Cloning of a retinoic acid-sensitive mRNA expressed in cartilage and during chondrogenesis
Cloning of a retinoic acid-sensitive mRNA expressed in cartilage and during chondrogenesis
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DOI：
10.1074/jbc.271.6.3311
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发表时间：
1996-02-09
影响因子：
4.8
通讯作者：
Sandell, LJ
中科院分区：
文献类型：
--
作者：
Dietz, UH;Sandell, LJ
已知视黄酸（RA）在体内骨骼发育的各个方面发挥作用，包括形态发生、生长板成熟和凋亡。在细胞培养中，软骨细胞的RA处理抑制以II型胶原和聚集蛋白聚糖的产生为特征的分化表型。为了发现参与软骨细胞表型调节或与发育过程如软骨发生相关的分子，用逆转录-聚合酶链反应（PCR）扩增了含和不含RA的牛软骨细胞的mRNA，并通过差异显示进行了比较，克隆了表达被RA抑制的PCR产物，其中一个cDNA编码一种我们称之为软骨衍生的视黄酸敏感蛋白（CD-RAP）的分子，并对其性质进行了描述。本研究采用cDNA末端快速扩增法扩增了牛CD-BAP基因的全长mRNA，并采用序列特异性引物逆转录-PCR扩增了大鼠CD-RAP基因的部分片段。牛CD-RAP基因含有一个130个氨基酸的开放阅读框，通过北方印迹分析和原位杂交检测了牛CD-RAP基因的表达，在体外RA对CD-RAP mRNA表达的抑制具有时间和剂量依赖性，并在10（-8）至10（-6）M的浓度范围内进行测试，基因组DNA的Southern印迹分析表明，CD-RAP由单拷贝基因编码，没有其他基因密切相关，最近从黑色素瘤细胞系中分离并克隆了似乎是CD-RAP的人同源物，并显示其作为生长抑制蛋白发挥作用（Blesch，A.，Boberhoff，A. K.，阿普费尔河，贝尔，C.，Hessdoerfer，B.，施密特，A.，Jachimcza，P.，Lottspeich，F.，比特纳河，和Bogdahn，U.（1994）Cancer Res，54，5695-5701）。无论是CD-RAP还是这种蛋白质都没有显示出与已知蛋白质的任何同源性，我们推测，在体内，CD-RAP在软骨发育和维持过程中发挥作用。
Retinoic acid (RA) is known to play a role in various aspects of skeletal development in vivo, including morphogenesis, growth plate maturation, and apoptosis, In cell culture, RA treatment of chondrocytes suppresses the differentiated phenotype characterized by production of type II collagen and aggrecan, In an effort to discover molecules involved in regulation of the chondrocyte phenotype or related to developmental processes such as chondrogenesis, mRNAs from bovine chondrocytes cultured with and without RA were amplified by reverse transcription-polymerase chain reaction (PCR) and compared by differential display, PCR products whose expression was inhibited by RA treatment were cloned, One cDNA encodes a molecule we call cartilage-derived retinoic acid-sensitive protein (CD-RAP), and its properties are described here. The full-length bovine CD-BAP mRNA was cloned after amplification by the rapid amplification of cDNA ends procedure, and a part of the rat CD-RAP mRNA was amplified by reverse transcription-PCR using sequence-specific primers, The bovine CD-RAP mRNA contains an open reading frame of 130 amino acids, CD-RAP mRNA expression, as determined by Northern blot analysis and in situ hybridization, was present only in cartilage primordia and cartilage, The inhibition of CD-RAP mRNA expression by RA in vitro was time- and dose-dependent and was tested over concentrations from 10(-8) to 10(-6) M, Southern blot analysis of genomic DNA indicated that CD-RAP was encoded by a single copy gene and that no other genes were closely related, What appears to be the human homologue of CD-RAP was recently isolated and cloned from a melanoma cell line and shown to function as a growth inhibitory protein (Blesch, A., Boberhoff, A.-K., Apfel, R., Behl, C., Hessdoerfer, B., Schmitt, A., Jachimcza, P., Lottspeich, F., Buettner, R., and Bogdahn, U. (1994) Cancer Res, 54, 5695-5701). Neither CD-RAP nor this protein showed any homology to known proteins, We speculate that, in vivo, CD-RAP functions during cartilage development and maintenance.