Cthrc1 is a positive regulator of osteoblastic bone formation

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-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1-null 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-null 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-null 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-null 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.

Figure 1. Generation of Cthrc1-null 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-null littermates. (D) Whole-mount X-gal staining of E16.5 heterozygous Cthrc1 embryo. WT: wild-type mice; KO: Cthrc1-null mice; +/−: Cthrc1 heterozygous mice.

Figure 2. Bone phenotypes of Cthrc1-null mice.

(A–D) Micro-CT analyses of tibiae in 2-month-old Cthrc1-null and wild-type mice (n = 6). Bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) are shown. (E and F) Bone histomorphometric analyses of vertebrae in 2-month-old Cthrc1-null and wild-type mice (n = 6). Osteoblast number/bone surface (Ob.N/BS) and osteoblast surface/bone surface (Ob.S/BS) are shown. (G) Analyses of osteoblast proliferation in calvaria of 1-week-old Cthrc1-null and wild-type mice by BrdU incorporation assays. Arrows indicate BrdU positive osteoblasts. (H) Calcein double-labeling of vertebrae in 2-month-old Cthrc1-null and wild-type mice. BFR : Bone formation rate. WT: wild-type mice; KO: Cthrc1-null mice. Data are shown as the mean±SEM (^* p<0.05).

Figure 3. Generation of Cthrc1 transgenic mice and analyses of bone phenotypes.

(A) Schematic representation of the Col1a1-Cthrc1 transgene. Poly A: SV40 polyadenylation signal. Arrows indicate the primers for genotyping. (B) Expression of 3×HA-tagged Cthrc1 protein in humeri of E16.5 Cthrc1 transgenic mice. (C–F) Micro-CT analyses of tibiae in 2-month-old Cthrc1 transgenic and wild-type mice (n = 6). Bone volume/total volume (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) are shown. (G and H) Bone histomorphometric analyses of vertebrae in 2-month-old Cthrc1 transgenic and wild-type mice (n = 6). Osteoblast number/bone surface (Ob.N/BS) and osteoblast surface/bone surface (Ob.S/BS) are shown. (I) Analyses of osteoblast proliferation in calvaria of 1-week-old Cthrc1 transgenic and wild-type mice by BrdU incorporation assays. Arrows indicate BrdU positive osteoblasts. (J) Calcein double-labeling of vertebrae in 2-month-old Cthrc1 transgenic and wild-type mice. BFR: Bone formation rate. WT: wild-type mice; Tg: Cthrc1 transgenic mice. Data are shown as the mean±SEM (^* p<0.05).

Figure 4. Effects of Cthrc1 on osteogenesis.

(A) Cell proliferation in primary osteoblasts harvested from Cthrc1-null 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-null mice (upper panel) and Cthrc1 transgenic mice (lower panel). (C) The total numbers of CFU-ALP in bone marrow cell cultures derived from Cthrc1-null mice (left) and Cthrc1 transgenic mice (right). (D) The mineralized area of CFU-O in bone marrow cell cultures derived from Cthrc1-null mice (left) and Cthrc1 transgenic mice (right). WT: wild-type mice; KO: Cthrc1-null mice; Tg: Cthrc1 transgenic mice. Data are shown as the mean±SEM (^* p<0.05).

Figure 5. OVX-induced bone loss is attenuated in Cthrc1 transgenic mice.

(A) micro-CT of tibiae from 3-month-old Cthrc1 transgenic and wild-type mice which were sham-operated or ovariectomized at 2 months of age (n = 5). (B–E) Bone volume/total volume (BV/TV) (B), trabecular number (Tb.N) (C), trabecular thickness (Tb.Th) (D) and bone formation rate (BFR) (E) were assessed by micro-CT or bone histomorphometry. WT: wild-type mice; Tg: Cthrc1 transgenic mice. Data are shown as the mean±SEM (^* p<0.05).