Tamamura Yoshihiro, Otani Tomohiro, Kanatani Naoko, Koyama Eiki, Kitagaki Jirota, Komori Toshihisa, Yamada Yoshihiko, Costantini Frank, Wakisaka Satoshi, Pacifici Maurizio, Iwamoto Masahiro, Enomoto-Iwamoto Motomi
Department of Orthopaedic Surgery, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 2005 May 13;280(19):19185-95. doi: 10.1074/jbc.M414275200. Epub 2005 Mar 10.
Studies have suggested that continuous Wnt/beta-catenin signaling in nascent cartilaginous skeletal elements blocks chondrocyte hypertrophy and endochondral ossification, whereas signaling starting at later stages stimulates hypertrophy and ossification, indicating that Wnt/beta-catenin roles are developmentally regulated. To test this conclusion further, we created transgenic mice expressing a fusion mutant protein of beta-catenin and LEF (CA-LEF) in nascent chondrocytes. Transgenic mice had severe skeletal defects, particularly in limbs. Growth plates were totally disorganized, lacked maturing chondrocytes expressing Indian hedgehog and collagen X, and failed to undergo endochondral ossification. Interestingly, the transgenic cartilaginous elements were ill defined, intermingled with surrounding connective and vascular tissues, and even displayed abnormal joints. However, when activated beta-catenin mutant (delta-beta-catenin) was expressed in chondrocytes already engaged in maturation such as those present in chick limbs, chondrocyte maturation and bone formation were greatly enhanced. Differential responses to Wnt/beta-catenin signaling were confirmed in cultured chondrocytes. Activation in immature cells blocked maturation and actually de-stabilized their phenotype, as revealed by reduced expression of chondrocyte markers, abnormal cytoarchitecture, and loss of proteoglycan matrix. Activation in mature cells instead stimulated hypertrophy, matrix mineralization, and expression of terminal markers such as metalloprotease (MMP)-13 and vascular endothelial growth factor. Because proteoglycans are crucial for cartilage function, we tested possible mechanisms for matrix loss. Delta-beta-catenin expression markedly increased expression of MMP-2, MMP-3, MMP-7, MMP-9, MT3-MMP, and ADAMTS5. In conclusion, Wnt/beta-catenin signaling regulates chondrocyte phenotype, maturation, and function in a developmentally regulated manner, and regulated action by this pathway is critical for growth plate organization, cartilage boundary definition, and endochondral ossification.
研究表明，在新生软骨骨骼元件中持续的Wnt/β-连环蛋白信号传导会阻止软骨细胞肥大和软骨内骨化，而在后期开始的信号传导则会刺激肥大和骨化，这表明Wnt/β-连环蛋白的作用受发育调控。为了进一步验证这一结论，我们构建了在新生软骨细胞中表达β-连环蛋白与淋巴样增强因子（LEF）融合突变蛋白（CA-LEF）的转基因小鼠。转基因小鼠存在严重的骨骼缺陷，尤其是在四肢。生长板完全紊乱，缺乏表达印度刺猬因子和胶原蛋白X的成熟软骨细胞，并且无法进行软骨内骨化。有趣的是，转基因软骨元件界限不清，与周围的结缔组织和血管组织相互交织，甚至出现异常关节。然而，当在已经参与成熟的软骨细胞（如鸡四肢中的软骨细胞）中表达活化的β-连环蛋白突变体（δ-β-连环蛋白）时，软骨细胞成熟和骨形成会大大增强。在培养的软骨细胞中证实了对Wnt/β-连环蛋白信号传导的不同反应。在未成熟细胞中激活会阻止成熟，并实际上使其表型不稳定，这表现为软骨细胞标志物表达降低、细胞结构异常以及蛋白聚糖基质丧失。相反，在成熟细胞中激活则会刺激肥大、基质矿化以及诸如金属蛋白酶（MMP）-13和血管内皮生长因子等终末标志物的表达。由于蛋白聚糖对软骨功能至关重要，我们测试了基质丧失的可能机制。δ-β-连环蛋白的表达显著增加了MMP-2、MMP-3、MMP-7、MMP-9、MT3-MMP和ADAMTS5的表达。总之，Wnt/β-连环蛋白信号传导以发育调控的方式调节软骨细胞表型、成熟和功能，并且该途径的调控作用对于生长板组织、软骨边界界定和软骨内骨化至关重要。
