Functional analysis of the cartilage-derived multifunctional growth factor ecogenin/CTGF by using mutant animals

1)我们建立了在小鼠IX型胶原启动子控制下过量产生ecogenin/CTGF的转基因小鼠。小鼠表现出侏儒症和骨密度下降。ctgf在cbfal-null小鼠体内的表达完全被消除，ctgf转基因小鼠恢复了cbfal-null小鼠的软骨分化。2)我们对与K. Layons合作提供的ctgf - null小鼠的表型进行了检测，发现它们不仅存在软骨内成骨障碍，而且存在牙齿发育障碍。3) Ecogenin/CTGF促进关节软骨细胞增殖和蛋白合成，但不促进关节软骨细胞肥大。修复关节软骨缺损，无钙化。Ecogenin/CTGF通过p38 MAPK诱导软骨细胞分化，通过ERK诱导软骨细胞增殖。促生成素/CTGF对软骨细胞的旁分泌作用离不开Perlecan。此外，ecogenin/CTGF与actin结合，过表达时可诱导细胞凋亡，提示细胞内功能。因此，ecogenin/CTGF在肥大软骨细胞中的高水平基因表达，除了作为旁分泌因子的作用外，可能与软骨内成骨末期细胞死亡有关。我们还发现ecogenin/CTGF是缺氧诱导的血管生成因子之一。我们还发现了调节软骨细胞中生态原素/CTGF的顺式元件。4)在骨折愈合过程中，ecogenin/CTGF不仅在增生性软骨组织中表达，在成骨细胞和成骨前细胞中也表达。在拔牙后牙槽骨再生过程中，在成骨细胞中也观察到该基因的表达。机械应力诱导成骨细胞和骨细胞中ecogenin/CTGF的基因表达。这些结果表明，ecogenin/CTGF是一种多功能生长因子，在软骨内骨化、膜内骨化、牙齿发育、关节软骨维持和修复、骨重塑等过程中发挥重要作用。

1) We established transgenic mice that overproduce ecogenin/CTGF under the control of mouse type IX collagen promoter. The mice showed dwarfism and decreases bone density. The expression of ctgf was completely abolished in cbfal -nul I mice, ctgf-transgenic mice rescued the cartilage differentiation of cbfal-null mice.2) We examined the phenotype of ctgf null mice provided in collaboration with K. Layons and found that they had the disorders of not only endochondral ossification but also tooth development.3) Ecogenin/CTGF promoted proliferation and proteogycan synthesis of articular chondrocytes but did not promote their hypertrophy. It also repaired articular cartilage defect without calcification. Ecogenin/CTGF induced chondrocyte differentiation though p38 MAPK, and chondrocyte proliferation through ERK. Perlecan was essential for the paracrine action of ecogenin/CTGF on chondrocytes. Moreover, ecogenin/CTGF was found to bind actin and to induce apoptosis when overexpressed, suggesting intracellular function. Therefore, the high level of gene expression of ecogenin/CTGF in hypertrophic chondrocytes may be related to their cell death at the end of endochondral ossification, in addition to the role as a paracrine factor. We also found tahat ecogenin/CTGF is one of hypoxia-induced angiogenesis factors. We also found cis-element which regulates ecogenin/CTGF in chondrocytes.4) Expression of ecogenin/CTGF was observed in not only in hypertrophic chpndrcoyes but also in osteoblas and pre-osteoblats during fracture healing. Its gene expression was also observed in osteoblasts during alveolar bone regeneration after tooth extraction. Mechanical stress induced gene expression of ecogenin/CTGF in osteoblasts and osteocytes.These findings indicates that ecogenin/CTGF is a multifunctional growth factor which play important roles in endochondral ossification, intramembranous ossification, tooth development, maintainance and repair of articular cartilage and bone remodeling.