Functional analysis of diastrophic dysplasia sulfate transporter - Its involvement in growth regulation of chondrocytes mediated by sulfated proteoglycans
Functional analysis of diastrophic dysplasia sulfate transporter - Its involvement in growth regulation of chondrocytes mediated by sulfated proteoglycans
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DOI：
10.1074/jbc.273.20.12307
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发表时间：
1998-05-15
影响因子：
4.8
通讯作者：
Hiraki, Y
中科院分区：
文献类型：
--
作者：
Satoh, H;Susaki, M;Hiraki, Y
硫酸异型发育不良转运蛋白（DTDST）基因突变构成了隐性遗传的骨软骨发育不良家族，包括1B型软骨发育不全、II型骨不全和异型发育不良。然而，该基因产物的功能特性尚未被阐明。我们从大鼠UMR-106成骨细胞中克隆大鼠DTDST cDNA。Northern blot分析表明，软骨和肠是DTDST mRNA的主要表达位点。基因组序列分析显示，大鼠DTDST基因至少由5个外显子组成。由于第三外显子的替代利用，两种不同的转录本在软骨细胞中表达，对应于cDNA的5'-未翻译区域的内部部分。向非洲爪蟾卵母细胞注射大鼠和人DTDST cRNA诱导Na+非依赖性硫酸盐转运。细胞外氯化物和碳酸氢盐显著抑制了表达DTDST的转运活性。相比之下，小管Na+独立的硫酸盐转运体Sat-1需要在注射crna的卵母细胞中存在细胞外氯化物。在细胞外存在各种阴离子的情况下，研究了生长板软骨细胞中硫酸盐运输的活性谱，发现与卵母细胞中表达的DTDST基本相同。因此，软骨细胞的硫酸盐转运主要依赖于DTDST系统。最后，我们证明了通过氯酸盐处理软骨细胞对蛋白多糖的低硫酸酸化显著损害了细胞对成纤维细胞生长因子的生长反应，这表明DTDST在软骨内骨形成中的作用。
Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene constitute a family of recessively inherited osteochondrodysplasias including achondrogenesis type 1B, atelosteogenesis type II, and diastrophic dysplasia. However, the functional properties of the gene product have yet to be elucidated. We cloned rat DTDST cDNA from rat UMR-106 osteoblastic cells. Northern blot analysis suggested that cartilage and intestine were the major expression sites for DTDST mRNA. Analysis of the genomic sequence revealed that the rat DTDST gene was composed of at least five exons. Two distinct transcripts were expressed in chondrocytes due to alternative utilization of the third exon, corresponding to an internal portion of the 5'-untranslated region of the cDNA. Injection of rat and human DTDST cRNA into Xenopus laevis oocytes induced Na+-independent sulfate transport. Transport activity of the expressed DTDST was markedly inhibited by extracellular chloride and bicarbonate. In contrast, canalicular Na+-independent sulfate transporter Sat-1 required the presence of extracellular chloride in the cRNA-injected oocytes. The activity profile of sulfate transport in growth plate chondrocytes was studied in the extracellular presence of various anions and found substantially identical to DTDST expressed in oocytes. Thus, sulfate transport of chondrocytes is dominantly dependent on the DTDST system. Finally, we demonstrate that undersulfation of proteoglycans by the chlorate treatment of chondrocytes significantly impaired growth response of the cells to fibroblast growth factor, suggesting a role for DTDST in endochondral bone formation.