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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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Effects of Cthrc1 on osteogenesis.(A) Cell proliferation in primary osteoblasts harvested from Cthrc1- mice (upper panel) and from Cthrc1 transgenic mice (lower panel) as shown by BrdU incorporation assays. (B) Expression of early and late osteoblast marker genes in primary osteoblasts harvested from Cthrc1- mice (upper panel) and Cthrc1 transgenic mice (lower panel). (C) The total numbers of CFU-ALP in bone marrow cell cultures derived from Cthrc1- mice (left) and Cthrc1 transgenic mice (right). (D) The mineralized area of CFU-O in bone marrow cell cultures derived from Cthrc1- mice (left) and Cthrc1 transgenic mice (right). WT: wild-type mice; KO: Cthrc1- mice; Tg: Cthrc1 transgenic mice. Data are shown as the mean±SEM (*p<0.05).
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pone-0003174-g004: Effects of Cthrc1 on osteogenesis.(A) Cell proliferation in primary osteoblasts harvested from Cthrc1- mice (upper panel) and from Cthrc1 transgenic mice (lower panel) as shown by BrdU incorporation assays. (B) Expression of early and late osteoblast marker genes in primary osteoblasts harvested from Cthrc1- mice (upper panel) and Cthrc1 transgenic mice (lower panel). (C) The total numbers of CFU-ALP in bone marrow cell cultures derived from Cthrc1- mice (left) and Cthrc1 transgenic mice (right). (D) The mineralized area of CFU-O in bone marrow cell cultures derived from Cthrc1- mice (left) and Cthrc1 transgenic mice (right). WT: wild-type mice; KO: Cthrc1- mice; Tg: Cthrc1 transgenic mice. Data are shown as the mean±SEM (*p<0.05).

Mentions: Both the loss-of-function and gain-of-function analyses using mouse genetics approaches indicate that Cthrc1 positively regulates osteoblastic bone formation in vivo. To clarify the functions of Cthrc1 in osteoblasts, we isolated osteoblasts from the calvaria of wild-type (Cthrc1+/+ osteoblasts) and Cthrc1- (Cthrc1−/− osteoblasts) newborn mice and analyzed the effects of Cthrc1 on osteoblast proliferation and differentiation in vitro. BrdU incorporation in Cthrc1−/− osteoblasts was 40% less than that in Cthrc1+/+ osteoblasts (Figure 4A). Real-time PCR analyses confirmed that the mRNA levels of the osteoblast marker genes, ALP, Col1a1, and Osteocalcin were all decreased in Cthrc1−/− osteoblasts, whereas the expression level of receptor activator of nuclear factor κB ligand (RANKL), a major determinant of osteoclastogenesis, did not differ (Figure 4B and Figure S4B). We further performed in vitro analyses of osteoprogenitor frequency and differentiation capacity by colony-forming unit (CFU) assays. CFU-ALP assays demonstrated that Cthrc1−/− bone marrow cells had dramatically reduced osteoprogenitors, and mineralized area of CFU-osteoblast (CFU-O) was also reduced by more than half in Cthrc1−/− bone marrow cells, consistent with decreased functional progenitors (Figure 4C and D). Likewise, we examined osteoblast proliferation and differentiation in osteoblasts derived from Cthrc1 transgenic mice (Tg-osteoblasts) in vitro. As shown in Figure 4A, BrdU incorporation in Tg-osteoblasts was 25% greater than that in wild-type osteoblasts. The mRNA levels of ALP, Col1a1, and Osteocalcin were all increased in Tg-osteoblasts, whereas the expression level of RANKL did not differ (Figure 4B and Figure S4D). CFU assays showed that the frequency of osteoprogenitors and mineralized area of CFU-O were significantly increased in Cthrc1 transgenic mice (Figure 4C and D). Thus, these results indicate that Cthrc1 accelerates bone formation by stimulating osteoblast proliferation and differentiation, resulting in increased postnatal bone volume.


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)

Effects of Cthrc1 on osteogenesis.(A) Cell proliferation in primary osteoblasts harvested from Cthrc1- mice (upper panel) and from Cthrc1 transgenic mice (lower panel) as shown by BrdU incorporation assays. (B) Expression of early and late osteoblast marker genes in primary osteoblasts harvested from Cthrc1- mice (upper panel) and Cthrc1 transgenic mice (lower panel). (C) The total numbers of CFU-ALP in bone marrow cell cultures derived from Cthrc1- mice (left) and Cthrc1 transgenic mice (right). (D) The mineralized area of CFU-O in bone marrow cell cultures derived from Cthrc1- mice (left) and Cthrc1 transgenic mice (right). WT: wild-type mice; KO: Cthrc1- mice; Tg: Cthrc1 transgenic mice. Data are shown as the mean±SEM (*p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2527134&req=5

pone-0003174-g004: Effects of Cthrc1 on osteogenesis.(A) Cell proliferation in primary osteoblasts harvested from Cthrc1- mice (upper panel) and from Cthrc1 transgenic mice (lower panel) as shown by BrdU incorporation assays. (B) Expression of early and late osteoblast marker genes in primary osteoblasts harvested from Cthrc1- mice (upper panel) and Cthrc1 transgenic mice (lower panel). (C) The total numbers of CFU-ALP in bone marrow cell cultures derived from Cthrc1- mice (left) and Cthrc1 transgenic mice (right). (D) The mineralized area of CFU-O in bone marrow cell cultures derived from Cthrc1- mice (left) and Cthrc1 transgenic mice (right). WT: wild-type mice; KO: Cthrc1- mice; Tg: Cthrc1 transgenic mice. Data are shown as the mean±SEM (*p<0.05).
Mentions: Both the loss-of-function and gain-of-function analyses using mouse genetics approaches indicate that Cthrc1 positively regulates osteoblastic bone formation in vivo. To clarify the functions of Cthrc1 in osteoblasts, we isolated osteoblasts from the calvaria of wild-type (Cthrc1+/+ osteoblasts) and Cthrc1- (Cthrc1−/− osteoblasts) newborn mice and analyzed the effects of Cthrc1 on osteoblast proliferation and differentiation in vitro. BrdU incorporation in Cthrc1−/− osteoblasts was 40% less than that in Cthrc1+/+ osteoblasts (Figure 4A). Real-time PCR analyses confirmed that the mRNA levels of the osteoblast marker genes, ALP, Col1a1, and Osteocalcin were all decreased in Cthrc1−/− osteoblasts, whereas the expression level of receptor activator of nuclear factor κB ligand (RANKL), a major determinant of osteoclastogenesis, did not differ (Figure 4B and Figure S4B). We further performed in vitro analyses of osteoprogenitor frequency and differentiation capacity by colony-forming unit (CFU) assays. CFU-ALP assays demonstrated that Cthrc1−/− bone marrow cells had dramatically reduced osteoprogenitors, and mineralized area of CFU-osteoblast (CFU-O) was also reduced by more than half in Cthrc1−/− bone marrow cells, consistent with decreased functional progenitors (Figure 4C and D). Likewise, we examined osteoblast proliferation and differentiation in osteoblasts derived from Cthrc1 transgenic mice (Tg-osteoblasts) in vitro. As shown in Figure 4A, BrdU incorporation in Tg-osteoblasts was 25% greater than that in wild-type osteoblasts. The mRNA levels of ALP, Col1a1, and Osteocalcin were all increased in Tg-osteoblasts, whereas the expression level of RANKL did not differ (Figure 4B and Figure S4D). CFU assays showed that the frequency of osteoprogenitors and mineralized area of CFU-O were significantly increased in Cthrc1 transgenic mice (Figure 4C and D). Thus, these results indicate that Cthrc1 accelerates bone formation by stimulating osteoblast proliferation and differentiation, resulting in increased postnatal bone volume.

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