Transcriptional regulation of the sodium-coupled neutral amino acid transporter (SNAT2) by 17β-estradiol

Abstract

The sodium-coupled neutral amino acid transporter 2 (SNAT2) translocates small neutral amino acids into the mammary gland to promote cell proliferation during gestation. It is known that SNAT2 expression increases during pregnancy, and in vitro studies indicate that this transporter is induced by 17β-estradiol. In this study, we elucidated the mechanism by which 17β-estradiol regulates the transcription of SNAT2. In silico analysis revealed the presence of a potential estrogen response element (ERE) in the SNAT2 promoter. Reporter assays showed an increase in SNAT2 promoter activity when cotransfected with estrogen receptor alpha (ER-α) after 17β-estradiol stimulation. Deletion of the ERE reduced estradiol-induced promoter activity by 63%. Additionally, EMSAs and supershift assays showed that ER-α binds to the SNAT2 ERE and that this binding competes with the interaction of ER-α with its consensus ERE. An in vivo ChIP assay demonstrated that the binding of ER-α to the SNAT2 promoter gradually increased in the mammary gland during gestation and that maximal binding occurred at the highest 17β-estradiol serum concentration. Liquid chromatography-elevated energy mass spectrometry and Western blot analysis revealed that the SNAT2 ER-α-ERE complex contained poly(ADP-ribose) polymerase 1, Lupus Ku autoantigen protein p70, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) proteins and that the silencing of each of these proteins nearly abolished 17β-estradiol-stimulated SNAT2 promoter activity. Nuclear levels of GAPDH increased progressively during gestation in the mammary gland, and GAPDH binding was nucleotide-specific for the SNAT2 ERE. Thus, this study provides new insights into how the mammary epithelium adapts to control amino acid uptake through the transcriptional regulation of the SNAT2 transporter via 17β-estradiol.coactivator | amino acid transport T he mammary gland has a high demand for amino acids during the gestation and lactation periods. It requires the uptake of amino acids for cell proliferation during pregnancy and for milk protein synthesis during lactation. Studies examining arteriovenous differences have demonstrated that glutamine and alanine are efficiently transported into the mammary gland at the start of lactation (1). These amino acids are primarily transported by amino acid transport system A, which is comprised of three subtypes known as sodium-coupled neutral amino acid transporter (SNAT) 1, 2, and 4 (2). SNAT2 is characterized as the classical system A transporter, is ubiquitous in mammalian cells, and plays various roles in different tissues and depending on specific physiological conditions (3-5). SNAT2 also provides efflux substrates for other amino acid transport systems, like the amino acid heteroexchanger system L, facilitating the uptake of branched-chain amino acids, particularly leucine that activates the TOR pathway involved in protein synthesis and cell proliferation (6). Up-regulation of SNAT2 gene expression is mediated by different signal transduction pathways. In the l...

Identification of estrogen-responsive genes based on the DNA binding properties of estrogen receptors using high-throughput sequencing technology

“…However, among them, only a small number have been shown to possess functional EREs within the transcription regulatory region. In mammals, these genes include transcription factors, such as JUN [32] , FOS [33] , PGR [34] , and TP53 [35] , intracellular signaling molecules, such as HRAS [36] , BCL2 [37] , and BRCA1 [38] , enzymes, such as CHAT [39] , NQO1 [40] , and CKB [41] , secreted proteins, such as LTF [42] , SCGB1A1 [43] , OVGP1 [44] , C3 [45] , and AGT [46] , hormones, such as LHB [47] , OXT [48] , PRL [49] , and AVP [50] , membrane proteins, such as SNAT2 [51] and VEGFA [52] , the motogen TFF1 [53] , and the protease CTSD [54] . These genes are assumed to directly mediate various estrogen actions in normal tissues, as well as in cancer and other diseases.…”

Prolactin and the dietary protein/carbohydrate ratio regulate the expression of SNAT2 amino acid transporter in the mammary gland during lactation

“…Recently, it has been demonstrated that upregulation of SNAT2 gene expression during gestation is mediated by the estrogen receptor α bound to the coactivator glyceraldehyde 3-phosphate dehydrogenase that specifically binds to an estrogen response element found in the SNAT2 gene promoter [13]. Interestingly, we have evidence that after gestation, there is a second increase of SNAT2 mRNA that reaches its maximal in the peak of lactation in the mammary gland [12].…”

Hypoxia-induced switch in SNAT2/SLC38A2 regulation generates endocrine-resistance in breast cancer

“…Previous studies showed that the expression of SNAT2 was increased in ER+ positive breast cancer cell lines after 17β-estradiol (E 2 ) stimulation and an estrogen response element (ERE) was described in the SNAT2 promoter in rat mammary glands during gestation (31, 32). Because hypoxia is related to adverse outcome in tamoxifen-treated breast cancer patients (14) and SNAT2 was among the 202 genes bound by HIF-1α and ERα and regulated by fulvestrant (14), we investigated the regulation of SNAT2 expression by estrogen and hypoxia in ER+ breast cancer cell lines.…”