Study Of Epidermal Growth Factor Receptor Deficiency On Endochondral Ossification And Its Molecular Mechanism

Vertebrate skeletal elements develop by two distinct mechanisms: intramembranous and endochondral ossification. Intramembranous ossification is the formation of bone on, or in, fibrous connective tissue which is formed from condensed mesenchyme cells. Intramembraneous ossification is the process used to make flat bones such as the clavicle, mandible and flat bones of the skull. Endochondral ossification is initiated by mesenchymal cells. Mesenchymal cells differentiate into chondroblasts which can generate hyaline cartilage to make the cartilage model, within which, chondroblasts develop, proliferate, differentiate and apoptosis. Eventually the cartilage is replaced by bone matrix with the invasion of blood vessels, accompanied by osteoclasts and osteoblasts. Endochondral ossification is the process used to make most of the bone in vertebrate, such as limb long bones, scapular and digit bones. Normal endochondral bone development requires the exquisite coordination of hypertrophic cartilage formation, vascular invasion, and the development and function of osteoclasts and osteoblasts. It is also regulated by many factors such as, systematic factors, local factors, different transcription factors et al.The epidermal growth factor receptor(EGFR) belongs to a family of tyrosine kinases that includes four members (EGFR/ErbB1,HER2/ErbB2,HER3/ErbB3 and HER4/ErbB4). EGFR and its ligands function in diverse celluar functions including cell proliferation, differentiation, motility, and survival. EGFR signaling is important for the development of many tissues, including skins, lungs, intestines, and the craniofacial skeleton. Recent studies showed: EGFR-deficient mice have abnormal craniofacial cartilage and intramembranous bone formation resulting in abnormal development with narrow, elongated snouts, underdeveloped jaw, and a high incidence of cleft palate. The study suggests that EGFR signaling pathway play an important role in bone development. We have now determined the role of EGFR signalling in endochondral ossification, which is the first report for the EGFR signalling in endochondral ossification.We analyzed long bone development in EGFR-deficient mice by Trichrome Masson staining, TRAP staining, gelatine substrate gel analysis, northern blot and in situ hybridization techniques in vivo, and by osteoclast proliferation and formation assays in vitro as well. We found that the primary ossification in EGFR+/? bones occurred normally with humeri at E16.5 showing completed invasion of capillaries into the calcified hypertrophic cartilage(HC), with the resultant removal of the middle section of the HCand replacement of this area with vascularized tissues. In contrast, in the EGFR-/- humeri, the middle section of the HC remained intact, indicating delayed primary endochondral ossification. At E18.5, ossification in the EGFR+/? continued in the longitudinal direction resulting in the formation of an area of trabecular bone and a growth plate that contained an area of hypertrophic cartilage of relatively small size. However, at E18.5, the EGFR-/- humeri showed a lengthened HC zone that had not proceeded very far longitudinally. This delay in ossification continued until birth, with continuing accumulation of HC at the growth plates in newborn EGFR-/- compared to EGFR+/?. Trabeculae bone formation was also impaired in EGFR-/- mice. In E18.5 EGFR+/? the primary spongiosa area was large and contained many long trabeculae, but in the EGFR-/- mice, there were only a few short trabeculae. This impairment in bone formation persisted until birth.We next determined the recruitment of osteoclast and osteoblast. TRAP staining showed that many TRAP+ cells within the middle section of the HC in the E16.5 EGFR+/?. In contrast, TRAP+ cells were found mainly at the periphery of the HC in the EGFR-/-. Quantification of the number of these cells on serial sections showed a significant difference in the number of TRAP+ cells inside versus outside the calcified HC between EGFR+/? and EGFR-/-. There were no apparent differencesin the size of the TRAP+ cells. The difference in the number of TRAP+ cells inside the calcified HC between EGFR-/- and EGFR+/? diminished by E18.5 and newborn mice. These resultsindicate that the delayed primary ossification of EGFR- deficient HC is coupled with a delay in osteoclast recruitment. At mean while, we assayed by in situ for the expression of the Cbfa and Col I, markers of osteoblast differentiation and for the expression of osteocalcin, a marker of mature osteoblasts. In E16.5 EGFR+/? many Cbfa and Col I positive cells were found in the middle of the HC zone, whereas in the EGFR-/- these cells were found more at the periphery. Similarly,osteocalcin-positive cells were found in the middle of the HC zone of E16.5 EGFR+/?, but they were largely limited to the periphery in the EGFR-/- bones. Thus the delay in primary ossification of EGFR-/- HC is also coupled to a delay in recruitment of osteoblasts. However, by E18.5, there were many osteocalcin-positive cells in EGFR-/- bones at the cartilage bone junction. These result suggested that fewer bony trabuculae in EGFR-/- may due to the delayed recruitment of osteoblast.?Yet, there were still few trabecular spicules in these bones, and few osteocalcin-positive cells on these spicules. The deficiency in trabecular spicules persisted until birth.To determine the mechanism of delay recruitment of osteoclast, we analyzed by in situ the expression of MMP-9, MMP-13 and MMP-14 which were highly related to osteoclast recruitment. We found that the distribution of these genes was different at E16.5 stage which showed the same pattern as that of osteoclast, however, there was no difference in the amount of these genes'mRNA per cell. These results indicated that the delayed recruitment of osteoclast was not due to the deficiency of MMPs expression. To further investigate the mechanism of delayed recruitment of osteoclast, bone marrow cells from CD1 mice were isolated and cultured in vitro. The addition of AG1478 in these cultures attenuated osteoclast proliferation and formation, which were detected by either MTT or TRAP staining. Addition of increasing concentrations of AG1478 decreased the number of multinucleated TRAP+ cells that were formed in a dose-dependent manner (P<0.05).Our study showed that EGFR deficiency caused delayed primary ossification of the cartilage anlage and delayed osteoclast and osteoblast recruitment, Consequencely, which resulted in an expanded area of hypertrophic cartilage, few bony trabeculae and low bone density. Inhibiton of EGFR tyrosine kinase activity decreased the proliferation and generation of osteoclast from bone marrow cells. The result suggusts an important role of EGFR signaling in endochondral ossifcation.This is the first report regarding the role of EGFR signaling in endochondral ossification and its function in osteoclast proliferation and formation. These results may have crucial impact on bone repair.