Iwamoto M, Higuchi Y, Enomoto-Iwamoto M, Kurisu K, Koyama E, Yeh H, Rosenbloom J, Pacifici M
Department of Oral Anatomy and Developmental Biology, Osaka University Faculty of Dentistry, Osaka, Japan.
Osteoarthritis Cartilage. 2001;9 Suppl A:S41-7. doi: 10.1053/joca.2001.0443.
Based on function and developmental fate, cartilage tissue can be broadly classified into two types: transient (embryonic or growth-plate) cartilage and permanent cartilage. Chondrocytes in transient cartilage undergo terminal differentiation into hypertrophic cells, induce cartilage-matrix mineralization, and eventually disappear and are replaced by bone. On the other hand, chondrocytes in permanent cartilage do not differentiate further, do not become hypertrophic, and persist throughout life at specific sites, including joints and tracheal rings. While many studies have described differences in structure, matrix composition and biological characteristics between permanent and transient cartilage, it is poorly understood how the fates of permanent and transient cartilage are determined. Previous studies demonstrated that chondrocytes isolated from permanent cartilage have the potential to express markers of the mature hypertrophic phenotype once grown in culture, suggesting that cell hypertrophy is an intrinsic property of all chondrocytes and must be actively silenced in permanent cartilage in vivo. These silencing mechanisms, however, are largely unknown. In this paper, we first review nature of chondrocytes in transient and permanent cartilages and then report the cloning and characterization of a novel variant of ets transcription factor chERG, hereafter called C-1-1, which might be involved in regulation of permanent cartilage development.
For cloning of a novel variant of chERG (C-1-1), we isolated RNA from the cartilaginous femur or tibiotarsus of Day 17 chick embryos and processed it for reverse transcription-polymerase chain reaction (RT-PCR) with the primers from sequences upstream and downstream of the 81 and 72 bp segments alternatively-spliced in mammals. For investigation of function of chERG and C-1-1, we over-expressed chERG or C-1-1 in cultured chick chondrocytes or the developing limb of chick embryo using a retrovirus (RCAS) system, and examined the phenotype changes in the infected chondrocytes or the infected limb elements.
C-1-1 is an alternative and novel variant lacking the 27 amino acids segment of chERG that has been reported previously. C-1-1 is preferentially expressed in developing articular cartilage, whereas chERG is preferentially expressed in growth plate cartilage. Growth of articular chondrocytes in culture was accompanied by decreasing C-1-1 expression after several passages, while expression of hypertrophic markers increased. Expression of C-1-1 in cultured chondrocytes inhibited cell hypertrophy, alkaline phosphatase activity, and cartilage matrix mineralization. In contrast, over-expression of chERG promoted chondrocyte maturation and mineralization.
Our data demonstrate for the first time that chERG and C-1-1 play distinct roles in skeletogenesis and may have crucial roles in the development and function of transient and permanent cartilages.
根据功能和发育命运，软骨组织可大致分为两类：暂时性（胚胎性或生长板）软骨和永久性软骨。暂时性软骨中的软骨细胞经历终末分化成为肥大细胞，诱导软骨基质矿化，最终消失并被骨替代。另一方面，永久性软骨中的软骨细胞不再进一步分化，不会变成肥大细胞，并在包括关节和气管环在内的特定部位终生存在。虽然许多研究描述了永久性和暂时性软骨在结构、基质组成和生物学特性方面的差异，但对于永久性和暂时性软骨的命运是如何决定的却知之甚少。先前的研究表明，从永久性软骨中分离出的软骨细胞一旦在培养中生长就有表达成熟肥大表型标志物的潜力，这表明细胞肥大是所有软骨细胞的固有特性，在体内的永久性软骨中必须被积极沉默。然而，这些沉默机制在很大程度上尚不清楚。在本文中，我们首先综述了暂时性和永久性软骨中软骨细胞的性质，然后报告了一种新的ets转录因子chERG变体（以下称为C-1-1）的克隆和特性，它可能参与永久性软骨发育的调控。
为了克隆chERG的新变体（C-1-1），我们从第17天鸡胚的软骨股骨或胫跗骨中分离RNA，并用来自在哺乳动物中交替剪接的81和72 bp片段上下游序列的引物对其进行逆转录-聚合酶链反应（RT-PCR）处理。为了研究chERG和C-1-1的功能，我们使用逆转录病毒（RCAS）系统在培养的鸡软骨细胞或鸡胚发育中的肢体中过表达chERG或C-1-1，并检查感染的软骨细胞或感染的肢体元件中的表型变化。
C-1-1是一种新的替代变体，缺少先前报道的chERG的27个氨基酸片段。C-1-1在发育中的关节软骨中优先表达，而chERG在生长板软骨中优先表达。培养的关节软骨细胞生长时，经过几次传代后C-1-1表达降低，而肥大标志物的表达增加。在培养的软骨细胞中C-1-1的表达抑制细胞肥大、碱性磷酸酶活性和软骨基质矿化。相反，chERG的过表达促进软骨细胞成熟和矿化。
我们的数据首次证明chERG和C-1-1在骨骼发生中发挥不同作用，并且可能在暂时性和永久性软骨的发育和功能中起关键作用。
