Hitomi Imachi

Menin, a product of the MENI gene, binds to estrogen receptor to enhance its activity in breast cancer cells: possibility of a novel predictive factor for tamoxifen resistance.

Multiple coactivator and corepressor complexes play an important role in endocrine processes and breast cancer; in particular, estrogen and estrogen receptor-α (ERα) promote the proliferation of breast cancer cells. Menin is a tumor suppressor encoded by Men1 that is mutated in the human-inherited tumor syndrome multiple endocrine neoplasia type 1 (MEN1); it also serves as a critical link in the recruitment of nuclear receptor-mediated transcription. Here, we show that menin expressed in breast cancer cell line MCF-7 is colocalized with ERα and functions as a direct coactivator of ER-mediated transcription in breast cancer cells. In MCF-7 cells, coexpression of menin and estrogen-response element-luciferase induced the activity of the latter in a hormone-dependent manner. Cells knocked down for ERα exhibited impaired ERE-luciferase activity induced by menin. Mammalian two-hybrid assay and GST pull-down assays indicated that menin could interact with the AF-2 domain of ERα. These results indicate that menin is a direct activator of ERα function. Tamoxifen inhibited the binding of menin to AF-2 in mammalian two-hybrid assay, but in menin-overexpressing clones, tamoxifen suppressed ERE-luciferase activity only to the levels of nontreated wild-type MCF-7. In a clinical study with 65 ER-positive breast cancer samples—all of which had been treated with tamoxifen for 2–5xa0years as adjuvant therapies—menin-positive tumors had a worse outcome than menin-negative ones. These indicated that menin can function as a transcriptional regulator of ERα and is a possible predictive factor for tamoxifen resistance.

Regulation of Scavenger Receptor Class BI Gene Expression by Angiotensin II in Vascular Endothelial Cells

High-density lipoprotein mediates a normal physiological process called reverse cholesterol transport. In this process, a scavenger receptor of the B class (SR-BI)/human homologue of SR-BI, CD36, and LIMPII analogous-1 (hSR-BI/CLA-1) facilitates the cellular uptake of cholesterol from high-density lipoprotein. In endothelial cells, high-density lipoprotein activates endothelial NO synthase via hSR-BI/CLA-1. Angiotensin II (Ang II) is a powerful accelerator of atherosclerosis and modulates the expression of endothelial NO synthase. In the present study, we have examined the role of Ang II on hSR-BI/CLA-1 expression in human umbilical vein endothelial cells. Our results showed that endogenous expression of hSR-BI/CLA-1 was suppressed by exposure to Ang II in human umbilical vein endothelial cells. Administration of the Ang II type-1 receptor blocker olmesartan inhibited Ang II–induced hSR-BI/CLA-1 protein repression. In Ang II–treated cells, high-density lipoprotein had no effect on endothelial NO synthase activation. Ang II decreased transcriptional activity of the hSR-BI/CLA-1 promoter. The inhibitory effect of Ang II on hSR-BI/CLA-1 promoter activity was abrogated by wortmannin and LY294002, specific inhibitors of phosphatidylinositol 3-kinase. Exposure of human umbilical vein endothelial cells to Ang II elicited a rapid phosphorylation of Akt and FoxO1, a known target of Akt signaling. Constitutively active Akt inhibits the activity of the hSR-BI/CLA-1 promoter, and a dominant-negative mutant of Akt or mutagenesis of a FoxO1 response element in the hSR-BI/CLA-1 abolished the ability of Ang II to suppress promoter activity. Together, these results indicate that the phosphatidylinositol 3-kinase/Akt/FoxO1 pathway participates in Ang II suppression of hSR-BI/CLA-1 expression and suggests that the endothelial receptor for hSR-BI/CLA-1 is downregulated by the renin–angiotensin system.

Prolactin regulatory element binding protein as a potential transcriptional factor for the insulin gene in response to glucose stimulation

Aims/hypothesisProlactin regulatory element binding (PREB) protein has been identified as a factor that regulates prolactin promoter activity in rat anterior pituitary. PREB is located not only in the anterior pituitary but also in pancreas; however its role in the pancreas is not known. We therefore examined the role of PREB in insulin gene expression.Materials and methodsTo analyse the effects of PREB on insulin gene transcription, we employed the luciferase reporter gene assay and electrophoretic mobility shift assay (EMSA). In cells expressing or knocked down for PREB, insulin expression and secretion were determined.ResultsPREB was located mainly in nuclei of rat pancreatic beta cells and its cell line, INS-1. A nuclear extract of INS-1 cells contained material that was recognised by PREB antiserum. This nuclear extract also showed insulin promoter binding activity that was super-shifted by PREB antiserum in EMSA studies. In the INS-1 cells, co-expression of PREB and the insulin promoter induced activity of the latter. The addition of glucose to the cells increased PREB expression. Deletional analysis of the insulin promoter showed that A3, a glucose-responsive cis-element in the insulin promoter, mediated the transcriptional effect of PREB. In addition, synthesised PREB bound the A3 element by EMSA, while a mutant of this motif in the insulin promoter abrogated the effect of PREB. Cells expressing or knocked down for PREB exhibited increased or decreased insulin expression, respectively.Conclusions/interpretationThese results demonstrate that PREB may contribute to the regulation of insulin gene transcription and insulin secretion in response to glucose stimulation.

Platelet derived growth factor regulates ABCA1 expression in vascular smooth muscle cells

The ATP‐binding cassette transporter A1 (ABCA1) regulates lipid efflux from peripheral cells to High‐density lipoprotein. The platelet‐derived growth factor (PDGF) is a potent mitogen that enables vascular smooth muscle cells to participate in atherosclerosis. In this report, we showed that PDGF suppressed endogenous expression of ABCA1 in cultured vascular smooth muscle cells. Exposure of CRL‐208 cells to PDGF elicited a rapid phosphorylation of a kinase downstream from PI3‐K, Akt. The constitutively active form of both p110, a subunit of PI3‐K, and Akt inhibited activity of the ABCA1 promoter. In conclusion, PI3‐K‐Akt pathways participate in PDGF‐suppression of ABCA1 expression.

Exendin-4 regulates pancreatic ABCA1 transcription via CaMKK/CaMKIV pathway.

ATP‐binding cassette transporter A1 (ABCA1) in pancreatic β cells influences insulin secretion and glucose homeostasis. This study investigates whether the long‐acting agonist of the glucagon‐like peptide 1, namely exendin‐4, which mediates stimulatory effects on ABCA1 gene expression, could interfere with the Ca2+/calmodulin (CaM)‐dependent protein kinase (CaMK) cascade. ABCA1 promoter activity was examined by reporter gene assay in rat insulin‐secreting INS‐1 cells incubated with exendin‐4. CaMKIV activity was assessed by detection of activation‐loop phosphorylation (Thr196) of CaMKIV. We investigated the influence of the constitutively active form (CaMKIVc) or CaMKIV knockdown on ABCA1 expression. Increased abundance of ABCA1 protein was noted in response to rising concentrations of exendin‐4 with maximum induction at 10 nM. Exendin‐4 also stimulated ABCA1 promoter activity, but failed to do so in the presence of STO‐609, a CaMKK inhibitor. Up‐regulation of CaMKIV phosphorylation (at Thr196) peaked after 10 min. of exposure to exendin‐4. CaMKIVc enhanced or up‐regulated ABCA1 promoter activity in INS‐1 cells. Furthermore, exendin‐4 induction of ABCA1 protein expression was significantly suppressed in cells treated with CaMKIV‐siRNA. Activation of the CaMKK/CaMKIV cascade by exendin‐4 stimulated ABCA1 gene transcription, indicating that exendin‐4 plays an important role in insulin secretion and cholesterol ester content in pancreatic β cells.

The Transcriptional Factor Prolactin Regulatory Element-Binding Protein Mediates the Gene Transcription of Adrenal Scavenger Receptor Class B Type I via 3′,5′-Cyclic Adenosine 5′-Monophosphate

Prolactin regulatory element-binding (PREB) protein is a transcription factor that regulates prolactin promoter activity in the rat anterior pituitary. The PREB protein is not only expressed in the anterior pituitary but also in the adrenal gland. However, the role of PREB in the adrenal gland is not clearly understood. Scavenger receptor class B type I (SR-BI) is a receptor for high-density lipoprotein that mediates the cellular uptake of high-density lipoprotein-cholesteryl ester and is a major route for cholesterol delivery to the steroidogenic pathway in the adrenal gland. In the present study, we have examined the role of PREB in regulating SR-BI. SR-BI expression was found to be regulated by cAMP, which stimulated the expression of PREB in a dose-dependent manner. Conversely, overexpression of PREB using a PREB-expressing adenovirus increased the expression of the SR-BI protein in the adrenocortical cell line Y-1. In addition, PREB induced the expression of the luciferase reporter protein that was under the control of the SR-BI promoter. EMSA showed that PREB mediates its transcriptional effect by binding to the PREB-responsive cis-element of the SR-BI promoter. Finally, we used small interfering RNA to inhibit PREB expression in the Y-1 cells and demonstrated that the knockdown of PREB expression attenuated the effects of cAMP on SR-BI expression. In summary, our data showed that in the adrenal gland, PREB regulates the transcription of the SR-BI gene via cAMP.

Exendin-4 regulates GLUT2 expression via the CaMKK/CaMKIV pathway in a pancreatic β-cell line

The GLUT2 glucose transporter plays an important role in glucose-induced insulin secretion in pancreatic β-cells by catalyzing the uptake of glucose into the cell. In this study, we investigated whether exendin-4, a long-acting agonist of glucagon-like peptide-1, mediates stimulatory effects on GLUT2 gene expression through the Ca²+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) cascade. GLUT2 expression was examined by real-time polymerase chain reaction, Western blot analysis, and a reporter gene assay in rat insulin-secreting INS-1 cells incubated with exendin-4. An increased expression level of GLUT2 protein was noted in response to increasing concentrations of exendin-4, with maximal induction at 10 nmol/L. Real-time polymerase chain reaction analysis similarly revealed a significant increase in the amount of GLUT2 messenger RNA by 10 nmol/L exendin-4. Exendin-4 also stimulated GLUT2 promoter activity in response to increasing exendin-4 concentrations, but failed to do so in the presence of STO-609, a CaMKK inhibitor. We also investigated the effect of the constitutively active form of CaMKK (CaMKKc) on GLUT2 promoter activity. The result is consistent with the observations that CaMKKc/CaMKIV enhanced or up-regulated GLUT2 promoter activity in INS-1 cells. Furthermore, exendin-4 induction of GLUT2 protein expression was significantly suppressed in the cells knocking down the CaMKIV. In summary, activation of the CaMKK/CaMKIV cascade might be required for exendin-4-induced GLUT2 gene transcription, indicating that exendin-4 plays an important role in insulin secretion in pancreatic β-cells.

PREB regulates transcription of pancreatic glucokinase in response to glucose and cAMP

Prolactin regulatory element binding (PREB) is a transcription factor that regulates prolactin promoter activity in rat anterior pituitary. The PREB protein is not only expressed in the anterior pituitary but also in the pancreas. We have recently reported that in pancreatic β‐cells, PREB regulates the transcription of the insulin gene in response to glucose stimulation. In the current study, we have examined the role of PREB in regulating glucokinase (GK) in pancreatic β‐cells. To analyse the effects of PREB on GK gene transcription, we employed a reporter gene assay. In the cells expressing or with knocked down PREB, GK expression was determined. GK expression was regulated by glucose and cAMP, and both glucose and cAMP stimulated the expression of PREB in a dose‐dependent manner. Conversely, overexpression of PREB using a PREB‐expressing adenovirus increased the expression of the GK protein. GK enzymatic activity was also significantly increased in the cells that stably expressed PREB. In addition, PREB induced GK promoter activity. Chromatin immunoprecipitation (ChIP) analyses showed that PREB mediated its transcriptional effect by binding to the PREB‐responsive cis‐element of the GK promoter. Finally, we used siRNA to inhibit PREB expression in cells and demonstrated that the knockdown of PREB attenuated the effects of glucose and cAMP on GK expression. Our data show that in pancreatic β‐cells, PREB regulates the transcription of the GK gene in response to glucose and cAMP.

Hyperglycemia Suppresses ABCA1 Expression in Vascular Smooth Muscle Cells

Hyperglycemia is a major risk factor for atherosclerotic disease. The ATP-binding cassette transporter A1 (ABCA1) functions as a pivotal regulator of lipid efflux from cells to apolipoproteins and is thus involved in lowering the risk of atherosclerosis. In this study, we have examined the glucose-mediated regulation of the ABCA1 gene expression in vascular smooth muscle cells. ABCA1 expression was examined by real-time polymerase chain reaction (PCR), Western blot analysis, and reporter gene assay. The results showed that the expression of the ABCA1 mRNA and protein decreased after the cells were treated with 22.4 mM glucose for 48 h. The transcriptional activity of the ABCA1 promoter paralleled the endogenous expression of the ABCA1 gene. Next, we used inhibitors of certain signal transduction pathways to demonstrate that the glucose-induced ABCA1 suppression is sensitive to the p38-mitogen-activated protein kinase (MAPK) inhibitors. The expression of a constitutively active form of p38-MAPK in the cells inhibited the ABCA1 promoter activity, irrespective of the presence of glucose. A dominant-negative mutant of p38-MAPK abrogated the inhibitory effect of glucose on the ABCA1 promoter activity. These results indicate that the glucose-induced suppression of ABCA1 expression is partially mediated by the activation of the p38-MAPK pathway.

Transcriptional factor prolactin regulatory element-binding protein-mediated gene transcription of ABCA1 via 3′,5′-cyclic adenosine-5′-monophosphate

OBJECTIVEnProlactin regulatory element-binding (PREB) protein is a transcription factor that regulates prolactin promoter activity in the rat anterior pituitary. The PREB protein is not only expressed in the anterior pituitary but also in the cardiovascular system, including vascular smooth muscle cells (SMCs). However, the role of PREB in SMCs is not clearly understood. The ATP-binding cassette transporter A1 (ABCA1) regulates lipid efflux from peripheral cells to apolipoproteins. In the present study, we have examined the role of PREB in regulating ABCA1 expression mediated by 3,5-cyclic adenosine-5-monophosphate (cAMP).nnnMETHODS AND RESULTSnPREB was expressed in the rats SMC line CRL-2018. ABCA1 expression was found to be regulated by cAMP, which stimulated the expression of PREB in a dose-dependent manner. Conversely, over-expression of PREB, which was induced by a PREB-expressing adenovirus, increased the expression of the ABCA1 protein in CRL-2018 cells. In addition, PREB stimulated the activity of the luciferase reporter protein that was under the control of the ABCA1 promoter. Chromatin immunoprecipitation assay showed that PREB mediates its transcriptional activity by directly binding to the ABCA1 promoter region. Finally, we used siRNA to inhibit PREB expression in the cells and demonstrated that the knockdown of PREB expression attenuated the effects of cAMP on ABCA1 expression.nnnCONCLUSIONSnIn summary, our data showed that PREB regulates the cAMP-mediated transcription of the ABCA1 gene in vascular SMCs.