Functional Analysis of Diastrophic Dysplasia Sulfate Transporter: ITS INVOLVEMENT IN GROWTH REGULATION OF CHONDROCYTES MEDIATED BY SULFATED PROTEOGLYCANS

Hideshi Satoh,Masakazu Susaki,Chisa Shukunami,Ken-Ichi Iyama,Takaharu Negoro,Yuji Hiraki

doi:10.1074/jbc.273.20.12307

Abstract

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.

Highlights

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
Using RT-polymerase chain reaction (PCR) with human DTDST-specific primers 5 and 6 (Table I), we amplified rat cDNA from total RNA isolated from rat UMR106 osteoblastic cells
The entire coding region and the 5Јand 3Ј-regions of the cDNA were isolated by RT-PCR and rapid amplification of cDNA ends (RACE) reactions

Summary

Introduction

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. Northern blot analysis suggested that cartilage and intestine were the major expression sites for DTDST mRNA. 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. The oocyte expression system has been proved to be a powerful tool for functional analysis of the DRA and Sat-1 gene products [6, 8]. We directly characterized the sulfate transport activity of rat DTDST by injection of its cRNA into Xenopus oocytes and compared it to that of normal rat costal chondrocytes. The activity profile of the gene product showed an ion dependence of transport distinct from that of Sat-1, suggesting that DTDST was a sulfate/chloride antiporter.

Methods

Results

Conclusion