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Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis.

Nagai N, Hosokawa M, Itohara S, Adachi E, Matsushita T, Hosokawa N, Nagata K - J. Cell Biol. (2000)

Bottom Line: However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo.The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes.When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47+/+ and Hsp47+/- cells were resistant, but those of Hsp47-/- cells were sensitive.

View Article: PubMed Central - PubMed

Affiliation: Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), and Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

ABSTRACT
Triple helix formation of procollagen after the assembly of three alpha-chains at the C-propeptide is a prerequisite for refined structures such as fibers and meshworks. Hsp47 is an ER-resident stress inducible glycoprotein that specifically and transiently binds to newly synthesized procollagens. However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo. Here, we describe the establishment of Hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of alpha1(I) collagen and fibril structures in mesenchymal tissues. The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes. The homozygous mice did not survive beyond 11.5 days postcoitus (dpc), and displayed abnormally orientated epithelial tissues and ruptured blood vessels. When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47+/+ and Hsp47+/- cells were resistant, but those of Hsp47-/- cells were sensitive. These results indicate for the first time that type I collagen is unable to form a rigid triple-helical structure without the assistance of molecular chaperone Hsp47, and that mice require Hsp47 for normal development.

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Targeted disruption of the Hsp47 gene and characterization of the  phenotype. a, Homologous recombination with the targeting vector deletes exon IV and part of exon V, and simultaneously inserts a neomycin-resistance gene. Arrows indicate the orientation of neomycin-resistance gene and DT-A cassettes. Arrowheads indicate the location of primers used in RT-PCR assay. B, BamHI; K, KpnI; X, XhoI. b, Southern blot analysis demonstrating the genotypes of the offspring. A 3′ flanking probe shown as FC in a detects a 9-kb BamHI fragment in wild-type genomic DNA and a 6-kb fragment in the targeted allele in mice. Targeted ES clones were confirmed using a 5′ external probe (not shown). c, RT-PCR analysis was performed for the offspring using primers shown in a. n, Denotes the lane of negative control in which reactions are performed without RNA. d, Immunoblot analysis of Hsp47 was performed for wild-type, heterozygotic, and homozygotic  mice. B shows the positive control lane with the extract of Balbc/3T3 cells. Lateral views of 9.5 dpc (e) and 10.5 dpc (f) Hsp47−/− homozygous embryos (right) and wild type (left). Embryos were observed before (f) and after (e) fixation with 10% formaldehyde. Bars, 1 mm.
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Figure 1: Targeted disruption of the Hsp47 gene and characterization of the phenotype. a, Homologous recombination with the targeting vector deletes exon IV and part of exon V, and simultaneously inserts a neomycin-resistance gene. Arrows indicate the orientation of neomycin-resistance gene and DT-A cassettes. Arrowheads indicate the location of primers used in RT-PCR assay. B, BamHI; K, KpnI; X, XhoI. b, Southern blot analysis demonstrating the genotypes of the offspring. A 3′ flanking probe shown as FC in a detects a 9-kb BamHI fragment in wild-type genomic DNA and a 6-kb fragment in the targeted allele in mice. Targeted ES clones were confirmed using a 5′ external probe (not shown). c, RT-PCR analysis was performed for the offspring using primers shown in a. n, Denotes the lane of negative control in which reactions are performed without RNA. d, Immunoblot analysis of Hsp47 was performed for wild-type, heterozygotic, and homozygotic mice. B shows the positive control lane with the extract of Balbc/3T3 cells. Lateral views of 9.5 dpc (e) and 10.5 dpc (f) Hsp47−/− homozygous embryos (right) and wild type (left). Embryos were observed before (f) and after (e) fixation with 10% formaldehyde. Bars, 1 mm.

Mentions: The Hsp47 gene was disrupted in murine ES cells by the use of the targeting vector shown in Fig. 1 a. Heterozygous mice, which appeared phenotypically normal, were intercrossed to generate homozygous Hsp47−/− mice. Homozygosity for the Hsp47 mutation resulted in embryonic lethality in both C57BL/6 × 129/Ola and ICR × 129/Ola genetic backgrounds. Although homozygous new born mice and embryos after 11.5 dpc were never obtained, the mutant embryos were recovered with the expected Mendelian ratios at 9.5 dpc and 10.5 dpc (data not shown). The homozygosity of Hsp47−/− mice was determined by Southern blotting using 9.5 dpc embryos (Fig. 1 b). The absence of Hsp47 expression in the mutant embryo was confirmed at the mRNA level by RT-PCR (Fig. 1 c), and at the protein level by Western blot analysis (Fig. 1 d). The number of somites was consistently smaller in Hsp47 homozygotes than in the wild/heterozygotic littermates from 9.5 dpc (Table ) and neural tube closure was delayed (data not shown), suggesting growth retardation in the knockout embryos. At 10.5 dpc, the mutant embryos were more translucent compared with their wild-type littermates (Fig. 1 f), probably reflecting the low cell density of their bodies, and were so fragile that they could not be manipulated with forceps. The body was shrunken, and the number of erythrocytes decreased before death (data not shown).


Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis.

Nagai N, Hosokawa M, Itohara S, Adachi E, Matsushita T, Hosokawa N, Nagata K - J. Cell Biol. (2000)

Targeted disruption of the Hsp47 gene and characterization of the  phenotype. a, Homologous recombination with the targeting vector deletes exon IV and part of exon V, and simultaneously inserts a neomycin-resistance gene. Arrows indicate the orientation of neomycin-resistance gene and DT-A cassettes. Arrowheads indicate the location of primers used in RT-PCR assay. B, BamHI; K, KpnI; X, XhoI. b, Southern blot analysis demonstrating the genotypes of the offspring. A 3′ flanking probe shown as FC in a detects a 9-kb BamHI fragment in wild-type genomic DNA and a 6-kb fragment in the targeted allele in mice. Targeted ES clones were confirmed using a 5′ external probe (not shown). c, RT-PCR analysis was performed for the offspring using primers shown in a. n, Denotes the lane of negative control in which reactions are performed without RNA. d, Immunoblot analysis of Hsp47 was performed for wild-type, heterozygotic, and homozygotic  mice. B shows the positive control lane with the extract of Balbc/3T3 cells. Lateral views of 9.5 dpc (e) and 10.5 dpc (f) Hsp47−/− homozygous embryos (right) and wild type (left). Embryos were observed before (f) and after (e) fixation with 10% formaldehyde. Bars, 1 mm.
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Figure 1: Targeted disruption of the Hsp47 gene and characterization of the phenotype. a, Homologous recombination with the targeting vector deletes exon IV and part of exon V, and simultaneously inserts a neomycin-resistance gene. Arrows indicate the orientation of neomycin-resistance gene and DT-A cassettes. Arrowheads indicate the location of primers used in RT-PCR assay. B, BamHI; K, KpnI; X, XhoI. b, Southern blot analysis demonstrating the genotypes of the offspring. A 3′ flanking probe shown as FC in a detects a 9-kb BamHI fragment in wild-type genomic DNA and a 6-kb fragment in the targeted allele in mice. Targeted ES clones were confirmed using a 5′ external probe (not shown). c, RT-PCR analysis was performed for the offspring using primers shown in a. n, Denotes the lane of negative control in which reactions are performed without RNA. d, Immunoblot analysis of Hsp47 was performed for wild-type, heterozygotic, and homozygotic mice. B shows the positive control lane with the extract of Balbc/3T3 cells. Lateral views of 9.5 dpc (e) and 10.5 dpc (f) Hsp47−/− homozygous embryos (right) and wild type (left). Embryos were observed before (f) and after (e) fixation with 10% formaldehyde. Bars, 1 mm.
Mentions: The Hsp47 gene was disrupted in murine ES cells by the use of the targeting vector shown in Fig. 1 a. Heterozygous mice, which appeared phenotypically normal, were intercrossed to generate homozygous Hsp47−/− mice. Homozygosity for the Hsp47 mutation resulted in embryonic lethality in both C57BL/6 × 129/Ola and ICR × 129/Ola genetic backgrounds. Although homozygous new born mice and embryos after 11.5 dpc were never obtained, the mutant embryos were recovered with the expected Mendelian ratios at 9.5 dpc and 10.5 dpc (data not shown). The homozygosity of Hsp47−/− mice was determined by Southern blotting using 9.5 dpc embryos (Fig. 1 b). The absence of Hsp47 expression in the mutant embryo was confirmed at the mRNA level by RT-PCR (Fig. 1 c), and at the protein level by Western blot analysis (Fig. 1 d). The number of somites was consistently smaller in Hsp47 homozygotes than in the wild/heterozygotic littermates from 9.5 dpc (Table ) and neural tube closure was delayed (data not shown), suggesting growth retardation in the knockout embryos. At 10.5 dpc, the mutant embryos were more translucent compared with their wild-type littermates (Fig. 1 f), probably reflecting the low cell density of their bodies, and were so fragile that they could not be manipulated with forceps. The body was shrunken, and the number of erythrocytes decreased before death (data not shown).

Bottom Line: However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo.The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes.When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47+/+ and Hsp47+/- cells were resistant, but those of Hsp47-/- cells were sensitive.

View Article: PubMed Central - PubMed

Affiliation: Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), and Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

ABSTRACT
Triple helix formation of procollagen after the assembly of three alpha-chains at the C-propeptide is a prerequisite for refined structures such as fibers and meshworks. Hsp47 is an ER-resident stress inducible glycoprotein that specifically and transiently binds to newly synthesized procollagens. However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo. Here, we describe the establishment of Hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of alpha1(I) collagen and fibril structures in mesenchymal tissues. The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes. The homozygous mice did not survive beyond 11.5 days postcoitus (dpc), and displayed abnormally orientated epithelial tissues and ruptured blood vessels. When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47+/+ and Hsp47+/- cells were resistant, but those of Hsp47-/- cells were sensitive. These results indicate for the first time that type I collagen is unable to form a rigid triple-helical structure without the assistance of molecular chaperone Hsp47, and that mice require Hsp47 for normal development.

Show MeSH
Related in: MedlinePlus