiderably shorter and twisted (Fig. 3). The ossified region ofthe humerus of cKO skeletal preparations were shorter by ,84%

compared with E18.5 control embryos. In addition, cKO embryoshad short snouts and partially uncalcified foreleg phalanges.However, the size of the cranial bone was similar to that of the

control and appeared to be normally mineralized (Fig. 3K).Taken together, endochondral ossification in Hsp47 cKO micewas substantially impaired. Tissue sections of E14.5–18.5 and

neonates revealed abnormal curvature of the spines in Hsp47

cKO mice; in particular, the cervical, lumbar and sacral spine

were severely twisted, leading to kyphosis (Fig. 4H). In Hsp47

cKO mice, the notochords remained as rod-like axial structures atE14.5 (Fig. 4B), whereas they formed nucleus pulposus and

disappeared in the vertebral bodies of sacral vertebrae in the

control mice (Fig. 4A).

In control mice, the elbow joints contained distinct acellularjoint cavities (Fig. 5A), whereas elbow joint cavities were not

observed in Hsp47 cKO mice (Fig. 5B). Similar phenotypes were

also observed in other joints of Hsp47 cKO mice. The long bones,

such as the humerus, of Hsp47 cKO mice were also severely

disorganized.

Conditional deletion of Hsp47 occurred in E14.5 Hsp47cKO cartilage

To examine the expression level of collagen and Hsp47 mRNAs,

we performed in situ hybridization using antisense riboprobes of

Col1a1, Col2a1 and Hsp47 on frozen sections of E14.5 lumbarvertebrae. The absence of Hsp47 mRNA from most chondrocytes

of E14.5 Hsp47 cKO mice was clear, and a small number of

Hsp47-positive cells was observed inside of perichondrium

(Fig. 6A). Col2a1 mRNA was strongly expressed in the

Fig. 2. Dysmorphism of endochondral bones in Hsp47 cKO mice. Control:

Hsp47flox/flox; Hsp47 cKO: Hsp47flox/flox; Col2a1-Cre. (A) Gross appearance

of mutant animals at E12.0, E18.5 and P0. All limbs are severely twisted and

some of the Hsp47 cKO mice have bled in the shoulder, elbow and knee joints

(arrowheads). (B) Micro-CT scans of newborn mice. Note in Hsp47 cKO

mice the severe ossification defects of endochondral bones, and there is no

well-organized vertebral column, rib cage or long bones of limbs.

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chondrocytes of control and Hsp47 cKO mice. Interestingly,

Col1a1 mRNA was strongly upregulated only in the vertebral

column of Hsp47 cKO mice. Additionally, the results of a

TUNEL assay revealed that apoptosis was induced in the

chondrocytes of Hsp47 cKO mice (Fig. 6B).

Collagen accumulation was lower in the cartilage invertebrae of cKO mice

Immunohistological staining was performed on frozen sagittal

sections of vertebrae from E18.5 embryos. Analysis of these

sections revealed severe chondrodysplasia in Hsp47 cKO mice.

Type II and type XI collagen were robustly stained in the annulus

fibrosus, IVDs and vertebral bodies of control mice at E18.5, and

thick collagenous fibrotic structures were well developed in these

tissues (Fig. 7C,G). Strong Hsp47 staining was also observed in

chondrocytes of the IVDs (Fig. 7A,E). By contrast, the

accumulation of type II and type XI collagen in the cartilage of

Hsp47 cKO mice was substantially reduced (Fig. 7D,H).

Extracellular accumulation of type II and type XI collagen was

impaired in the chondrocytes of Hsp47 cKO mice, and previous

results suggested that these collagens accumulated in the ER

(Ishida et al., 2009). This was confirmed by measuring the

induction of the molecular chaperone BiP, an indicator of ER

stress (Haze et al., 1999; Bertolotti et al., 2000), in the IVDs of

Hsp47 cKO mice (Fig. 4J).

Effect of Hsp47 disruption on collagen fiber formation

When laser light is focused on highly organized birefringent

materials, such as correctly aligned type I collagen fibers or

myosin filaments in animals, laser light with double the frequency

is emitted from the material, a phenomenon called optical second-

harmonic generation (SHG) (Williams et al., 2005). However, an

SHG signal is barely detectable when scattered from nonlinear

media such as heat-denatured or degraded collagen fibers

(Theodossiou et al., 2006). Recently, SHG imaging has been

used to study collagen fibers in cartilage, hard tissue and some

tumors (Brown et al., 2003; Yeh et al., 2005).

There were two possible effects of the disruption of Hsp47

gene expression in chondrocytes on collagen secretion and/or

accumulation in the extracellular matrix. One was a decrease in

the accumulation of extracellular collagen as a result of the

retention of misfolded procollagen in the ER, and the other was

the disordered alignment of fibrillar collagen in the extracellular

matrix as a result of the structurally abnormal collagen, as occurs

with type I collagen (Ishida et al., 2006). SHG imaging was used

to determine whether the fibrillar alignment of collagen in the

bone and fibrocartilage was disordered. Frozen serial sections

from the IVDs and spongy bones of control neonates generated

strong SHG signals (Fig. 7M). However, the SHG signal emitted

from the intervertebral disc (IVD) of Hsp47 cKO mice was

,99% lower (Fig. 7N, see the red boxed region and bar graph)

than that of the control with equal amounts of type I collagen

(Fig. 7K,L). Thus, SHG imaging revealed that collagen

molecules in the IVDs of Hsp47 cKO mice were severely

misaligned and failed to form robust connective tissue. This

clearly shows for the first time that disruption of Hsp47 impairs

fibrinogenesis of collagens in fibrocartilage tissues during

development.

Fig. 3. Severe, generalized chondrodysplasia in Hsp47 cKO mice. Skeletal preparations of (A,B) E15.0 and (C–L) E18.5 embryos. (A) Hsp47flox/flox control,

(B) Hsp47 cKO (Hsp47flox/flox; Col2a1–Cre), (C, E, G and left part of I–L) control and (D,F, H, right part of I–L) Hsp47 cKO mice. Skeletons stained with Alcian

Blue and Alizarin Red. (A–D) Gross morphology. (E,F) A ventral view of the sacral regions. Note there is no ossified sacral vertebral body judging from Alizarin

Red S stain in the Hsp47 cKO embryo (arrow in F), suggesting defects in mineralization of endochondral bones. (G,H) Lateral views of the sacral vertebrae.

Vertebral arches of cKO mice failed to fuse (arrowhead in H). (I) skulls; (J) rib cages; (K) left forelimbs; (L) left hindlimbs. Inactivation of Hsp47 results in

considerably shortened radius, ulna, scapula, fibula, tibia and femur; and also results in gross morphological defects in cartilage (right part of J–L). Fe, femur; Fi,

fibula; H, humerus; np, nucleus pulposus; R, radius; S, scapula; Ti, tibia U, ulna; vb, vertebral body.

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Secretion of type II collagen was decreased in primarychondrocytes of Hsp47 cKO mice

The effects of Hsp47 disruption on the secretion of cartilage-specific collagen was investigated in cultured primary

chondrocytes. First, immunohistological analysis of culturedcells was performed to identify changes in Hsp47 protein levels.No Hsp47 was detected in chondrocytes from Hsp47 cKO

embryos, whereas type II collagen was observed (Fig. 8C).Localization of type II collagen was analyzed by double stainingwith antibodies against marker proteins. PDI and Golgi matrixprotein GM130 were used as ER and Golgi markers, respectively.

Type II collagen localized in the ER because it was well mergedwith PDI in the control and Hsp47 cKO chondrocytes (Fig. 8A),but only a small amount of type II collagen accumulated in the

Golgi complex of control and Hsp47-null chondrocytes (Fig. 8B).Western blot analysis revealed that the secretion and extracellularaccumulation of type II collagen was markedly less in Hsp47

cKO chondrocytes (Fig. 8D) than in the control, and that therewas less intracellularly.

The ultrastructure of distended rough endoplasmicreticulum of chondrocytes and cartilage collagen fibers

Next, ultrastructural analysis was performed to more closelyexamine phenotypic changes in the connective tissues. Cartilageand hypertrophic cartilage in E18.5 Hsp47flox heterozygousembryos carrying Col2a1–Cre did not show any abnormalities

compared with control (Hsp47flox/flox) embryos. Normally,cartilage tissues in the vertebrae is composed of proteoglycansand long collagen fibers, including type II collagen (arrows in

Fig. 9C), whereas the ECM proteins in Hsp47 cKO mice werenon-uniform and lacked long collagenous fibers (Fig. 9D).Instead, dot-like proteoglycan structures were detected in both

control and cKO mice. These results suggested that secretedcartilage collagens did not accumulate like normal collagen fibersin the ECM of the vertebrae of cKO mice. Chondrocytes inHsp47 cKO embryos had dilated rough endoplasmic reticulum

(rER) filled with granular material (Fig. 9B). Moreover,cytoplasmic projections were poorly developed in chondrocytesin cKO embryos (Fig. 9B), suggesting defects in the secretion of

substrates and/or interaction between cells and the ECM matrix.In addition, normal type I collagen fibers with a 67 nm periodbanding pattern were observed in the cranial bones of control,

Hsp47flox/wt and Hsp47 cKO mice (supplementary material Fig.S2), indicating that the maturation of type I collagen secretedfrom osteoblasts of cranial bones might be normal even in Hsp47

cKO mice.

DiscussionHsp47 is essential for osteochondrogenesis

The expression of collagen correlated with that of Hsp47 (Clarkeet al., 1993). During development, fibroblasts synthesize largeamounts of Hsp47; amounts are much lower in aged individuals.

The expression pattern of Hsp47 during development mirroredthat of various types of collagen. Interestingly, in variouspathological conditions where collagen synthesis is abnormally

stimulated, such as fibrosis, cicatrization and keloid formation,the expression of Hsp47 is also upregulated (Naitoh et al., 2001).In the case of liver fibrosis, the synthesis of both type I collagen

and Hsp47 is increased, particularly in the activatedmyofibroblastic Ito cells (Masuda et al., 1994). In additionto these correlative expressions of collagen and Hsp47 in

Fig. 5. Defective joint formation in Hsp47 cKO embryos. (A,B) Fast

Green–Safranin O staining of sections of the elbow joints of E18.5 mice;

(A) Control and (B) Hsp47 cKO. The control elbow joint has prominent and

distinct acellular joint cavities (arrowhead in A), whereas the Hsp47 cKO has

no cavities (B), and there are also morphological defects of the humerus and

the joint. Scale bars: 200 mm.

Fig. 4. Loss of the notochord and formation of the vertebral column were

defective in Hsp47 cKO embryos. Sagittal sections of the sacral vertebral

column of E14.5–18.5 and neonatal (P0) Hsp47flox/flox control (A,C,E,G) and

Hsp47 cKO (Hsp47flox/flox; Col2a1–Cre) (B,D,F,H) mice were stained with

Hematoxylin and Eosin. In control mice, the notochord gradually expanded at

the intervertebral disc areas to form a nucleus pulposus (np) and vanished

within the vertebral bodies (vb; A,C,E,G). In Hsp47 cKO mice the notochord

was still present at E14.5 (B), and disorganized vertebral discs and vertebral

bodies were observed at later stages (F,H). n, notochord; np, nucleus

pulposus; vb, vertebral body. The arrowhead indicates the acellular

notochordal tube. Scale bar: 100 mm.

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Fig. 6. Upregulation of Col1a1 mRNA and induced apoptosis in Hsp47 cKO cartilage. (A) Differential expression patterns of Col2a1, Col1a1 and Hsp47

mRNA. These expression patterns were analyzed in semi-serial sections of lumbar regions of E14.5 mouse embryos by in situ hybridization. (B) TUNEL staining

of semi-serial sections of E14.5 lumbar regions. Note Col2a1 mRNA was expressed in the cartilage of lumbar regions in both Hsp47flox/flox control and Hsp47 cKO

(Hsp47flox/flox; Col2a1–Cre) mice. Interestingly, Col1a1 mRNA was markedly upregulated in cKO mice (A). TUNEL stained cells were detected in cKO cartilage,

whereas very little signal was detected in control cartilage. Scale bars: 200 mm.

Fig. 7. Decrease in cartilage collagens and loss of

fibril formation of collagen in intervertebral discs in

Hsp47 cKO mice. Immunohistochemical analysis was

performed on frozen sections of E18.5 embryos of

(A,C,E,G,I,K) Hsp47flox/flox control mice and

(B,D,F,H,J,L) Hsp47 cKO mice (Hsp47flox/flox;

Col2a1–Cre). (M,N) Second harmonic generation

(SHG) imaging of intervertebral discs was also

performed on frozen sections of E18.5 control (M) and

Hsp47 cKO (N) mice. There is abundant Hsp47 and

type II and type XI collagen in the intervertebral discs

of control (A,C,G), but the amount of type II and type

XI collagen is markedly less, and Bip is upregulated in

the Hsp47 cKO (D,H,J) mice. (K–N) Exceptionally,

there is the very narrow region surrounding the annulus

fibrosus (af), in which the amount of type I and type II

collagen is very similar, judging from the intensity of

the fluorescence (near the red boxes in K and L). A

moderately strong SHG signal is present in the control

intervertebral disc (M), whereas the SHG signal of

Hsp47 cKO mice is much less in spite of the existence

of fibrillar collagen (N and bar graph), suggesting that

there is less collagen fibril formation in the cartilage of

Hsp47 cKO mice. In the bar graph, the intensity of the

SHG signal and the fluorescence of type I collagen

(measured in the red boxed regions) of the control was

regarded as 100%. ***P,0.01. af, annulus fibrosus; np

nucleus pulposus; vb, vertebral body. Scale bars:

100 mm (A–H,K–N), 20 mm (I,J).

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collagen-related diseases, the downregulation of Hsp47 by

treatment with antisense RNA or stellate cell-specific Hsp47

siRNA dramatically decreased the progression of fibrosis and

represents a promising therapeutic strategy for fibrotic diseases

(Sato et al., 2008). Recent studies also revealed that a mutation in

Hsp47 resulted in the development of OI in humans and dogs

(Drogemuller et al., 2009; Christiansen et al., 2010).

Previously, we reported that the disruption of Hsp47 in mice

caused severe defects in procollagen maturation in the ER,

resulting in embryonic lethality at E11.5 (Nagai et al., 2000). In

Hsp47-null mice, type I collagen fiber formation in the

mesenchyme and the formation of basement membranes

consisting of type IV collagen are impaired, which clearly

establishes that Hsp47 is a molecular chaperone necessary for the

proper molecular maturation of type I and type IV procollagen in

the ER. However, no information was available as to whether

Hsp47 was also required for the maturation of other types of

collagen. To address this question, we developed a strategy for

the tissue-specific disruption of Hsp47 using the Cre–loxP

system. By crossing Hsp47 floxed mice with mice harboring

Cre gene driven by the regulatory regions of collagen II alpha 1,

we were able to disrupt Hsp47 expression in a cartilage-specific

manner.

Generally, chondrocytes have two major functions: endochondral

ossification for proper skeletal development, and articular

cartilage maintenance for joint movement. Chondrogenic cell-

line-specific Hsp47 disruption caused perinatal lethality in mice,

with severely defective cartilage and endochondral bone

formation. In these mice, type II collagen secretion and

accumulation in the ECM was impaired, indicating that Hsp47

is essential for the molecular maturation of type II procollagen in

the ER, and thus for proper cartilage formation and endochondral

bone formation.

Fig. 8. Hsp47 deficiency reduces accumulation and secretion of type II collagen by the chondrocytes. (A–C) Primary chondrocyte of E18.5 embryos

isolated from rib cartilage of control and Hsp47 cKO mice were maintained for 7 days. Localization of type II collagen was analyzed by double staining

with antibodies against localization marker proteins. PDI (A) and GM130 (B) were used as ER and Golgi markers, respectively. (D) Western blotting

analysis of type II collagen using total lysates prepared from 7-day cultured primary chondrocytes isolated from rib cartilage in control and Hsp47

cKO mice.

Fig. 9. Electron micrographs of vertebral cartilage showing defects in

collagen fibrillar formation in Hsp47 cKO mice. (A,B) Chondrocytes of

vertebrae from E18.5 Hsp47flox/flox control mice (A) and Hsp47 cKO

(Hsp47flox/flox; Col2a1–Cre; B) mice. Arrowheads in A indicate well-

developed cytoplasmic projections. (C,D) The matrix structure of cartilage

from control (C) and Hsp47 cKO (D) mice. Note the fibrillar structure is not

evident in the extracellular matrix of Hsp47 cKO cartilage (D), whereas the

long collagenous fibers are well formed in control cartilage (arrows in C). The

rough endoplasmic reticulum (rER) consist of narrow well-defined membrane

structures in control chondrocytes (showed magnified in the inset in A),

whereas distended rER is present in the Hsp47 cKO chondrocytes (inset in B).

N, nucleus. Scale bars: 2 mm (A,B), 200 nm (C,D).

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Hsp47 conditional knockout mice revealed defects of theremoval of the notochord

A recent study showed that type II collagen has essential roles inthe removal of notochord (Aszodi et al., 1998). In wild-typemice, notochord was removed at approximately E14.5. However,

in mice deficient in type II collagen the notochord of sacralvertebrae did not disappear until P0.

In the case of Hsp47 cKO mice, Hsp47 was absent at E14.5,

and the notochord was retained until this stage. The acellularnotochordal tube was observed even at E18.5, which was notthe case in control mice (Fig. 4). These results indicates that

defects of production of type II collagen in Hsp47 cKO miceresults in a delay in the developmental step of dismantling thenotochord.

Comparison with human collagen diseases and othermouse models of chondrodysplasia

Chondrodysplasias and related phenotypes that arise from themisfolding and loss of procollagens in humans and mice havebeen shown to be caused by mutations in various collagen genes,

including COL2A1, COL9A1–3, COL11A1 and COL11A2

(Spranger et al., 1994; Jackson et al., 2010; Li et al., 1995;Vikkula et al., 1995; Mortier et al., 2000). Many mutations result

from the substitution of essential residues in the Gly–x–y tripletrepeats that form the mandatory repetitive structure of the triplehelical domains of a-chains (Mortier et al., 2000). In addition to

mutations in collagen genes, the mutation of other genes can alsoresult in the development of cartilage-related diseases. Forexample, multiple epiphyseal dysplasia is due to mutations in

matrilin-3 (Chapman et al., 2001) or in COMP (Briggs et al.,1995). Achondroplasia is caused by mutations in the gene forfibroblast growth factor receptor 3 (Shiang et al., 1994), andmetaphyseal chondrodysplasia is caused by mutations in the gene

for the parathyroid hormone-related peptide receptor (Schipaniet al., 1995). These connective tissue disorders can be dividedinto three groups: (1) structural defects or the incomplete

maturation of collagen molecules; (2) defects in the productionof ECM components other than collagen; and (3) defects in thedifferentiation of chondrocytes. Mutations in collagen genes or

genes necessary for collagen modification lead to disorders in thefirst group.

Chondrocyte-specific Hsp47 cKO mice and other models of

mammalian chondrodysplasia resulting from mutation ofcollagen genes show marked similarities. The skeletal defectsin Hsp47 cKO mice revealed by m-CT examination or direct

staining of skeletal preparations were similar to those observedin human patients with achondrogenesis type II that harbormutations in COL2A1, and other mouse models forchondrodysplasia that harbor abnormalities in the type II

collagen gene causing short limbs, a small thorax and abnormalgrowth plates (Metsaranta et al., 1992; Chan et al., 1995; Li et al.,1995). Ultrastructural observation revealed other common

features, including distended rERs in the chondrocytes ofHsp47 cKO mice and human patients with hypochondrogenesisor spondyloepiphyseal dysplasia caused by mutation of COL2A1

(Freisinger et al., 1994). It is worth noting that the disruption ofcollagen-specific molecular chaperones and mutation of thesubstrate gene for type II collagen, result in similar phenotypes,

consistent with previous results demonstrating that Hsp47 isnecessary for the correct folding and/or secretion of procollagens.Some patients and mice with chondrodysplasias exhibit deformed

cartilage and compensatory upregulation of type I and IIIcollagen in chondrocytes (Freisinger et al., 1994; Chan et al.,1995; Aszodi et al., 1998), leading to the abnormal fibrillation ofcartilage. In the case of chondrocytes in Hsp47 cKO mouse,

Col1a1 mRNA was upregulated in E14.5 embryos (Fig. 6),whereas the immunofluorescence was lower. Additionally, theSHG signal was dramatically decreased in fibrocartilage of

Hsp47 cKO mice (Fig. 7). Taken together the ablation of Hsp47

might cause the compensatory production of type I collagen fromchondrocytes although they do not make a fibrous structures.

Recently, a missense mutation (c.233TC.Leu78Pro) inSERPINH1 was reported to cause an autosomal dominant

disorder affecting the OI condition (Christiansen et al., 2010).HSP47 expression in the fibroblasts of this patient was low,perhaps because of increased instability of the protein. The OI-like phenotype might have been caused by defective maturation

of type I procollagen and possibly other types of procollagen,including types II, IX, X and/or XI. Therefore, examination ofother types of collagen, such as type XI collagen, a minor species

of collagen in cartilage, should be performed in Hsp47 cKOmice. Unfortunately, we were unable to succeed in addressingthis issue because of the technical difficulty in detecting

qualitative changes in FACIT collagens. Also, an issue to beaddressed in the future is whether Hsp47 ablation causes thechondrocyte differentiation indirectly for example by global

defects in collagen folding.

Hsp47 acts as a molecular chaperone for many types ofcollagen in vivoPreviously, we reported that Hsp47 could bind to collagenous

triple helical domains with consensus amino acid sequencescontaining the Gly-x-Arg triplet as the minimal requirement(Koide et al., 2002). This suggests that Hsp47 binds to many

types of collagen, and we subsequently reported that Hsp47 couldbind to collagen types I–V in vitro (Natsume et al., 1994). Wealso showed that Hsp47 has an essential role as a collagen-

specific molecular chaperone in the ER, necessary for the correctfolding and secretion of at least type I and type IV procollagen(Nagai et al., 2000; Matsuoka et al., 2004). However, it wasunclear whether Hsp47 can act as a molecular chaperone for

other types of procollagen in vivo.

In the current study, we clearly demonstrated that Hsp47 has

an indispensable role as a molecular chaperone in the secretion oftype II collagen in chondrocytes. From these studies, the generalrole of Hsp47 as molecular chaperone could apply to a wider

spectrum of collagen species including major constituents of theECM, fibrillar collagen (type I, II, III and XI) and basementmembrane collagen (type IV), all of which contain a commontriple helical structure. However, we have no direct evidence of

whether Hsp47 is necessary for the molecular maturation ofnetwork-forming collagen, FACIT collagen and/or other typesof collagen which have transmembrane domains.

Fibrinogenesis in fibrocartilage observed by SHG imaging

The recently developed SHG imaging method is applicable forthe examination of pathological conditions in which collagen

synthesis is abnormally stimulated, such as the fibrillation thatoccurs during osteoarthritis or the development of certain tumors(Brown et al., 2003; Yeh et al., 2005).

To examine the fibrinogenesis of collagen in tissues, includingfibrocartilage, we used SHG imaging and found that SHG signals

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from the fibrocartilage of Hsp47 cKO mice were much lowerthan in controls. Although type I collagen was observed in the

IVDs, the accumulation of type I collagen in the fibrocartilage ofHsp47 cKO mice did not have well-aligned fibrillar structuresthat would provide a strong SHG signal. That was also verifiedby ultrastructural observation of ECM structures by electron

microscopy. Thus, we were able to clearly show that inactivationof the Hsp47 gene in fibrocartilage results in considerabledisruption of collagen fiber formation in the ECM. We also

successfully demonstrated that SHG imaging could be useful forthe detailed analysis of fibrogenesis in collagen-related disorders.

Materials and MethodsGeneration of Hsp47 floxed mice and conditional Hsp47 knockout miceAn Hsp47 clone including all exons was isolated from a genomic BAC libraryderived from the 129/Sv strain. This clone was modified with two loxP sequences(59-ATAACTTCGTATAATGTATGCTATACGAAGTTAT-39), a neomycin (Neo)selection cassette and a diphtheria toxin A (DTA) selection cassette using pGKNEO-F2L2DTA as a targeting vector (Fig. 1A). The targeting vector was electroporatedinto the K.Y1.1 mouse embryonic stem cell line. After neomycin resistance (Neo)positive selection with G418, stem cell clones that had undergone homologousrecombination were screened by PCR, Southern blotting analysis and partial genomesequencing, including the loxP site. Chimeric mice were generated from fourhomologous recombinant clones by aggregation, as described previously (Kondohet al., 1999), and the chimeras obtained were bred with C57BL/6 mice to generateHsp47 floxed heterozygous mice. Hsp47 floxed heterozygous mice were then bred tohomozygosity. The Col2a1–Cre strain was acquired from the Jackson Laboratory(Bar Harbor, ME) (Ovchinnikov et al., 2000). In an initial cross, Col2a1–Cretransgenic mice were mated with mice heterozygous for the Hsp47 floxed allele. Theoffspring harboring the Col2a1–Cre transgene and an Hsp47 floxed allele were thenmated with Hsp47 floxed heterozygous mice or homozygous mice to obtain embryosharboring the Col2a1–Cre transgene with two Hsp47 floxed alleles.

Routine mouse genotyping was performed by PCR. The following primer pairswere used for floxed Hsp47 alleles and the Cre transgene: (59-GAGTGG-GCTGAGCCCTCTCAAGAAAATCC-39) and (59-CTTCGGTCAGGCCCAGT-CCTGCCAGATG-39); and (59-TCCAATTTACTGACCGTACACCAAAATT-TG-39) and (59-CCTGATCCTGGCAATTTCGGCTATAC-39), respectively.

Histological analysisMicro-computed tomography (m-CT) analysis was performed using CT (SMX-100CT-SV3- type, Shimazu, Kyoto, Japan), as described previously (Ikeguchiet al., 2006). Whole-mount staining of skeletons with Alcian Blue and AlizarinRed used to visualize cartilage and ossified bone was performed essentially asdescribed previously (McLeod, 1980). For histological analysis, neonates werefixed in 4% paraformaldehyde and embedded in paraffin. Sections (4 mm thick)were used for Hematoxylin and Eosin staining, Safranin-O–Fast Green stainingand Alcian Blue–Nuclear Fast Red staining, Von Kossa reaction and Nuclear FastRed staining were performed using established protocols (Matsumoto et al., 2009).

For immunofluorescence analysis and SHG imaging, embryos were first fixed in4% paraformaldehyde, which was changed to 30% sucrose in PBS after 20%EDTA decalcification at 4 C. Immunohistological analysis was performed using16 mm thick frozen sections and cultured primary chondrocytes as describedbelow. The following antibodies were used: mouse monoclonal anti-Hsp47antibody (Stressgen Biotechnologies; 1:600), rabbit polyclonal anti-type I collagen(LSL, Rochdale, UK; 1:200), mouse monoclonal anti-type II collagen (Lab VisionCorporation; 1:400), rabbit polyclonal anti-type II collagen (Rockland,Gilbertsville, PA; 1:300), rabbit polyclonal anti-type XI collagen (LSL; 1:200),rabbit polyclonal anti-Grp78 (Affinity Bioreagent; 1:400), mouse monoclonal anti-PDI (Assay Designs, Mona Vale NSW, Australia; 1:400) and mouse monoclonalanti-GM130 (BD Biosciences; 1:400). Alexa-Fluor-488-conjugated anti-mouse orrabbit IgG and Alexa-Fluor-594-conjugated anti-mouse or rabbit IgG (Invitrogen;1:400) were used as secondary antibodies. We checked the recognition specificityof the anti-type I and anti-type II antibodies using with the limb sections of controlmice (see supplementary material Fig. S3). In addition, cell nuclei were stainedwith Hoechst 33342.

In situ hybridization was performed using 7 mm thick frozen sections. Antisenseand sense RNA probes for each gene to be examined were transcribed from theirrespective plasmids using a digoxigenin (DIG) RNA labeling kit (Roche Diagnostics,Penzberg, Germany). For mouse RNA probes, a 0.9-kb cDNA fragment of mouseCol1a1 were subcloned into pGEM4Z as previously reported (Masuda et al., 1994), a1.6-kb cDNA fragment of mouse Col2a1 were subcloned into pBluesript2 KS(+) anda 0.5-kb cDNA fragment of mouse Hsp47 was subcloned into pBluescript2 SK(+);they were used for in situ hybridization following established protocols (Shukunamiet al., 2008). TUNEL analysis was performed on frozen 7 mm sections using afluorescein-conjugated in situ cell detection kit (Roche).

SHG imaging

Frozen sections of E18.5 embryos were used for SHG imaging. Sample preparationwas performed at 30 C to reduce the denaturation and degradation of collagenmolecules as much as possible. Images were scanned using a custom-builtmultiphoton laser scanning microscope. Images of the SHG signal were obtainedusing a 120 femtosecond laser pulse centered at 840 nm excitation and 405–435 nm bandpass filters. Spectra were generated using a focal spectrum analyzeron a multiphoton laser scanning microscope. The short wavelength signal from thefibrillar structures in vertebrae was located at exactly half the excitationwavelength and had an extremely narrow bandwidth. The fact that it waslocated at half the excitation wavelength shifted was consistent with SHG asdescribed previously (Brown et al., 2003).

Transmission electron microscopy

For ultrastructural observation, sacral vertebrae and tibiae from embryos at E18.5were fixed with 1% acrolein, 2% paraformaldehyde and 3% glutaraldehyde in0.04 M cacodylate buffer (pH 7.4). After decalcification in 10% EDTA, thesamples were post-fixed with 1% OsO4 in 0.1 M cacodylate buffer and embeddedin Epon 812 (TAAB, Aldermaston, UK). Ultrathin sections were examined byTEM (H-7600; Hitachi Co., Tokyo, Japan) at an accelerating voltage of 8 V.

Cell culture

Primary chondrocytes were prepared from the rib cartilage of E18 embryos using amodification of previously published protocols (Muramatsu et al., 2007).Chondrocytes were isolated using 0.15% collagenase (Sigma) for 1.5 hours at37 C after adherent connective tissue had been removed with 0.1% trypsin(Nacalai, Kyoto, Japan) pretreatment for 40 minutes at 37 C. Isolated chondrocyteswere maintained in DMEM–F12 (Gibco) supplemented with 10% FCS andascorbic acid (50 mg/ml) and medium were changed every 24 hours. After 7 daysin culture, immunofluorescence analysis was performed as described previously(Ishida et al., 2006) using the antibodies listed above.

Western blotting analysis was performed as described previously (Fernandeset al., 2007), using the following primary antibodies: mouse monoclonal anti-typeII collagen alpha1 (Lab Vision Corporation; 1:1000) and mouse monoclonal anti-GAPDH (HyTest, Turku, Finland; 1:1000). Anti-mouse IgG alkaline phosphataseconjugate (Zymed; 1:1000), anti-rabbit IgG HRS conjugate (BiosourceInternational, Camarillo, CA; 1:1000) and anti-goat IgG alkaline phosphataseconjugate (Biosource; 1:1000) were used as secondary antibodies.

Statistical analysis

Results are presented as means 6 s.d. Statistical differences were determined usingStudent’s t-test. Significance was accepted at P-values less than 0.01.

AcknowledgementsWe are very grateful to J. Takeda (Osaka University) for providingKY1.1 ES cells. We also thank T. Matsushita (Kyoto University), D.Okui (Kyoto University), J. Nakamura (Kyoto Sangyo University),K. Yasuda (Nagasaki University), C. Syukunami (Kyoto University),A. Kitamura (Hokkaido University) and Y. Ishida (Shionogi & Co.,Ltd.) for professional help and discussion.

FundingY. Masago was supported by a fellowship from the Japan Society forthe Promotion of Science for Young Scientists. This work wassupported by a Grant-in-Aid for JSPS Fellows, Ground-basedResearch Program for Space Utilization from the Japan SpaceForum, Grants-in-aid for Creative Scientific Research 19GS0314,Grant-in-Aid for Scientific Research on Priority Areas 19058008 andHuman Frontier Science Program.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.089748/-/DC1

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