Haploinsufficiency of osterix in chondrocytes impairs skeletal growth in mice

Abstract

(Osx) is essential for both intramembranous or endochondral bone formation. Osteoblast-specific ablation of Osx using Col1␣1-Cre resulted in osteopenia, because of impaired osteoblast differentiation in adult mice. Since Osx is also known to be expressed in chondrocytes, we evaluated the role of Osx expressed in chondrocytes by examining the skeletal phenotype of mice with conditional disruption of Osx in Col2␣1-expressing chondrocytes. Surprisingly, Cre-positive mice that were homozygous for Osx floxed alleles died after birth. Alcian blue and alizarin red staining revealed that the lengths of skeleton, femur, and vertebrae were reduced by 21, 26, and 14% (P Ͻ 0.01), respectively, in the knockout (KO) compared with wild-type mice. To determine if haploid insufficiency of Osx in chondrocytes influenced postnatal skeletal growth, we compared skeletal phenotype of floxed heterozygous mice that were Cre-positive or Cre-negative. Body length was reduced by 8% (P Ͻ 0.001), and areal BMD of total body, femur, and tibia was reduced by 5, 7, and 8% (P Ͻ 0.05), respectively, in mice with conditional disruption of one allele of Osx in chondrocytes. Micro-CT showed reduced cortical volumetric bone mineral density and trabecular bone volume to total volume in the femurs of Osx flox/ϩ ;col2␣1-Cre mice. Histological analysis revealed that the impairment of longitudinal growth was associated with disrupted growth plates in the Osx flox/ϩ ;col2␣1-Cre mice. Primary chondrocytes isolated from KO embryos showed reduced expression of chondral ossification markers but elevated expression of chondrogenesis markers. Our findings indicate that Osx expressed in chondrocytes regulates bone growth in part by regulating chondrocyte hypertrophy. osterix; chondrocyte; endochondral bone formation; col2␣1-Cre; haploinsufficiency ENDOCHONDRAL BONE FORMATION is a process that involves tightly controlled proliferation and differentiation of chondrocytes at the growth plates of long bones. During long bone development, the cartilage anlagen surrounded by the perichondrium derived from mesenchymal cells are first laid down and gradually replaced by bone (17, 28). The cartilage anlagen elongate and expand in width by proliferation of chondrocytes as well as by deposition of collagen II (Col2) rich cartilage matrix. Subsequently, chondrocytes in the central region of the cartilage undergo further differentiation to hypertrophic chondrocytes and synthesize collagen X (Col10)-rich extracellular matrix, which is subsequently mineralized by calcium deposition. The calcified cartilage is then resorbed by chondroclasts and replaced by bone matrix via osteoblasts that are brought in by invading blood vessels. Thus, in the growth plate, a coordinated sequence of chondrocyte proliferation, hypertrophy, and apoptosis results in longitudinal growth of long bones (16,17,28). During endochondral bone formation, chondrocyte differentiation plays a critical role in extracellular matrix formation, mineralization, and the stimulation of osteoblast differentiation. Ch...

Epiphyseal Chondrocyte Secondary Ossification Centers Require Thyroid Hormone Activation of Indian Hedgehog and Osterix Signaling

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Conditional Disruption of the Prolyl Hydroxylase Domain-Containing Protein 2 (Phd2) Gene Defines Its Key Role in Skeletal Development

“…For Western blot analysis, Osx antibody (ab22552; Abcam Inc.) and Phd2 antibody (#4835; Cell Signaling Technology, Inc., Danvers, MA, USA) were used at a 1:1000 dilution. Immunohistochemistry was carried out with paraffin sections of 5‐week femoral metaphysis as described . Osx antibody was used at a dilution of 1:500 and osteocalcin (OC) antibody (ab93876; Abcam Inc., Cambridge, MA, USA) at a dilution of 1:300.…”

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