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. 2004 Oct;15(10):4467–4475. doi: 10.1091/mbc.E04-01-0050

Insufficient Folding of Type IV Collagen and Formation of Abnormal Basement Membrane-like Structure in Embryoid Bodies Derived from Hsp47-Null Embryonic Stem CellsD⃞

Yasuhiro Matsuoka *, Hiroshi Kubota *,†, Eijiro Adachi ‡, Naoko Nagai *,§, Toshihiro Marutani *, Nobuko Hosokawa *,†, Kazuhiro Nagata *,†,∥
Editor: Peter Walter
PMCID: PMC519141  PMID: 15282337

Abstract

Hsp47 is a molecular chaperone that specifically recognizes procollagen in the endoplasmic reticulum. Hsp47-null mouse embryos produce immature type I collagen and form discontinuous basement membranes. We established Hsp47-/- embryonic stem cell lines and examined formation of basement membrane and production of type IV collagen in embryoid bodies, a model for postimplantation egg-cylinder stage embryos. The visceral endodermal cell layers surrounding Hsp47-/- embryoid bodies were often disorganized, a result that suggested abnormal function of the basement membrane under the visceral endoderm. Rate of type IV collagen secretion by Hsp47-/- cells was fourfold lower than that of Hsp47+/+ cells. Furthermore, type IV collagen secreted from Hsp47-/- cells was much more sensitive to protease digestion than was type IV collagen secreted from Hsp47+/+ cells, which suggested insufficient or incorrect triple helix formation in type IV collagen in the absence of Hsp47. These results indicate for the first time that Hsp47 is required for the molecular maturation of type IV collagen and suggest that misfolded type IV collagen causes abnormal morphology of embryoid bodies.

INTRODUCTION

Collagen is one of the most abundant proteins of the extracellular matrix (Kuhn et al., 1987); 26 subclasses of collagen molecules, types I through XXVI, have been identified (Kuhn et al., 1987; Prockop and Kivirikko, 1995; Sato et al., 2002). Although the fibril-forming types I and III collagen are abundant in most extracellular matrices, type IV collagen, a nonfibrillar network-forming collagen (Timpl et al., 1981; Kuhn et al., 1987), is the major component of basement membrane (BM). Collagen molecules contain helical domains, known as collagenous domains, that are composed of X-Y-Gly triplet repeats; often, the Y residue is hydroxyproline (Kuhn et al., 1987; van der Rest M, 1991). The type IV procollagen molecule consists of three domains; 7S, a short, N-terminal triple-helical domain; NC1, a noncollagenous, C-terminal C-propeptide domain; and a long triple-helical collagenous domain centrally (Kuhn et al., 1987).

During collagen biosynthesis, procollagen chains interact with several endoplasmic reticulum (ER)-resident molecular chaperones and protein folding catalysts (Lamande and Bateman, 1999), including the 47-kDa heat shock protein (Hsp47) (Nagata, 1996), the 78-kDa glucose-regulated protein (GRP78, also called BiP) (Chessler and Byers, 1993), protein disulfide isomerase (PDI) (Wilson et al., 1998), and prolyl 4-hydroxylase (P4H) (Chessler and Byers, 1992; Walmsley et al., 1999). Formation of triple helixes within collagenous domains proceeds from the C terminus to the N terminus (Bachinger et al., 1980; Engel and Prockop, 1991; Bulleid et al., 1997), and hydroxylation of proline residues at the Y positions stabilizes the triple helixes of procollagen (Uitto and Prockop, 1974). After triple helix formation, procollagen is exported through the general secretion pathway and then mature collagen forms higher order complexes in the extracellular matrix (Timpl et al., 1981; Kuhn et al., 1987).

Hsp47 is a collagen-binding protein (Nagata and Yamada, 1986) that assists in the molecular maturation of procollagen (Nagai et al., 2000; Tasab et al., 2000). Hsp47 can bind to procollagen in vivo (Nakai et al., 1990) and in vitro (Natsume et al., 1994; Koide et al., 2002) and preferentially binds the triple-helical region of procollagen (Koide et al., 2000; Tasab et al., 2000). In vitro analyses have indicated that Hsp47 recognizes ProPro-Gly triplet repeats (Koide et al., 2000) and preferentially binds to ProArg-Gly triplet repeats (Koide et al., 2002; Tasab et al., 2002). Hsp47 dissociates from procollagen in the ER-Golgi intermediate compartment or in the cis-Golgi during transport to the Golgi apparatus (Nakai et al., 1992; Satoh et al., 1996). We have found that Hsp47 knockout mouse embryos cannot survive beyond 11.5 d postcoitus and that they are severely deficient in fibrillar collagen within the extracellular matrix of mesenchymal tissues (Nagai et al., 2000). Fibroblasts established from Hsp47-/- mice produce immature type I collagen without a well-defined triple-helical structure (Nagai et al., 2000).

Although Hsp47 binds types I through V collagen in vitro (Natsume et al., 1994), little is known about its role as a molecular chaperone in type IV collagen maturation in vivo. We established embryonic stem (ES) cell lines which lack the Hsp47 gene to examine the role of Hsp47 in the molecular maturation of type IV collagen in developing embryoid bodies (EBs), an in vitro model for postimplantation egg-cylinder–stage embryos. The morphology of the visceral endodermal (VE) cell layer is disorganized in EBs of Hsp47-/- ES cells, a finding that suggests impaired function of the BM. Hsp47-/- cells secreted type IV collagen at a much slower rate and produced misfolded type IV collagen that was susceptible to protease digestion. We discuss the essential role of Hsp47 in the productive folding of type IV collagen in vivo.

MATERIALS AND METHODS

Cell Culture

ES cells were cultured on mitomycin C-treated STO feeder cells in DMEM with high glucose and supplemented with 15% fetal bovine serum (Bioserum; CSL Limited, Victoria, Australia), 0.1 mM 2-mercaptoethanol, and 1000 U/ml leukemia inhibitory factor (LIF) (Chemicon International, Temecula, CA), as described previously (Robertson, 1987). ES cells were treated with 1 mM retinoic acid and 0.5 mM dibutyryladenosine 3′,5′-cyclic phosphate for 3 d to induce differentiation of monolayer endodermal cells.

For EB formation, ES cells were first cultured on tissue culture plates for 3 d with ES culture medium to remove residual feeder cells and then seeded on bacteriological petri dishes with ES culture medium without LIF. Cell aggregates were disrupted by hanging and dropping and were transferred to new petri dishes to maintain suspension cultures. The day on which primary cell aggregates were resuspended in new petri dishes was designated as day 0.

Isolation of Homozygous Hsp47 Knockout ES Cells

Hsp47+/- ES cells (Nagai et al., 2000) were seeded onto feeder cells at a concentration of 2.0 × 106 cells per 10-cm dish. On the following day, these Hsp47+/- ES cells were cultured in medium that contained 12.5–17.5 mg/ml G-418 (Calbiochem-Novabiochem, San Diego, CA); cells were maintained in this medium for 8 d. Drug-resistant colonies were picked, expanded, and screened using Southern blotting to obtain clones that had undergone a second recombination event that yielded Hsp47-/- cells.

Antibodies

For Western blotting or immunoprecipitation, we used rabbit polyclonal antibodies raised against mouse Hsp47 and rat type I collagen (LSL, Tokyo, Japan), laminin (provided by Dr. Hayashi, University of Tokyo, Japan), and mouse plasma fibronectin (H6660/4731; provided by Dr. Hancock, National Institutes of Health, Bethesda, MD); rat monoclonal antibody (mAb) to the α2 chain of human type IV collagen (H22) (Sado et al., 1995); and mouse mAb to chicken gizzard actin (C4) (Chemicon International). Goat anti-rabbit IgG (Biomedical Technologies, Stoughton, MA), and anti-mouse IgG and anti-rat IgG (Organon Teknika, Durham, United Kingdom) were used as secondary antibodies.

Western Blot Analysis

Proteins were extracted from ES cells, EBs, and BALB/3T3 (control) cells in cell extraction buffer that contained 0.05 M Tris-HCl (pH 8.0), 0.15 M NaCl, 5.0 mM EDTA, 1% NP-40, and protease inhibitors [2.0 mM N-ethylmaleimide, 2.0 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, and 1 μg/ml leupeptin and pepstatin] at 4°C. After centrifugation, soluble protein in the extract was quantified according to the method of Bradford (Bradford, 1976). Proteins were separated using 8% SDS-PAGE (Laemmli, 1970) and were blotted onto nitrocellulose filters. Filters were blocked in Dulbecco's phosphate-buffered saline containing 5% skim milk and 3% bovine serum albumin (BSA). Specific antibody binding was detected using the enhanced chemiluminescence system (Amersham Biosciences UK, Little Chalfont, Buckinghamshire, England).

Immunostaining

EBs were fixed in 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde at 4°C. Paraffin sections (4 μm) were treated with 0.3% hydrogen peroxide/methanol at room temperature for 30 min. After blocking nonspecific protein binding using 5% horse serum for 30 min, sections were incubated with rabbit antibodies against Hsp47, type IV collagen, or laminin (1:200), followed by incubation with biotinylated anti-rabbit IgG (Elite ABC kit PK-6100; Vector Laboratories, Burlingame, CA). Specific antibody binding was visualized using Elite ABC reagent (Vector Laboratories) and the Envision kit/HRP (DakoCytomation California, Carpinteria, CA). The percentage of EBs that contained a normal VE cell layer was estimated using the following four criteria for normal: 1) EB diameter was >100 μm; 2) extracellular thin layer (between the outer VE cell layer and the epiblast/inner cell mass) stained strongly with antitype IV collagen antibody; 3) VE cell layer showed normal simple epithelial morphology; and 4) >20% of the outer surface of the EB was surrounded by VE cells.

Metabolic Labeling

Ascorbic acid phosphate (136 μg/ml) was added to monolayer endodermal cells 16 h before metabolic labeling. Cells were incubated with 3.9 MBq/ml 35S-labeled Met and Cys (Express 35S protein labeling mixture; PerkinElmer Life and Analytical Sciences, Boston, MA) in medium containing ascorbic acid phosphate without fetal calf serum, Met, Cys, or LIF for 30 min. For pulse-labeling and -chase experiments, labeled cells were chased for appropriate periods of time in medium containing excess unlabeled Met and Cys. Soluble proteins were extracted in cell extraction buffer (described previously). For immunoprecipitation, anti-mouse type IV collagen antibody was added to cell extracts and culture media, and anti-mouse plasma fibronectin antibody was added to culture media; both were incubated at 4°C overnight. Protein A-Sepharose 4 Fast Flow resin (Pharmacia Biotechnology, Wikstroms, Sweden) was added to the mixture and resin was recovered by centrifugation. Immunocomplexes bound to the resin were washed in cell extraction buffer that had been modified by increasing the NaCl concentration to 0.4 M. Proteins were separated using 5% SDS-PAGE; gels were fixed in saturated trichloroacetic acid (TCA), soaked in 1 M sodium salicylic acid, and exposed to x-ray film.

Protease Digestion of Secreted Type IV Collagen

Monolayer endodermal cells were cultured in medium that contained ascorbic acid phosphate (136 μg/ml) and dialyzed 10% fetal calf serum in the presence of 3.7 MBq/ml l-[2,3-[3H]Pro (Amersham Biosciences UK) for 10 h. Aliquots of medium containing equal amounts of TCA-insoluble radioactivity were treated with a protease mixture consisting of 100 μg/ml trypsin and 250 μg/ml chymotrypsin in 0.05 M Tris-HCl (pH 7.4), 0.2 M NaCl, and 0.25 M glucose at 37°C or 4°C. Digests of type IV collagen were analyzed using 5% SDS-PAGE; proteins were fixed using 30% methanol/10% acetic acid, and gels were soaked in EN3HANCE (PerkinElmer Life and Analytical Sciences) and exposed to x-ray film.

Binding of Type IV Collagen to Fibronectin

Secreted type IV collagen was labeled with l-[2,3-[3H]Pro (described previously) by using a shortened labeling time of 6 h. Bovine plasma fibronectin (Itoham Foods, Hyogo, Japan) was coupled with cyanogen bromide-activated-Sepharose 4B (Pharmacia Biotechnology). Fibronectin-coupled beads (20 μl) were mixed with cell culture medium (200 μl), incubated for 2 h at 4°C with gentle mixing, and washed in 0.05 M HEPES (pH 7.5) containing 0.15 M NaCl and 2.5 mM EDTA. Bound proteins were extracted by boiling for 5 min in Laemmli's sample buffer supplemented with 0.1 M dithiothreitol. Extracted proteins were separated using 5% SDS-PAGE; proteins were fixed using 30% methanol/10% acetic acid, and gels were treated with EN3HANCE and exposed to x-ray films. Type IV collagen radioactivity was quantified using the software program NIH Image, version 1.62.

Northern Blot Analysis

Total RNA was extracted from cultured cells by the acid guanidium-phenol-chloroform method (Chomczynski and Sacchi, 1987) by using TRIzol (Invitrogen, Carlsbad, CA) and separated using formaldehyde/agarose gel electro-phoresis. RNA was blotted onto nylon filters (GeneScreen Plus; PerkinElmer Life and Analytical Sciences). Filters were hybridized with a 32P-labeled human proα1(IV) cDNA fragment (Pihlajaniemi et al., 1985) or a β-actin cDNA fragment (Gunning et al., 1983), washed in two times in SSC at 65°C, and exposed to x-ray films.

RESULTS

Abnormal Morphology of VE Cell Layer in EBs of Hsp47-/- ES Cells

BM has been reported to be discontinuously disrupted in Hsp47-null mouse embryos (Nagai et al., 2000). We first examined the morphology of Hsp47-/- EBs by immunostaining of sections (Figure 1) and found that the morphology of VE cell layers composing outer surface of EBs are abnormal in Hsp47-/- EBs. At day 8, anti-Hsp47 antibody strongly stained VE cell layers in Hsp47+/+ EBs (Figure 1A), whereas no staining was observed in Hsp47-/- EBs, confirming the absence of Hsp47 protein (Figure 1, B and C). Hsp47+/+ and Hsp47-/- EBs both contained BM-like structures that separate the outer VE cell layer from the inner epiblast cells (Figure 1, D–K). In both genotypes, most EBs formed cavities (Figure 1, D–K), which suggested that differentiation of EBs were not affected by absence of Hsp47 at this stage of development.

Figure 1.

Figure 1.

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VE cell layer shows disorganized morphology in EBs of Hsp47-/- ES cells. The distribution of Hsp47 (A–C), type IV collagen (D–F, J, and K), and laminin (G–I) in EBs differentiated from Hsp47-/- and Hsp47+/+ ES cells at day 8 were analyzed by immunostaining. (A, D, G, and J) Hsp47+/+. (B, E, H, and K) Hsp47-/- clone 7. (C, F, and I) Hsp47-/- clone 10. (L) Percentage of EBs that contained normal morphology of VE cell layers adjacent to BM-like structures (see Materials and Methods for details) was determined. Bars, 100 μm (A–I); and 10 μm (J and K). EE, epiblast epithelium; asterisk, cavity.

However, we found significant difference in the morphology of VE cell layer situated along the BM-like structures. In most Hsp47-/- EBs, the VE cell layers exhibited disorganized morphology, and weak dispersed staining with antitype IV collagen and anti-laminin antibodies was observed at the extracellular regions (Figure 1, E, F, H, I, and K). In contrast, the VE cell layers exhibited smooth simple epithelium-like morphology in Hsp47+/+ EBs (Figure 1, D, G, and J). Most Hsp47+/+ EBs contained thin BM-like structures strongly and continuously stained with antitype IV collagen and anti-laminin antibodies. In two Hsp47-/- ES clones, normal VE cell layer epithelial morphology was seen only in 13% (12 of 95) and 15% (21 of 144) of EBs, whereas normal morphology was seen in 57% (158 of 276) of Hsp47+/+ EBs (Figure 1L). Because the BM is important for maintaining epithelial cell layers, the abnormal morphology of the VE cell layer adjacent to the BM in Hsp47-/- EBs may be due to a defect in BM function.

Expression of Type IV Collagen Is Not Affected by the Absence of Hsp47

Because the morphology of VE cell layers along the BM-like structures was disordered in Hsp47-/- EBs (Figure 1) and Hsp47 is known to recognize type IV collagen in vitro (Natsume et al., 1994), we next examined expression levels of type IV collagen, a component of BM, in Hsp47-/- cells during differentiation (Figure 2). Using Western blotting, we found Hsp47 in Hsp47+/+ monolayer endodermal cells at day 3 and in Hsp47+/+ EBs at day 8. The level of Hsp47 and type IV collagen both increased over the course of differentiation (Figure 2A), a result that is consistent with previous observations that indicated Hsp47 and type IV collagen are both expressed in early-stage mouse embryos and in differentiating F9 cells (Leivo et al., 1980; Takechi et al., 1992; Nagai et al., 2000). In contrast, type I collagen was not detected at any stage. These results suggest that the up-regulation of Hsp47 during ES cell differentiation may play a role in the production of type IV collagen.

Figure 2.

Figure 2.

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Expression of Hsp47 and type IV collagen in differentiating ES cells and EBs. (A) Expression of Hsp47 correlates with that of type IV collagen in monolayer endodermal cells and in EBs differentiated from Hsp47+/+ ES cells. Total soluble proteins in monolayers of differentiating Hsp47+/+ ES cells, Hsp47+/+ EBs, and BALB/3T3 cells was analyzed by Western blotting by using specific antibodies. (B) Type IV collagen in soluble proteins extracted from monolayers of differentiating Hsp47+/+ and Hsp47-/- ES cells were analyzed. (C) Northern blot analysis of type IV collagen mRNA in monolayers of differentiating Hsp47+/+ and Hsp47-/- ES cells.

The level of type IV collagen and of type IV collagen mRNA was similar between Hsp47-/- and Hsp47+/+ cells (Figure 2, B and C). In both cell types, most type IV collagen was present in the NP-40–soluble fraction (unpublished data), which suggests that type IV collagen does not aggregate in these cells. These results indicate that disruption of the Hsp47 gene does not affect the level of expression of type IV collagen.

Type IV Collagen Secretion Rate Is Significantly Slower in Hsp47-/- Cells

To examine whether the secretion of type IV collagen is affected by the absence of Hsp47, we determined the secretion rate of type IV collagen by pulse-label and -chase experiments by using 35S-Met and 35S-Cys. Intracellular and secreted type IV collagens were detected by immunoprecipitation by using the specific antibody (Figure 3). The decrease in the intracellular amounts of type IV collagen directly corresponded to the increase in the amounts of type IV collagen secreted into the medium both in Hsp47+/+ and Hsp47-/- cells (Figure 3, A–C). The level of labeled intracellular type IV collagen decreased during chase periods, and the rate of decrease was fourfold slower in Hsp47-/- cells than in Hsp47+/+ cells (Figure 3D), which was consistent with the rate of increase in the type IV collagen secreted into the medium, that is, the rate of secretion was significantly higher in Hsp47+/+ cells compared with that in Hsp47-/- cells (Figure 3, A–C). In contrast, the secretion of fibronectin analyzed as a control was not affected by the absence of Hsp47 (Figure 3E), which indicated that general secretion pathways were not impaired by the disruption of Hsp47 gene. These results indicate that the absence of Hsp47 specifically caused a marked decrease in the rate of secretion of type IV collagen, a finding that is consistent with the previous observation that the secretion of type IV collagen was enhanced in Hsp47 overexpress cells (Tomita et al., 1999; Rocnik et al., 2002).

Figure 3.

Figure 3.

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Rate of type IV collagen secretion is significantly slower in Hsp47-/- cells than in Hsp47+/+ cells, as determined by pulse-chase experiment. (A–C) Radioactivity of intracellular and secreted type IV collagen of Hsp47+/+ cells (A), Hsp47-/- clone 7 cells (B), and Hsp47-/- clone 10 cells (C). (D) Relative radioactivity of intracellular type IV collagen was plotted in a logarithmic scale (n = 3). (E) Radioactivity of fibronectin secreted into the medium.

Type IV Collagen Secreted by Hsp47-/- Cells Is Susceptible to Protease Digestion

Unfolded collagen molecules are more sensitive to protease digestion than are those with correct triple-helical structures (Dolz et al., 1988; Nagai et al., 2000). We compared the protease sensitivity of type IV collagen derived from differentiating ES cells by digesting the collagen by using a mixture of trypsin and chymotrypsin to address the molecular features of secreted collagen (Figure 4). Type IV collagen molecules of 210 and 200 kDa secreted by Hsp47+/+ cells yielded a 190-kDa fragment probably consisting of triple-helical domain after protease treatment at 37°C for 5 min. In contrast, type IV collagen molecules secreted by Hsp47-/- cells that were treated under the same conditions were undetectable by SDS-PAGE (Figure 4). Complete digestion of type IV collagen secreted by Hsp47-/- cells also was observed after treatment at 4°C for 5 min (unpublished data). In contrast, protease sensitivity for intracellular type IV collagen of Hsp47-/- cells was similar to that of Hsp47+/+ cells (Supplemental Data 1), suggesting that properly folded type IV collagen is rapidly secreted from the ER and only minor portion was remained in the Hsp47+/+ cells. These results indicate that type IV collagen secreted by Hsp47-/- cells is not in the form with a correctly folded triple-helix, even at temperatures well below the melting point of normal collagen. These results clearly show that Hsp47 is essential for triple-helix formation of type IV collagen.

Figure 4.

Figure 4.

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Type IV collagen secreted from Hsp47-/- cells is susceptible to protease digestion. Differentiating ES cells were cultured in the presence of l-[2,3-[3H]Pro, and aliquots of medium containing equivalent amounts of TCA-insoluble radioactivity were treated with a mixture of trypsin and chymotrypsin at 37°C for indicated periods. The medium before and after protease digestion was analyzed by SDS-PAGE.

Type IV Collagen Secreted from Hsp47-/- Cells Binds with High Affinity to Fibronectin

Fibronectin is an extracellular matrix protein that interacts with various types of collagen (Engvall et al., 1978; Jilek and Hormann, 1978). Interestingly, fibronectin preferentially binds via ionic interaction (Vuento et al., 1982) to denatured, unfolded collagen (gelatin) rather than to native collagen (Engvall and Ruoslahti, 1977; Engvall et al., 1982). We therefore used fibronectin as a high-affinity indicator of denatured or misfolded collagen. Binding of secreted type IV collagen to fibronectin was examined using fibronectin-coupled Sepharose beads (Figure 5). Fibronectin-Sepharose bound a much higher (10-fold) quantity of type IV collagen, which had been secreted by Hsp47-/- cells compared with that secreted by Hsp47+/+ cells (Figure 5, A and C). Similarly, heat-denatured type IV collagen exhibited marked increase (14-fold) in binding to fibronectin-Sepharose (Figure 5, B and D), a result that confirmed that fibronectin binds to denatured type IV collagen with high affinity. These results support our earlier observation; type IV collagen secreted by Hsp47-/- cells is not correctly folded.

Figure 5.

Figure 5.

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Type IV collagen secreted from Hsp47-/- cells exhibits higher affinity to fibronectin than that secreted from Hsp47+/+ cells. Culture medium containing proteins labeled with l-[2,3-[3H]Prowas mixed with fibronectin-Sepharose beads. After washing, proteins bound to the beads were analyzed by SDS-PAGE. (A) Binding of type IV collagen secreted from Hsp47+/+ or Hsp47-/- cells to fibronectin. (B) Binding of type IV collagen secreted from Hsp47+/+ cells after heat treatment. The input applied to SDS-PAGE was 10% of total sample. (C and D) Quantification of type IV collagen binding to fibronectin beads (including means and standard deviations of four experiments). Type IV collagen binding to BSA-Sepharose is shown by solid bars, and type IV collagen binding to fibronectin-Sepharose is shown by open bars. HT, heat-treatment; I, input; B, fraction bound to BSA-Sepharose; F, fraction bound to fibronectin-Sepharose.

DISCUSSION

In this study, we found that type IV collagen secreted by Hsp47-/- cells is much more susceptible to protease digestion than that secreted by Hsp47+/+ cells (Figure 4), a finding that is consistent with our previous results for type I collagen (Nagai et al., 2000). Type IV collagen molecules secreted by Hsp47-/- cells were completely digested by trypsin/chymotrypsin at 37°C and at 4°C, whereas type IV collagen secreted by Hsp47+/+ cells were not digested completely, suggesting that collagen secreted by Hsp47-/- cells is unfolded, even at temperatures that are well below the melting point of normal collagen. These results clearly show that Hsp47 plays an essential role in the productive folding of type IV collagen.

Type IV collagen secreted by Hsp47-/- cells readily binds fibronectin at an affinity similar to that of fibronectin binding to heat-denatured type IV collagen (Figure 5). Because fibronectin preferentially binds denatured forms of collagen such as gelatin (Engvall and Ruoslahti, 1977; Engvall et al., 1982), these observations support the idea that type IV collagen secreted by Hsp47-/- cells is unfolded configuration. It is noteworthy that the α2 chain secreted from Hsp47-/- cells preferentially bound to fibronectin, whereas the α1 and α2 chains of heat-denatured collagen exhibited a similar binding activity to fibronectin (Figure 5, A and B). Folding and/or denaturing states may be different in the type IV collagen secreted from Hsp47-/- cells from those of heat-denatured one.

We also revealed here that secretion of type IV collagen was significantly delayed in the absence of Hsp47 (Figure 3), although the absence of Hsp47 does not affect the expression of type IV collagen (Figure 1). Delayed secretion is consistent with previous observations that indicated that secretion of type I collagen is enhanced with overexpression of Hsp47 (Tomita et al., 1999; Rocnik et al., 2002). Quality control in the ER is accomplished by cooperation among several ER-resident proteins (Hammond and Helenius, 1995; Ellgaard et al., 1999) that interact with unfolded proteins and retain them in the ER until they adopt correct conformation. During collagen synthesis, PDI associates with the C-propeptides of monomeric procollagen chains before initial chain assembly (Wilson et al., 1998), which may maintain them in the unfolded state until the trimer formation is initiated in the C-propeptide region (Bottomley et al., 2001). Thus, these ER chaperones, including PDI, may cause delayed secretion of type IV collagen in the absence of Hsp47.

Triple helix formation occurs from the C terminus to the N terminus (Bachinger et al., 1980; Engel and Prockop, 1991; Bulleid et al., 1997), and hydroxylation of proline residues at the Y position of X-Y-Gly repeats by P4H helps to stabilize the triple-helical structure (Berg, 1973). Hsp47 preferentially binds the triple-helical form of procollagen rather than the monomeric α-chain (Koide et al., 2000; Tasab et al., 2000, 2002). Hsp47 dissociates from procollagen during transport from the ER to the Golgi apparatus, presumably at the ER-Golgi intermediate compartment or at the cis-Golgi (Nakai et al., 1992; Satoh et al., 1996). Although Hsp47 is reported to prevent formation of collagen fibrils and aggregation of collagen molecules in vitro (Thomson and Ananthanarayanan, 2000; Tasab et al., 2002), we suggest that Hsp47 has another role in facilitating productive folding of collagen in the ER.

We previously reported that Hsp47 knockout mouse embryos cannot survive beyond 11.5 d postcoitus (Nagai et al., 2000). These embryos are severely deficient in collagen fibril formation, and Hsp47-/- fibroblasts established from Hsp47 knockout mouse embryos produce immature type I collagen that does not adopt an appropriate triple-helical conformation. In addition to these abnormalities, BMs of Hsp47-/- embryonic tissues are discontinuous. Hsp47 is therefore an important molecular chaperone during murine development. However, the molecular mechanisms that produce disrupted BMs were not clarified. From the data presented in this study, we suggest that the collagen fibril deficiency is caused by misfolding of type IV collagen. The BM that contains misfolded type IV collagen may be more fragile than one that contains properly folded type IV collagen.

Expression of Hsp47 correlates with expression of various types of collagen (Nagata and Yamada, 1986; Nakai et al., 1990; Takechi et al., 1992; Masuda et al., 1994); here, we showed that during differentiation of ES cells, expression of Hsp47 correlated with that of type IV collagen but not with that of type I collagen during the differentiation of ES cells (Figure 2), consistent with previous observations that both Hsp47 and type IV collagen are expressed in early-stage mouse embryos and in differentiating F9 cells (Leivo et al., 1980; Takechi et al., 1992; Nagai et al., 2000). The up-regulation of Hsp47 during ES cell differentiation may facilitate the folding and assembly of type IV collagen.

Although histochemical analysis indicated that Hsp47-/- EBs contained type IV collagen in the BM-like structure beneath the VE cell layer, the VE cell layer adjacent to this structure exhibited disorganized morphology (Figure 1). The percentage of Hsp47-/- EBs with normal VE cell morphology was only one-fourth that of Hsp47+/+ EBs. These results suggest that the BM-like structures in Hsp47-/- EBs are functionally abnormal, which is consistent with the observation that BMs are discontinuous in Hsp47-/- mouse embryos (Nagai et al., 2000), because EBs are an in vitro model for postimplantation egg-cylinder stage embryos. The type IV collagen that is secreted by Hsp47-/- cells into the extracellular matrix may not be competent to form a mesh-work in BMs.

Type IV collagen is important for BM function. For example, Alport syndrome is a genetic disease caused by any of >50 different mutations in the gene encoding the type IV collagen α5 chain, mutations that include single-base mutations and large deletions (Hudson et al., 1993). These mutations cause abnormal structure and function of the type IV collagen, resulting in derangement of BMs and defects in kidney function. Moreover, mouse embryos that lack the laminin γ1 subunit lack BMs and die by day 5.5 postcoitus (Smyth et al., 1999).

The results of this report combined with those of our previous report (Nagai et al., 2000) clearly show that Hsp47 in vivo is indispensable for productive folding of collagen types I and IV. Although Hsp47 can bind to collagen types I through V in vitro (Natsume et al., 1994), the in vivo chaperone function of Hsp47 for other types of collagen has not yet been established, because early-stage embryos die. To address this issue, we are now analyzing the role of Hsp47 in vivo by making mice with conditionally disrupted Hsp47 gene by adopting Cre-LoxP system.

Supplementary Material

[Supplemental Figure]
mbc_E04-01-0050_index.html (1,022B, html)

Acknowledgments

We thank Dr. M. Hosokawa for helpful discussion, and Drs. Sado, Hancock, and Hayashi for antibodies to type IV collagen, fibronectin, and laminin, respectively.

Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E04–01–0050. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E04–01–0050.

Abbreviations used: BM, basement membrane; EB, embryoid body; ER, endoplasmic reticulum; ES, embryonic stem; LIF, leukemia inhibitory factor; PDI, protein disulfide isomerase; TCA, trichloroacetic acid; VE, visceral endoderm.

D⃞

The online version of this article contains supplemental material accessible through http://www.molbiolcell.org.

References

  1. Bachinger, H.P., Bruckner, P., Timpl, R., Prockop, D.J., and Engel, J. (1980). Folding mechanism of the triple helix in type-III collagen and type-III pN-collagen. Role of disulfide bridges and peptide bond isomerization. Eur. J. Biochem. 106, 619-632. [DOI] [PubMed] [Google Scholar]
  2. Berg, R.A., P.D. (1973). The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen. Biochem. Biophys. Res. Commun. 52, 115-120. [DOI] [PubMed] [Google Scholar]
  3. Bottomley, M.J., Batten, M.R., Lumb, R.A., and Bulleid, N.J. (2001). Quality control in the endoplasmic reticulum: PDI mediates the ER retention of unassembled procollagen C-propeptides. Curr. Biol. 11, 1114-1118. [DOI] [PubMed] [Google Scholar]
  4. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. [DOI] [PubMed] [Google Scholar]
  5. Bulleid, N.J., Dalley, J.A., and Lees, J.F. (1997). The C-propeptide domain of procollagen can be replaced with a transmembrane domain without affecting trimer formation or collagen triple helix folding during biosynthesis. EMBO J. 16, 6694-6701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chessler, S.D., and Byers, P.H. (1992). Defective folding and stable association with protein disulfide isomerase/prolyl hydroxylase of type I procollagen with a deletion in the pro alpha 2(I) chain that preserves the Gly-X-Y repeat pattern. J. Biol. Chem. 267, 7751-7757. [PubMed] [Google Scholar]
  7. Chessler, S.D., and Byers, P.H. (1993). BiP binds type I procollagen pro alpha chains with mutations in the carboxyl-terminal propeptide synthesized by cells from patients with osteogenesis imperfecta. J. Biol. Chem. 268, 18226-18233. [PubMed] [Google Scholar]
  8. Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-159. [DOI] [PubMed] [Google Scholar]
  9. Dolz, R., Engel, J., and Kuhn, K. (1988). Folding of collagen IV. Eur. J. Biochem. 178, 357-366. [DOI] [PubMed] [Google Scholar]
  10. Ellgaard, L., Molinari, M., and Helenius, A. (1999). Setting the standards: quality control in the secretory pathway. Science 286, 1882-1888. [DOI] [PubMed] [Google Scholar]
  11. Engel, J., and Prockop, D.J. (1991). The zipper-like folding of collagen triple helices and the effects of mutations that disrupt the zipper. Annu. Rev. Biophys. Biophys. Chem. 20, 137-152. [DOI] [PubMed] [Google Scholar]
  12. Engvall, E., Bell, M.L., Carlsson, R.N., Miller, E.J., and Ruoslahti, E. (1982). Nonhelical, fibronectin-binding basement-membrane collagen from endodermal cell culture. Cell 29, 475-482. [DOI] [PubMed] [Google Scholar]
  13. Engvall, E., and Ruoslahti, E. (1977). Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int. J. Cancer 20, 1-5. [DOI] [PubMed] [Google Scholar]
  14. Engvall, E., Ruoslahti, E., and Miller, E.J. (1978). Affinity of fibronectin to collagens of different genetic types and to fibrinogen. J. Exp. Med. 147, 1584-1595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gunning, P., Ponte, P., Okayama, H., Engel, J., Blau, H., and Kedes, L. (1983). Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol. Cell. Biol. 3, 787-795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hammond, C., and Helenius, A. (1995). Quality control in the secretory pathway. Curr. Opin. Cell Biol. 7, 523-529. [DOI] [PubMed] [Google Scholar]
  17. Hudson, B.G., Reeders, S.T., and Tryggvason, K. (1993). Type IV collagen: structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis. J. Biol. Chem. 268, 26033-26036. [PubMed] [Google Scholar]
  18. Jilek, F., and Hormann, H. (1978). Cold-insoluble globulin (fibronectin), IV[1-35 affinity to soluble collagen of various types. Hoppe Seylers Z. Physiol. Chem. 359, 247-250. [DOI] [PubMed] [Google Scholar]
  19. Koide, T., Aso, A., Yorihuzi, T., and Nagata, K. (2000). Conformational requirements of collagenous peptides for recognition by the chaperone protein HSP47. J. Biol. Chem. 275, 27957-27963. [DOI] [PubMed] [Google Scholar]
  20. Koide, T., Takahara, Y., Asada, S., and Nagata, K. (2002). Xaa-Arg-Gly triplets in the collagen triple helix are dominant binding sites for the molecular chaperone HSP47. J. Biol. Chem. 277, 6178-6182. [DOI] [PubMed] [Google Scholar]
  21. Kuhn, K., et al. (1987). Structure and function of collagen types, San Diego: Academic Press.
  22. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. [DOI] [PubMed] [Google Scholar]
  23. Lamande, S.R., and Bateman, J.F. (1999). Procollagen folding and assembly: the role of endoplasmic reticulum enzymes and molecular chaperones. Semin. Cell Dev. Biol. 10, 455-464. [DOI] [PubMed] [Google Scholar]
  24. Leivo, I., Vaheri, A., Timpl, R., and Wartiovaara, J. (1980). Appearance and distribution of collagens and laminin in the early mouse embryo. Dev. Biol. 76, 100-114. [DOI] [PubMed] [Google Scholar]
  25. Masuda, H., Fukumoto, M., Hirayoshi, K., and Nagata, K. (1994). Coexpression of the collagen-binding stress protein HSP47 gene and the alpha 1(I) and alpha 1(III) collagen genes in carbon tetrachloride-induced rat liver fibrosis. J. Clin. Investig. 94, 2481-2488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nagai, N., Hosokawa, M., Itohara, S., Adachi, E., Matsushita, T., Hosokawa, N., and Nagata, K. (2000). Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis. J. Cell Biol. 150, 1499-1506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nagata, K. (1996). Hsp 47, a collagen-specific molecular chaperone. Trends Biochem. Sci. 21, 22-26. [DOI] [PubMed] [Google Scholar]
  28. Nagata, K., and Yamada, K.M. (1986). Phosphorylation and transformation sensitivity of a major collagen-binding protein of fibroblasts. J. Biol. Chem. 261, 7531-7536. [PubMed] [Google Scholar]
  29. Nakai, A., Hirayoshi, K., and Nagata, K. (1990). Transformation of BALB/3T3 cells by simian virus 40 causes a decreased synthesis of a collagen-binding heat-shock protein (hsp47). J. Biol. Chem. 265, 992-999. [PubMed] [Google Scholar]
  30. Nakai, A., Satoh, M., Hirayoshi, K., and Nagata, K. (1992). Involvement of the stress protein HSP47 in procollagen processing in the endoplasmic reticulum. J. Cell Biol. 117, 903-914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Natsume, T., Koide, T., Yokota, S., Hirayoshi, K., and Nagata, K. (1994). Interactions between collagen-binding stress protein HSP47 and collagen. Analysis of kinetic parameters by surface plasmon resonance biosensor. J. Biol. Chem. 269, 31224-31228. [PubMed] [Google Scholar]
  32. Pihlajaniemi, T., Tryggvason, K., Myers, J.C., Kurkinen, M., Lebo, R., Cheung, M.C., Prockop, D.J., and Boyd, C.D. (1985). cDNA clones coding for the pro-alpha1(IV) chain of human type IV procollagen reveal an unusual homology of amino acid sequences in two halves of the carboxyl-terminal domain. J. Biol. Chem. 260, 7681-7687. [PubMed] [Google Scholar]
  33. Prockop, D.J., and Kivirikko, K.I. (1995). Collagens: molecular biology, diseases, and potentials for therapy. Annu. Rev. Biochem. [DOI] [PubMed]
  34. Robertson, E.J. (1987). Embryo-derived stem cell lines. IRL Press: Oxford, 19-112.
  35. Rocnik, E.F., van der Veer, E., Cao, H., Hegele, R.A., and Pickering, J.G. (2002). Functional linkage between the endoplasmic reticulum protein Hsp47 and procollagen expression in human vascular smooth muscle cells. J. Biol. Chem. 277, 38571-38578. [DOI] [PubMed] [Google Scholar]
  36. Sado, Y., Kagawa, M., Kishiro, Y., Sugihara, K., Naito, I., Seyer, J.M., Sugimoto, M., Oohashi, T., and Ninomiya, Y. (1995). Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different alpha chains of human type IV collagen. Histochem. Cell Biol. 104, 267-275. [DOI] [PubMed] [Google Scholar]
  37. Sato, K., Yomogida, K., Wada, T., Yorihuzi, T., Nishimune, Y., Hosokawa, N., and Nagata, K. (2002). Type XXVI collagen, a new member of the collagen family, is specifically expressed in the testis and ovary. J. Biol. Chem. 277, 37678-37684. [DOI] [PubMed] [Google Scholar]
  38. Satoh, M., Hirayoshi, K., Yokota, S., Hosokawa, N., and Nagata, K. (1996). Intracellular interaction of collagen-specific stress protein HSP47 with newly synthesized procollagen. J. Cell Biol. 133, 469-483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smyth, N., Vatansever, H.S., Murray, P., Meyer, M., Frie, C., Paulsson, M., and Edgar, D. (1999). Absence of basement membranes after targeting the LAMC1 gene results in embryonic lethality due to failure of endoderm differentiation. J. Cell Biol. 144, 151-160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Takechi, H., Hirayoshi, K., Nakai, A., Kudo, H., Saga, S., and Nagata, K. (1992). Molecular cloning of a mouse 47-kDa heat-shock protein (HSP47), a collagen-binding stress protein, and its expression during the differentiation of F9 teratocarcinoma cells. Eur. J. Biochem. 206, 323-329. [DOI] [PubMed] [Google Scholar]
  41. Tasab, M., Batten, M.R., and Bulleid, N.J. (2000). Hsp 47, a molecular chaperone that interacts with and stabilizes correctly-folded procollagen. EMBO J. 19, 2204-2211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tasab, M., Jenkinson, L., and Bulleid, N.J. (2002). Sequence-specific recognition of collagen triple helices by the collagen-specific molecular chaperone HSP47. J. Biol. Chem. 277, 35007-35012. [DOI] [PubMed] [Google Scholar]
  43. Thomson, C.A., and Ananthanarayanan, V.S. (2000). Structure-function studies on hsp 47, pH-dependent inhibition of collagen fibril formation in vitro. Biochem. J. 349, 877-883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Timpl, R., Wiedemann, H., van Delden, V., Furthmayr, H., and Kuhn, K. (1981). A network model for the organization of type IV collagen molecules in basement membranes. Eur. J. Biochem. 120, 203-211. [DOI] [PubMed] [Google Scholar]
  45. Tomita, M., Yoshizato, K., Nagata, K., and Kitajima, T. (1999). Enhancement of secretion of human procollagen I in mouse HSP47-expressing insect cells. J. Biochem. 126, 1118-1126. [DOI] [PubMed] [Google Scholar]
  46. Uitto, J., and Prockop, D.J. (1974). Intracellular hydroxylation of non-helical protocollagen to form triple-helical procollagen and subsequent secretion of the molecule. Eur. J. Biochem. 43, 221-230. [DOI] [PubMed] [Google Scholar]
  47. van der Rest, M., G.R. (1991). Collagen family of proteins. FASEB J. 13, 2814-2823. [PubMed] [Google Scholar]
  48. Vuento, M., Salonen, E., Osterlund, K., and Stenman, U.H. (1982). Essential charged amino acids in the binding of fibronectin to gelatin. Biochem. J. 201, 1-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Walmsley, A.R., Batten, M.R., Lad, U., and Bulleid, N.J. (1999). Intracellular retention of procollagen within the endoplasmic reticulum is mediated by prolyl 4-hydroxylase. J. Biol. Chem. 274, 14884-14892. [DOI] [PubMed] [Google Scholar]
  50. Wilson, R., Lees, J.F., and Bulleid, N.J. (1998). Protein disulfide isomerase acts as a molecular chaperone during the assembly of procollagen. J. Biol. Chem. 273, 9637-9643. [DOI] [PubMed] [Google Scholar]

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