Epiphyseal abnormalities, trabecular bone loss and articular chondrocyte hypertrophy develop in the long bones of postnatal Ext1-deficient mice

Abstract

Long bones are integral components of the limb skeleton. Recent studies have indicated that embryonic long bone development is altered by mutations in Ext genes and consequent heparan sulfate (HS) deficiency, possibly due to changes in activity and distribution of HS-binding/growth plate-associated signaling proteins. Here we asked whether Ext function is continuously required after birth to sustain growth plate function and long bone growth and organization. Compound transgenic Ext1f/f;Col2CreERT mice were injected with tamoxifen at postnatal day 5 (P5) to ablate Ext1 in cartilage and monitored over time. The Ext1-deficient mice exhibited growth retardation already by 2 weeks post-injection, as did their long bones. Mutant growth plates displayed a severe disorganization of chondrocyte columnar organization, a shortened hypertrophic zone with low expression of collagen X and MMP-13, and reduced primary spongiosa accompanied, however, by increased numbers of TRAP-positive osteoclasts at the chondro-osseous border. The mutant epiphyses were abnormal as well. Formation of a secondary ossification center was significantly delayed but interestingly, hypertrophic-like chondrocytes emerged within articular cartilage, similar to those often seen in osteoarthritic joints. Indeed, the cells displayed a large size and round shape, expressed collagen X and MMP-13 and were surrounded by an abundant Perlecan-rich pericellular matrix not seen in control articular chondrocytes. In addition, ectopic cartilaginous by EXT mutations and HS deficiency. In sum, the data do show that Ext1 is continuously required for postnatal growth and organization of long bones as well as their adjacent joints. Ext1 deficiency elicits defects that can occur in human skeletal conditions including trabecular bone loss, osteoarthritis and HME.

Interactions of signaling proteins, growth factors and other proteins with heparan sulfate: mechanisms and mysteries

“…It has remained unclear and controversial as to whether and how the HS deficiency causes exostosis formation, why the exostoses form near but not inside the growth plates, whether the exostoses are formed by aberrant function of growth plate chondrocytes themselves or involve perichondrial progenitors, and what growth factors trigger and sustain exostosis outgrowth ( 4,53 ). In studies we and others carried out previously, we found that mutant mice lacking one allele of Ext1 or Ext2 did not reproduce the human HME skeletal phenotype, while double heterozygous Ext1 +/− ; Ext2 +/− mice or conditionally ablated Ext1 −/− mice did ( 55, 56 ). The data strongly indicated that a partial decrease in HS levels such as that elicited by a simple heterozygous EXT mutation is insufficient for exostosis formation and HME progression, but a deeper decrease is required.…”

Aberrant perichondrial BMP signaling mediates multiple osteochondromagenesis in mice

“…The penetrance of the phenotypes is largely comparable between the two models, except for the higher occurrence of osteochondromas in the distal humerus, proximal radius, and distal femur and the lack of scoliosis in the Fsp1-Ext1 CKO model. Additionally, unlike Col2a1-Ext1 CKO mice, which exhibit enhanced osteoclastogenesis (26), there was no increase in the number of osteoclasts in Fsp1-Ext1 CKO mice (Supplemental Figure 3). Overall, these results demonstrate that loss of Ext1 restricted to perichondrial progenitor cells is sufficient to induce MHE-like osteochondromagenesis in various bones.…”

Osteoblastic heparan sulfate regulates osteoprotegerin function and bone mass

“…Meanwhile, it remains to be determined whether the latter phenotype is secondary to osteopenia or a primary developmental defect independent of osteopenia, as abnormalities in secondary ossification centers are not a common feature seen in a number of severely osteopenic mouse lines. Interestingly, a chondrocyte-targeted Ext1 knockout induced by a Col2-based Cre driver causes a delay in secondary ossification center formation (46), suggesting that HS may be functionally involved in the development of a secondary ossification center. This chondrocyte-targeted knockout mouse model also exhibits a reduction in trabecular bone volume (46).…”

“…Interestingly, a chondrocyte-targeted Ext1 knockout induced by a Col2-based Cre driver causes a delay in secondary ossification center formation (46), suggesting that HS may be functionally involved in the development of a secondary ossification center. This chondrocyte-targeted knockout mouse model also exhibits a reduction in trabecular bone volume (46). Considering that the Col2-Cre transgene exhibits a leaky expression in osteochondroprogenitors (47), this phenotype may be due to Ext1 ablation in the osteoblastic lineage.…”