Amino Acid Transporter SLC6A14 Research Articles

Abstract 4340: The amino acid transporter SLC6A14 is an effective drug target for treatment of pancreatic cancer

Abstract Pancreatic cancer is the deadliest of all cancers with worst outcome and poor survival rate. Chemotherapy with gemcitabine works well for early stage cancer, but becomes ineffective for advanced-stage cancer. As such, there is a dire need for new approaches to treat this cancer. The metabolism of tumor cells is very different from that of normal cells. In particular, the differences in amino acid metabolism are gaining increasing attention in cancer biology. Selective amino acid transporters are upregulated in cancer in response to the increased demands for amino acids in tumor cells. Such tumor-selective amino acid transporters are logical druggable targets for cancer therapy. As such, pharmacologic blockade of such upregulated transporters would lead to cell death selectively in tumor cells by depriving the tumor cells of essential nutrients. With this in mind, we analyzed 8 different publically available microarray datasets in Gene Expression Omnibus for the amino acid transporters that are upregulated in pancreatic cancer. This led to the identification of three transporters (SLC1A5, SLC7A5, and SLC6A14) that are overexpressed in tumors compared to normal tissue. Among these, the fold-increase in expression was the highest for SLC6A14 in all datasets (range for fold-increase, 1.3 - 163.3; range for p value, 0.05 - 2x10-14). The fold-increase for the other two transporters is 2 at the maximum. We confirmed these findings using primary pancreatic cancer tissues and cancer cell lines. First, we used paired samples of pancreatic cancer tissues and the corresponding adjacent normal tissues. SLC6A14 mRNA was increased at least by 7-fold in tumor versus normal tissue. The same was true with three additional unpaired cancer and normal pancreatic tissues. We then evaluated the expression of the transporter at mRNA and protein level in a normal pancreatic cell line (HPNE) and five different pancreatic cancer cell lines. The transporter expression was higher in cancer cell lines compared to the normal cell line. This was confirmed by immunofluorescence by comparing the transporter expression between the normal cell line HPNE and the cancer cell line BxPC-3. Blockade of the transporter function in BxPC-3 cells with α-methyltryptophan (-MT) induced amino acid starvation, activated autophagy, altered cell cycle distribution and inhibited mTOR and colony formation. Decreased invasion and migration observed with α-MT in tumor cells was associated with a decrease in stabilization of HIF-1α. Xenograft studies in nude mice showed that administration of α-MT decreased the growth of BxPC-3 and Mia PaCa-2 cells into tumor. To conclude, SLC6A14 is upregulated in pancreatic cancer and blockade of the transporter with α-MT shows differential and selective detrimental effects on pancreatic tumor cells, both in vitro and in vivo. These studies identify SLC6A14 as a novel and effective drug target for pancreatic cancer treatment. Citation Format: Yangzom D. Bhutia, Veena Coothankandaswamy, Sha Cao, Ying Xu, Vadivel Ganapathy. The amino acid transporter SLC6A14 is an effective drug target for treatment of pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4340. doi:10.1158/1538-7445.AM2014-4340

Abstract 3928: Genetic deletion or pharmacologic blockade of the amino acid transporter Slc6a14 in mice suppresses breast cancer induced by Polyoma middle T oncogene

Abstract Tumor cells have an increased need for amino acids. Mammalian cells cannot synthesize essential amino acids; they must obtain these amino acids via specific transporters. Glutamine, though a non-essential amino acid, is critical for tumor cells (glutamine addiction). Entry of amino acids into tumor cells is enhanced by upregulation of specific transporters. If we can interfere with the entry of amino acids into tumor cells, we should be able to starve these cells to death. If the transporters that are specifically induced in tumor cells are identified, blockade of the induced transporters would constitute a logical strategy for cancer treatment. Mammalian cells express ∼40 amino acid transporters, expressed in different combinations and in a cell type-specific manner. Among them, SLC6A14 is unique in that it transports all essential amino acids as well as glutamine, and is expressed only at low levels in normal tissues, but induced in colon cancer and in ER+ breast cancer. Tumor cells in these cancers upregulate SLC6A14 to meet their increased demand for essential amino acids and glutamine, indicating that SLC6A14 drives their “glutamine addiction.” We have now established the potential of this transporter as a drug target for breast cancer treatment using genetic and pharmacologic approaches. For this, we first generated Slc6a14-/- mouse. The Slc6a14 gene is located on X chromosome. Deletion of the gene is not lethal and there is no overt phenotype in hemizygous males (-/y) or in homozygous females (-/-). We then examined the progression of breast cancer in Polyoma middle T antigen (Py-MT) Tg mouse on Slc6a14+/+ and Slc6a14-/- background. Deletion of Slc6a14 markedly suppressed breast cancer and lung metastasis induced by the Py-MT oncogene. We have also identified -methyl-L-tryptophan (-MLT) as a selective blocker of Slc6a14. We investigated the consequences of pharmacologic blockade of Slc6a14 on Py-MT-induced breast cancer with oral administration of -MLT (1 mg/ml in drinking water). The blocker was able to suppress breast cancer to a marked extent. Py-MT-induced breast tumors showed robust upregulation of Slc6a14; however, the tumors also showed upregulation of Slc7a5/Slc3a2, another amino acid transporter. Therefore, we examined the interaction of -MLT with Slc7a5/Slc3a2 and found that while -MLT is a blocker of Slc6a14, it is a transportable substrate for Slc7a5/Slc3a2, demonstrating that only the function of Slc6a14 is selectively blocked by -MLT. We conclude that blockade of Slc6a14 is a viable strategy for treatment of certain specific subtypes of breast cancer (e.g., ER-positive) that are associated with upregulation of the transporter. Citation Format: Babu Ellappan, Yangzom D. Bhutia, Muthusamy Thangaraju, Puttur D. Prasad, Vadivel Ganapathy. Genetic deletion or pharmacologic blockade of the amino acid transporter Slc6a14 in mice suppresses breast cancer induced by Polyoma middle T oncogene. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3928. doi:10.1158/1538-7445.AM2014-3928

Downregulation of the Creatine Transporter SLC6A8 by JAK2

Janus-activated kinase-2 (JAK2) participates in the regulation of the Na⁺-coupled glucose transporter SGLT1 and the Na⁺-coupled amino acid transporter SLC6A19. Concentrative cellular creatine uptake is similarly accomplished by Na⁺-coupled transport. The carrier involved is SLC6A8 (CreaT). The present study thus explored whether JAK2 regulates the activity of SLC6A8. To this end, cRNA encoding SLC6A8 was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, constitutively active (V617F)JAK2 or inactive (K882E)JAK2. Electrogenic creatine transport was determined in those oocytes by dual-electrode voltage-clamp experiments. In oocytes injected with cRNA encoding SLC6A8 but not in oocytes injected with water or with cRNA encoding JAK2 alone, addition of 1 mM creatine to the extracellular bath generated an inward current (I (crea)). In SLC6A8 expressing oocytes I (crea) was significantly decreased by coexpression of JAK2 or (V617F)JAK2 but not by coexpression of (K882E)JAK2. According to kinetic analysis, coexpression of JAK2 decreased the maximal transport rate without significantly modifying the affinity of the carrier. In oocytes expressing SLC6A8 and (V617F)JAK2 I (crea) was gradually increased by the JAK2 inhibitor AG490 (40 μM). In SLC6A8 and JAK2 coexpressing oocytes the decline of I (crea) following disruption of carrier insertion with brefeldin A (5 μM) was similar in the absence and presence of JAK2. In conclusion, JAK2 is a novel regulator of the creatine transporter SLC6A8, which downregulates the carrier, presumably by interference with carrier protein insertion into the cell membrane.

Up-Regulation of Amino Acid Transporter SLC6A19 Activity and Surface Protein Abundance by PKB/Akt and PIKfyve

Background: The amino acid transporter B0AT1 (SLC6A19) accomplishes concentrative cellular uptake of neutral amino acids. SLC6A19 is stimulated by serum- & glucocorticoid-inducible kinase (SGK) isoforms. SGKs are related to PKB/Akt isoforms, which also stimulate several amino acid transporters. PKB/Akt modulates glucose transport in part by phosphorylating and thus activating phosphatidylinositol-3-phosphate-5-kinase (PIKfyve), which fosters carrier protein insertion into the cell membrane. The present study explored whether PKB/Akt and/or PIKfyve stimulate SLC6A19. Methods: SLC6A19 was expressed in Xenopus oocytes with or without wild-type PKB/Akt or inactive ^T308A/S473APKB/Akt without or with additional expression of wild-type PIKfyve or PKB/Akt-resistant ^S318APIKfyve. Electrogenic amino acid transport was determined by dual electrode voltage clamping. Results: In SLC6A19-expressing oocytes but not in water-injected oocytes, the addition of the neutral amino acid L-leucine (2 mM) to the bath generated a current (I_le), which was significantly increased following coexpression of PKB/Akt, but not by coexpression of ^T308A/S473APKB/Akt. The effect of PKB/Akt was augmented by additional coexpression of PIKfyve but not of ^S318APIKfyve. Coexpression of PKB/Akt enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The decline of I_le following inhibition of carrier insertion by brefeldin A (5 µM) was similar in the absence and presence of PKB/Akt indicating that PKB/Akt stimulated carrier insertion into rather than inhibiting carrier retrieval from the cell membrane. Conclusion: PKB/Akt up-regulates SLC6A19 activity, which may foster amino acid uptake into PKB/Akt-expressing epithelial and tumor cells.

Stimulation of the amino acid transporter SLC6A19 by JAK2

JAK2 (Janus kinase-2) is expressed in a wide variety of cells including tumor cells and contributes to the proliferation and survival of those cells. The gain of function mutation V617FJAK2 mutant is found in the majority of myeloproliferative diseases. Cell proliferation depends on the availability of amino acids. Concentrative cellular amino acid uptake is in part accomplished by Na+ coupled amino acid transport through SLC6A19 (B(0)AT). The present study thus explored whether JAK2 activates SLC6A19. To this end, SLC6A19 was expressed in Xenopus oocytes with or without wild type JAK2, V617FJAK2 or inactive K882EJAK2 and electrogenic amino acid transport determined by dual electrode voltage clamp. In SLC6A19-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of leucine (2mM) to the bath generated a current (Ile), which was significantly increased following coexpression of JAK2 or V617FJAK2, but not by coexpression of K882EJAK2. Coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. Exposure of the oocytes to the JAK2 inhibitor AG490 (40μM) resulted in a gradual decline of Ile. According to chemiluminescence JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of Ile following inhibition of carrier insertion by brefeldin A (5μM) was similar in the absence and presence of JAK2 indicating that JAK2 stimulates carrier insertion into rather than inhibiting carrier retrival from the cell membrane. In conclusion, JAK2 up-regulates SLC6A19 activity which may foster amino acid uptake into JAK2 expressing cells.

The Serum and Glucocorticoid Inducible Kinases SGK1-3 Stimulate the Neutral Amino Acid Transporter SLC6A19

The neutral amino acid transporter SLC6A19 (B⁰AT1) plays a decisive role in transport of neutral amino acids in the kidney and intestine. Recently, mutations in SLC6A19 were identified that result in severe neutral aminoaciduria known as Hartnup disorder. SLC6A19 expression and function is controlled by the brush-border angiotensin-converting enzyme 2 (ACE2). Beyond that the mechanisms regulating SLC6A19 function are unknown. The SLC6A19 sequence contains a conserved putative phosphorylation site for the serum and glucocorticoid inducible kinase isoforms SGK1-3, kinases known to regulate a variety of channels and transporters. The present study explored the role of SGK1-3 in the regulation of SLC6A19. As shown by two-electrode voltage clamp in the Xenopus oocyte expression system, leucine-induced currents in SLC6A19 expressing oocytes were activated by the protein kinases SGK1-3. The putative phosphorylation site on the transporter is not essential for SLC6A19 regulation by the kinases. As determined by quantitative immunoassay and electrophysiology, the kinases increase SLC6A19 currents by increasing the cell surface expression of the protein without altering the affinity of the carrier. Following inhibition of carrier insertion into the cell membrane by treatment with brefeldin A (BFA), the leucine-induced current declined significantly slower in Xenopus oocytes expressing SLC6A19 together with SGK1 than in oocytes expressing SLC6A19 alone, a finding pointing to SGK-mediated transporter stabilization in the plasma membrane. Coexpression of ACE2 markedly increased leucine-induced currents in SLC6A19 expressing oocytes that were further enhanced by SGK1-3 kinases. In conclusion, SGK isoforms are novel potent stimulators of SLC6A19 and may thus participate in the regulation of neutral amino acid transport in vivo.

Iminoglycinuria and hyperglycinuria are discrete human phenotypes resulting from complex mutations in proline and glycine transporters

Iminoglycinuria (IG) is an autosomal recessive abnormality of renal transport of glycine and the imino acids proline and hydroxyproline, but the specific genetic defect(s) have not been determined. Similarly, although the related disorder hyperglycinuria (HG) without iminoaciduria has been attributed to heterozygosity of a putative defective glycine, proline, and hydroxyproline transporter, confirming the underlying genetic defect(s) has been difficult. Here we applied a candidate gene sequencing approach in 7 families first identified through newborn IG screening programs. Both inheritance and functional studies identified the gene encoding the proton amino acid transporter SLC36A2 (PAT2) as the major gene responsible for IG in these families, and its inheritance was consistent with a classical semidominant pattern in which 2 inherited nonfunctional alleles conferred the IG phenotype, while 1 nonfunctional allele was sufficient to confer the HG phenotype. Mutations in SLC36A2 that retained residual transport activity resulted in the IG phenotype when combined with mutations in the gene encoding the imino acid transporter SLC6A20 (IMINO). Additional mutations were identified in the genes encoding the putative glycine transporter SLC6A18 (XT2) and the neutral amino acid transporter SLC6A19 (B0AT1) in families with either IG or HG, suggesting that mutations in the genes encoding these transporters may also contribute to these phenotypes. In summary, although recognized as apparently simple Mendelian disorders, IG and HG exhibit complex molecular explanations depending on a major gene and accompanying modifier genes.

The SLC6 orphans are forming a family of amino acid transporters

Transporters in the human genome are grouped in solute carrier families (SLC). The SLC6 family is one of the biggest transporter families in the human genome comprising 20 members. It is usually referred to as the neurotransmitter transporter family because its founding members encode transporters for the neurotransmitters GABA, noradrenaline, serotonin and dopamine. The family also includes a number of ‘orphan’ transporters, the function of which has remained elusive until recently. Identification of the broadly specific neutral amino acid transporter SLC6A19 (also called B 0AT1) suggested that all orphan transporters may in fact be amino acid transporters. This was subsequently confirmed by the identification of SLC6A20 as the long-sought IMINO system, a proline transporter found in kidney, intestine and brain. Very recently, SLC6A15 was identified as the neutral amino acid transporter B 0AT2. All amino acid transporters appear to cotransport only 1Na + together with the amino acid substrate. Both, B 0AT1 and B 0AT2 are chloride independent, whereas IMINO is chloride dependent. The amino acid transporters of the SLC6 family are functionally and sequence related to the recently crystallized leucine transporter from Aquifex aeolicus. The structure elegantly explains many of the mechanistic features of the SLC6 amino acid transporters.

Hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter SLC6A19.

Hartnup disorder (OMIM 234500) is an autosomal recessive abnormality of renal and gastrointestinal neutral amino acid transport noted for its clinical variability. We localized a gene causing Hartnup disorder to chromosome 5p15.33 and cloned a new gene, SLC6A19, in this region. SLC6A19 is a sodium-dependent and chloride-independent neutral amino acid transporter, expressed predominately in kidney and intestine, with properties of system B(0). We identified six mutations in SLC6A19 that cosegregated with disease in the predicted recessive manner, with most affected individuals being compound heterozygotes. The disease-causing mutations that we tested reduced neutral amino acid transport function in vitro. Population frequencies for the most common mutated SLC6A19 alleles are 0.007 for 517G --> A and 0.001 for 718C --> T. Our findings indicate that SLC6A19 is the long-sought gene that is mutated in Hartnup disorder; its identification provides the opportunity to examine the inconsistent multisystemic features of this disorder.

CD9 is involved in extravillous trophoblast invasion

Prenatal exposure to excessive glucocorticoids (GCs) leads to intrauterine growth retardation and fetal programming of adult health and disease through deregulation of placental functions. Placental secretion of insulin-like growth factor-I (IGF-I) plays a critical role in the regulation of placental development and function. However, it remains elusive whether GCs affect placental functions through glucocorticoid receptor (GR)-mediated transcriptional regulation of IGF-I gene. In this study, human placental choriocarcinoma (BeWo) cells before and after syncytialization were used as cytotrophoblast and syncytiotrophoblast models, respectively, to explore the effects of dexamethasone (Dex) on transcriptional regulation of IGF-I gene at both stages. Dex significantly inhibited (P < 0.05) cell proliferation in cytotrophoblasts and down-regulated amino acid transporter SLC7A5 in syncytiotrophoblasts. Concurrently, the abundance of IGF-I mRNA and its transcript variants, together with IGF-I level in culture media, were significantly reduced, in association with significantly enhanced (P < 0.05) GR phosphorylation. GR antagonist RU486 was able to abolish all these effects. Two glucocorticoid response elements (GREs) were predicted in the promoter regions of IGF-I gene. GR binding to GRE1 was significantly enriched (P < 0.05) in both cytotrophoblasts and syncytiotrophoblasts, but that to GRE2 was significantly diminished (P < 0.05) in cytotrophoblasts but not in syncytiotrophoblasts, in response to Dex treatment. IGF-I supplementation completely rescued Dex-induced cell cycle arrest but not SLC7A5 down-regulation, indicating different regulatory mechanisms. Taken together, our results suggest that GR-mediated transcriptional regulation of IGF-I is involved in Dex-induced inhibition of placental cell proliferation and function.

Establishment of radioimmunoassay for fetal antigen 1 and it's immunohistochemical study

Prenatal exposure to excessive glucocorticoids (GCs) leads to intrauterine growth retardation and fetal programming of adult health and disease through deregulation of placental functions. Placental secretion of insulin-like growth factor-I (IGF-I) plays a critical role in the regulation of placental development and function. However, it remains elusive whether GCs affect placental functions through glucocorticoid receptor (GR)-mediated transcriptional regulation of IGF-I gene. In this study, human placental choriocarcinoma (BeWo) cells before and after syncytialization were used as cytotrophoblast and syncytiotrophoblast models, respectively, to explore the effects of dexamethasone (Dex) on transcriptional regulation of IGF-I gene at both stages. Dex significantly inhibited (P < 0.05) cell proliferation in cytotrophoblasts and down-regulated amino acid transporter SLC7A5 in syncytiotrophoblasts. Concurrently, the abundance of IGF-I mRNA and its transcript variants, together with IGF-I level in culture media, were significantly reduced, in association with significantly enhanced (P < 0.05) GR phosphorylation. GR antagonist RU486 was able to abolish all these effects. Two glucocorticoid response elements (GREs) were predicted in the promoter regions of IGF-I gene. GR binding to GRE1 was significantly enriched (P < 0.05) in both cytotrophoblasts and syncytiotrophoblasts, but that to GRE2 was significantly diminished (P < 0.05) in cytotrophoblasts but not in syncytiotrophoblasts, in response to Dex treatment. IGF-I supplementation completely rescued Dex-induced cell cycle arrest but not SLC7A5 down-regulation, indicating different regulatory mechanisms. Taken together, our results suggest that GR-mediated transcriptional regulation of IGF-I is involved in Dex-induced inhibition of placental cell proliferation and function.

Inhibition of HCG production by retinoids (acitretin) using cultured trophoblast cells

Prenatal exposure to excessive glucocorticoids (GCs) leads to intrauterine growth retardation and fetal programming of adult health and disease through deregulation of placental functions. Placental secretion of insulin-like growth factor-I (IGF-I) plays a critical role in the regulation of placental development and function. However, it remains elusive whether GCs affect placental functions through glucocorticoid receptor (GR)-mediated transcriptional regulation of IGF-I gene. In this study, human placental choriocarcinoma (BeWo) cells before and after syncytialization were used as cytotrophoblast and syncytiotrophoblast models, respectively, to explore the effects of dexamethasone (Dex) on transcriptional regulation of IGF-I gene at both stages. Dex significantly inhibited (P < 0.05) cell proliferation in cytotrophoblasts and down-regulated amino acid transporter SLC7A5 in syncytiotrophoblasts. Concurrently, the abundance of IGF-I mRNA and its transcript variants, together with IGF-I level in culture media, were significantly reduced, in association with significantly enhanced (P < 0.05) GR phosphorylation. GR antagonist RU486 was able to abolish all these effects. Two glucocorticoid response elements (GREs) were predicted in the promoter regions of IGF-I gene. GR binding to GRE1 was significantly enriched (P < 0.05) in both cytotrophoblasts and syncytiotrophoblasts, but that to GRE2 was significantly diminished (P < 0.05) in cytotrophoblasts but not in syncytiotrophoblasts, in response to Dex treatment. IGF-I supplementation completely rescued Dex-induced cell cycle arrest but not SLC7A5 down-regulation, indicating different regulatory mechanisms. Taken together, our results suggest that GR-mediated transcriptional regulation of IGF-I is involved in Dex-induced inhibition of placental cell proliferation and function.

Localization of oxidized glutathione-catalyzing enzymes in the human placenta

Prenatal exposure to excessive glucocorticoids (GCs) leads to intrauterine growth retardation and fetal programming of adult health and disease through deregulation of placental functions. Placental secretion of insulin-like growth factor-I (IGF-I) plays a critical role in the regulation of placental development and function. However, it remains elusive whether GCs affect placental functions through glucocorticoid receptor (GR)-mediated transcriptional regulation of IGF-I gene. In this study, human placental choriocarcinoma (BeWo) cells before and after syncytialization were used as cytotrophoblast and syncytiotrophoblast models, respectively, to explore the effects of dexamethasone (Dex) on transcriptional regulation of IGF-I gene at both stages. Dex significantly inhibited (P < 0.05) cell proliferation in cytotrophoblasts and down-regulated amino acid transporter SLC7A5 in syncytiotrophoblasts. Concurrently, the abundance of IGF-I mRNA and its transcript variants, together with IGF-I level in culture media, were significantly reduced, in association with significantly enhanced (P < 0.05) GR phosphorylation. GR antagonist RU486 was able to abolish all these effects. Two glucocorticoid response elements (GREs) were predicted in the promoter regions of IGF-I gene. GR binding to GRE1 was significantly enriched (P < 0.05) in both cytotrophoblasts and syncytiotrophoblasts, but that to GRE2 was significantly diminished (P < 0.05) in cytotrophoblasts but not in syncytiotrophoblasts, in response to Dex treatment. IGF-I supplementation completely rescued Dex-induced cell cycle arrest but not SLC7A5 down-regulation, indicating different regulatory mechanisms. Taken together, our results suggest that GR-mediated transcriptional regulation of IGF-I is involved in Dex-induced inhibition of placental cell proliferation and function.

Rat cytotrophoblast antigen defined by monoclonal antibodies

Prenatal exposure to excessive glucocorticoids (GCs) leads to intrauterine growth retardation and fetal programming of adult health and disease through deregulation of placental functions. Placental secretion of insulin-like growth factor-I (IGF-I) plays a critical role in the regulation of placental development and function. However, it remains elusive whether GCs affect placental functions through glucocorticoid receptor (GR)-mediated transcriptional regulation of IGF-I gene. In this study, human placental choriocarcinoma (BeWo) cells before and after syncytialization were used as cytotrophoblast and syncytiotrophoblast models, respectively, to explore the effects of dexamethasone (Dex) on transcriptional regulation of IGF-I gene at both stages. Dex significantly inhibited (P < 0.05) cell proliferation in cytotrophoblasts and down-regulated amino acid transporter SLC7A5 in syncytiotrophoblasts. Concurrently, the abundance of IGF-I mRNA and its transcript variants, together with IGF-I level in culture media, were significantly reduced, in association with significantly enhanced (P < 0.05) GR phosphorylation. GR antagonist RU486 was able to abolish all these effects. Two glucocorticoid response elements (GREs) were predicted in the promoter regions of IGF-I gene. GR binding to GRE1 was significantly enriched (P < 0.05) in both cytotrophoblasts and syncytiotrophoblasts, but that to GRE2 was significantly diminished (P < 0.05) in cytotrophoblasts but not in syncytiotrophoblasts, in response to Dex treatment. IGF-I supplementation completely rescued Dex-induced cell cycle arrest but not SLC7A5 down-regulation, indicating different regulatory mechanisms. Taken together, our results suggest that GR-mediated transcriptional regulation of IGF-I is involved in Dex-induced inhibition of placental cell proliferation and function.