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Cthrc1 is a positive regulator of osteoblastic bone formation.

Kimura H, Kwan KM, Zhang Z, Deng JM, Darnay BG, Behringer RR, Nakamura T, de Crombrugghe B, Akiyama H - PLoS ONE (2008)

Bottom Line: This remodeling process is regulated by many systemic and local factors.Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo.Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedics, Kyoto University, Kyoto, Japan.

ABSTRACT

Background: Bone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors.

Methodology/principal findings: We identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1- mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1- mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1- mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1- mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1- mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss.

Conclusions/significance: Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis.

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Related in: MedlinePlus

Generation of Cthrc1- mice.(A) Structure of the genomic Cthrc1 locus, targeting vector, and targeting allele. Exons are depicted as closed boxes, and intronic sequences are shown as solid lines. IRES-LacZ-pA-loxP-flanked PGK-neo-bpA cassettes are depicted as open boxes. S, SacI; B, BamHI. (B) Southern blot analysis of fetal genomic DNA. Genomic DNA isolated from the skin was digested with SacI and then hybridized with the 5′ or 3′ probe. The wild-type and the mutant allele were detected with the 5′ probe as 13.1-kb and 6.5-kb fragments and with the 3′ probe as 13.1-kb and 9.1-kb fragments, respectively. (C) RT-PCR analysis of Cthrc1 transcript in E16.5 wild-type and Cthrc1- littermates. (D) Whole-mount X-gal staining of E16.5 heterozygous Cthrc1 embryo. WT: wild-type mice; KO: Cthrc1- mice; +/−: Cthrc1 heterozygous mice.
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pone-0003174-g001: Generation of Cthrc1- mice.(A) Structure of the genomic Cthrc1 locus, targeting vector, and targeting allele. Exons are depicted as closed boxes, and intronic sequences are shown as solid lines. IRES-LacZ-pA-loxP-flanked PGK-neo-bpA cassettes are depicted as open boxes. S, SacI; B, BamHI. (B) Southern blot analysis of fetal genomic DNA. Genomic DNA isolated from the skin was digested with SacI and then hybridized with the 5′ or 3′ probe. The wild-type and the mutant allele were detected with the 5′ probe as 13.1-kb and 6.5-kb fragments and with the 3′ probe as 13.1-kb and 9.1-kb fragments, respectively. (C) RT-PCR analysis of Cthrc1 transcript in E16.5 wild-type and Cthrc1- littermates. (D) Whole-mount X-gal staining of E16.5 heterozygous Cthrc1 embryo. WT: wild-type mice; KO: Cthrc1- mice; +/−: Cthrc1 heterozygous mice.

Mentions: To investigate the physiological role of Cthrc1 in bone, we inactivated the Cthrc1 gene in mouse embryonic stem cells by homologous recombination. In the target strategy, an IRES-lacZ-pA-loxP-flanked neomycin resistance expression cassette was introduced into exon 2 which codes a short Gly-X-Y collagen triple helix repeat domain (Figure 1 A and B). As shown in Figure 1C, Cthrc1 RNA was absent in E16.5 Cthrc1- embryo, indicating that the mutation was a mutation. Homozygous Cthrc1 mutant mice were born at the expected Mendelian ratio and grew normally in comparison with their wild-type littermates (data not shown). X-gal staining of heterozygous Cthrc1 mutant embryos revealed the expression of Cthrc1 in bone-forming tissues in E12.5 embryos and in bones and periarticular cartilages in E16.5 embryos, which corresponded to the results of in situ hybridization (Figure 1D and Figure S2A). Skeletal preparations showed no obvious skeletal phenotypes in Cthrc1- newborn mice (Figure S2B). In situ hybridization analyses showed comparable expression of osteoblast marker genes, Runx2 and Col1a1, and chondrocyte marker genes, Col2a1 and Col10a1 between E16.5 Cthrc1- embryos and their wild-type littermates (Figure S3A), suggesting that Cthrc1 has no apparent effect on skeletal development. However, histological analyses of decalcified adult bone tissues revealed that the number and thickness of trabecular bones were reduced in Cthrc1- mice, compared with their wild-type littermates (data not shown). This result suggested that Cthrc1 may regulate bone remodeling postnatally. To test this hypothesis, we performed bone histomorphometric analyses and microcomputed tomography (micro-CT) analyses of 2-month-old Cthrc1- and wild-type mice. Micro-CT analyses of tibiae showed that the trabecular bone mass in Cthrc1- mice was approximately 70% of that in wild-type mice (Figure 2 A and B). Bone histomorphometric and micro-CT analyses showed a significant decrease in trabecular number (Tb.N) and osteoblast number (Ob.N/BS) in Cthrc1- mice, while trabecular thickness (Tb.Th) and tartrate-resistant acid phosphatase (TRAP)-positive osteoclast number (Oc.N/BS) had no change in Cthrc1- mice (Figure 2C–E and Figure S4A). In addition, the osteoblast surface (Ob.S/BS) in Cthrc1- mice, which represents the proportion of the bone surface covered with osteoblasts, was approximately 40% less than that in wild-type mice, and the osteoclast surface (Oc.S/BS), which represents the proportion of the bone surface covered with osteoclasts, showed no significant difference between Cthrc1- and wild-type mice (Figure 2F and Figure S4A). To analyze osteoblast proliferation in vivo, we performed BrdU incorporation assays in the calvaria of Cthrc1- and wild-type littermates, and found that the percentage of proliferating cells was decreased by approximately 30% in Cthrc1- mice (Figure 2G). Moreover, double-labeling analyses with calcein, a marker of newly formed bone, showed that the bone formation rate was significantly decreased in Cthrc1- mice (Figure 2H). Thus, these results indicate that the decreased bone mass in Cthrc1- mice is due to the suppression of osteoblastic bone formation, not due to an acceleration of osteoclastic bone resorption.


Cthrc1 is a positive regulator of osteoblastic bone formation.

Kimura H, Kwan KM, Zhang Z, Deng JM, Darnay BG, Behringer RR, Nakamura T, de Crombrugghe B, Akiyama H - PLoS ONE (2008)

Generation of Cthrc1- mice.(A) Structure of the genomic Cthrc1 locus, targeting vector, and targeting allele. Exons are depicted as closed boxes, and intronic sequences are shown as solid lines. IRES-LacZ-pA-loxP-flanked PGK-neo-bpA cassettes are depicted as open boxes. S, SacI; B, BamHI. (B) Southern blot analysis of fetal genomic DNA. Genomic DNA isolated from the skin was digested with SacI and then hybridized with the 5′ or 3′ probe. The wild-type and the mutant allele were detected with the 5′ probe as 13.1-kb and 6.5-kb fragments and with the 3′ probe as 13.1-kb and 9.1-kb fragments, respectively. (C) RT-PCR analysis of Cthrc1 transcript in E16.5 wild-type and Cthrc1- littermates. (D) Whole-mount X-gal staining of E16.5 heterozygous Cthrc1 embryo. WT: wild-type mice; KO: Cthrc1- mice; +/−: Cthrc1 heterozygous mice.
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Related In: Results  -  Collection

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pone-0003174-g001: Generation of Cthrc1- mice.(A) Structure of the genomic Cthrc1 locus, targeting vector, and targeting allele. Exons are depicted as closed boxes, and intronic sequences are shown as solid lines. IRES-LacZ-pA-loxP-flanked PGK-neo-bpA cassettes are depicted as open boxes. S, SacI; B, BamHI. (B) Southern blot analysis of fetal genomic DNA. Genomic DNA isolated from the skin was digested with SacI and then hybridized with the 5′ or 3′ probe. The wild-type and the mutant allele were detected with the 5′ probe as 13.1-kb and 6.5-kb fragments and with the 3′ probe as 13.1-kb and 9.1-kb fragments, respectively. (C) RT-PCR analysis of Cthrc1 transcript in E16.5 wild-type and Cthrc1- littermates. (D) Whole-mount X-gal staining of E16.5 heterozygous Cthrc1 embryo. WT: wild-type mice; KO: Cthrc1- mice; +/−: Cthrc1 heterozygous mice.
Mentions: To investigate the physiological role of Cthrc1 in bone, we inactivated the Cthrc1 gene in mouse embryonic stem cells by homologous recombination. In the target strategy, an IRES-lacZ-pA-loxP-flanked neomycin resistance expression cassette was introduced into exon 2 which codes a short Gly-X-Y collagen triple helix repeat domain (Figure 1 A and B). As shown in Figure 1C, Cthrc1 RNA was absent in E16.5 Cthrc1- embryo, indicating that the mutation was a mutation. Homozygous Cthrc1 mutant mice were born at the expected Mendelian ratio and grew normally in comparison with their wild-type littermates (data not shown). X-gal staining of heterozygous Cthrc1 mutant embryos revealed the expression of Cthrc1 in bone-forming tissues in E12.5 embryos and in bones and periarticular cartilages in E16.5 embryos, which corresponded to the results of in situ hybridization (Figure 1D and Figure S2A). Skeletal preparations showed no obvious skeletal phenotypes in Cthrc1- newborn mice (Figure S2B). In situ hybridization analyses showed comparable expression of osteoblast marker genes, Runx2 and Col1a1, and chondrocyte marker genes, Col2a1 and Col10a1 between E16.5 Cthrc1- embryos and their wild-type littermates (Figure S3A), suggesting that Cthrc1 has no apparent effect on skeletal development. However, histological analyses of decalcified adult bone tissues revealed that the number and thickness of trabecular bones were reduced in Cthrc1- mice, compared with their wild-type littermates (data not shown). This result suggested that Cthrc1 may regulate bone remodeling postnatally. To test this hypothesis, we performed bone histomorphometric analyses and microcomputed tomography (micro-CT) analyses of 2-month-old Cthrc1- and wild-type mice. Micro-CT analyses of tibiae showed that the trabecular bone mass in Cthrc1- mice was approximately 70% of that in wild-type mice (Figure 2 A and B). Bone histomorphometric and micro-CT analyses showed a significant decrease in trabecular number (Tb.N) and osteoblast number (Ob.N/BS) in Cthrc1- mice, while trabecular thickness (Tb.Th) and tartrate-resistant acid phosphatase (TRAP)-positive osteoclast number (Oc.N/BS) had no change in Cthrc1- mice (Figure 2C–E and Figure S4A). In addition, the osteoblast surface (Ob.S/BS) in Cthrc1- mice, which represents the proportion of the bone surface covered with osteoblasts, was approximately 40% less than that in wild-type mice, and the osteoclast surface (Oc.S/BS), which represents the proportion of the bone surface covered with osteoclasts, showed no significant difference between Cthrc1- and wild-type mice (Figure 2F and Figure S4A). To analyze osteoblast proliferation in vivo, we performed BrdU incorporation assays in the calvaria of Cthrc1- and wild-type littermates, and found that the percentage of proliferating cells was decreased by approximately 30% in Cthrc1- mice (Figure 2G). Moreover, double-labeling analyses with calcein, a marker of newly formed bone, showed that the bone formation rate was significantly decreased in Cthrc1- mice (Figure 2H). Thus, these results indicate that the decreased bone mass in Cthrc1- mice is due to the suppression of osteoblastic bone formation, not due to an acceleration of osteoclastic bone resorption.

Bottom Line: This remodeling process is regulated by many systemic and local factors.Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo.Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedics, Kyoto University, Kyoto, Japan.

ABSTRACT

Background: Bone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors.

Methodology/principal findings: We identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1- mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1- mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1- mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1- mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1- mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss.

Conclusions/significance: Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis.

Show MeSH
Related in: MedlinePlus