Transcriptional regulation of solute carrier family 6 member 9 gene.

BACKGROUND

Since the endoplasmic reticulum (ER) stress response is found in almost all tissues, its regulation and downstream factors have been vigorously explored.

METHODS AND RESULTS

In this study, we performed a comprehensive microarray analysis of genes induced by tunicamycin (Tm) stimulation in the rat cardiac fibroblast cell line H9c2. Tm treatment induced a marked increase in solute carrier family 6 member 9, Slc6a9, mRNA as well as typical ER stress-inducible factors GADD153 and GRP78 mRNA. Slc6a9 mRNA in H9c2 cells was also increased by ER stress inducers thapsigargin (Tg) and brefeldin A (BFA) other than Tm. The effects of inhibitors on the ER-localized stress sensors IRE1, ATF6, and PERK were examined, and the PERK inhibitor GSK2606414 significantly suppressed Tm-induced Slc6a9 mRNA expression. Analysis of Slc6a9 mRNA expression in PERK and ATF4-deficient HEK293 cells established by genome editing showed that Slc6a9 mRNA expression by Tm was suppressed in these cells. Furthermore, the long variant of Slc6a9 mRNA was increased in Tm-treated HEK293, while the short variant was negligible. Analysis of the nucleotide sequence of the 5'-flanking region of the Slc6a9 gene revealed an ATF4-binding sequence in exon1 is highly conserved among many species. As expected, our luciferase reporter assay containing the ATF4 binding sequence near the transcription start site showed a marked increase in promoter activity upon Tm stimulation and ATF4 co-expression.

CONCLUSIONS

The ATF4-binding sequence found near the transcription start site of the Slc6a9 gene is highly conserved among species and the element is functional in the PERK-ATF4 system. This study will help elucidate Slc6a9-mediated regulation of intracellular and extracellular amino acid homeostasis under pathophysiological conditions.