Prolactin regulatory element-binding protein is involved in suppression of the adiponectin gene in vivo.

PURPOSE: Prolactin regulatory element-binding protein (PREB), a member of the WD-repeat protein family, has been recognized as a transcriptional factor that regulates prolactin promoter activity in the anterior pituitary of rats. PREB is expressed not only in the pituitary but also in various other tissues, including the adipose tissue. Previous studies have shown that PREB acts as a transcriptional regulator and suppresses the expression of the adiponectin gene in cultured 3T3L1 preadipocytes. The aim of this study was to further examine the potential role of PREB in adipose tissue in vivo. METHODS: Transgenic mice that overexpressing PREB (PREB transgenic mice) were generated. Insulin resistance was evaluated in PREB transgenic mice using glucose and insulin tolerance tests. Adiponectin expression in the adipose tissue was examined by western blot analysis and quantitative polymerase chain reaction (qPCR). The expression levels of stearoyl-CoA desaturase (Scd) and adiponectin receptor 2(ADIPOR2) were quantified by qPCR. RESULTS: Glucose and insulin tolerance tests revealed insulin resistance in PREB transgenic mice. Serum adiponectin and leptin concentrations were decreased. Adiponectin gene expression was decreased in the adipose tissue, which was confirmed by the downregulation of the adiponectin-dependent hepatic Scd gene and upregulation of the ADIPOR2 gene in the liver of PREB transgenic mice. We also found that pioglitazone, an agonist for the peroxisome proliferator-activated receptor-r, improved the insulin resistance in the PREB transgenic mice after a 10-day feeding period. CONCLUSIONS: These results demonstrated that PREB might contribute to the regulation of adiponectin gene expression in vivo.

Insulin-like Growth Factor 1 Regulates the Expression of ATP-Binding Cassette Transporter A1 in Pancreatic Beta Cells.

ATP-binding cassette transporter A1 (ABCA1) in pancreatic beta cells influences insulin secretion and cholesterol homeostasis. The present study investigates whether insulin-like growth factor 1 (IGF-1), which mediates stimulation of ABCA1 gene expression, could also interfere with the phosphatidylinositol 3-kinase (PI3-K) cascade.ABCA1 expression was examined by real-time polymerase chain reaction (PCR), Western blot analysis, and a reporter gene assay in rat insulin-secreting INS-1 cells incubated with IGF-1. The binding of forkhead box O1 (FoxO1) protein to the ABCA1 promoter was assessed by a chromatin immunoprecipitation (ChIP) assay. ABCA1 protein levels increased in response to rising concentrations of IGF-1. Real-time PCR analysis showed a significant increase in ABCA1 mRNA expression. However, both effects were suppressed after silencing the IGF-1 receptor. In parallel with its effect on endogenous ABCA1 mRNA levels, IGF-1 induced the activity of a reporter construct containing the ABCA1 promoter, while it was abrogated by LY294002, a specific inhibitor of PI3-K. Constitutively active Akt stimulated activity of the ABCA1 promoter, and a dominant-negative mutant of Akt or mutagenesis of the FoxO1 response element in the ABCA1 promoter abolished the ability of IGF-1 to stimulate promoter activity. A ChIP assay showed that FoxO1 mediated its transcriptional activity by directly binding to the ABCA1 promoter region. The knockdown of FoxO1 disrupted the effect of IGF-1 on ABCA1 expression. Furthermore, IGF-1 promoted cholesterol efflux and reduced the pancreatic lipotoxicity. These results demonstrate that the PI3-K/Akt/FoxO1 pathway contributes to the regulation of ABCA1 expression in response to IGF-1 stimulation.

Raloxifene inhibits menin-dependent estrogen receptor activation in breast cancer cells.

BACKGROUND: Menin is a tumor suppressor encoded by Men1 that is mutated in the human-inherited tumor syndrome--multiple endocrine neoplasia type 1. Menin binds to estrogen receptors (ER) to enhance estrogen activity in breast cancer cells. AIM: Our clinical study showed that the outcome in the case of menin-positive tumors was worse than in the case of menin-negative tumors. We examined the role of raloxifene on the cell growth in a menin-positive breast cancer cell line. MATERIAL AND METHODS: To examine the mechanism of raloxifene on menin-dependent activation of ER, we employed the mammalian two-hybrid system. We have established a breast cancer cell line that stably expresses menin. Using these cells, we have examined the effect of raloxifene and tamoxifen on cell growth of menin-transfected cells. RESULTS: The expression of activation function (AF)-2 enhanced menin-mediated luciferase expression in the mammalian two-hybrid assay. Raloxifene attenuated the effect of menin on estrogen response element-luciferase activation, indicating that raloxifene inhibited the binding of menin to AF-2. Raloxifene significantly inhibited the growth of menin-transfected cells in a dose-dependent manner. Tamoxifen also inhibited menin-transfected MCF-7 cells; however, this inhibition was much less than that of raloxifene. CONCLUSION: Raloxifene inhibits the binding of menin to the AF-2 domain of ERα, suggesting that raloxifene is one of the therapeutic options for menin-positive breast cancer.

Exendin-4 regulates the expression of the ATP-binding cassette transporter A1 via transcriptional factor PREB in the pancreatic ß cell line.

BACKGROUND: PRL regulatory element-binding (PREB) protein is a transcription factor that regulates insulin promoter activity in the rat anterior pituitary. The PREB protein is expressed not only in the anterior pituitary but also in pancreatic ß cells. Previously, we have reported that PREB plays an important role in glucose-mediated insulin gene expression in pancreatic ß cells. The ATP-binding cassette transporter A1 (ABCA1) in pancreatic ß cells influences insulin secretion and glucose homeostasis. Exendin-4 (Ex-4), a longacting agonist of the glucagon-like peptide 1, stimulates ABCA1 expression in pancreatic ß cells. AIMS: In this study, we examined the role played by PREB in Ex-4-induced ABCA1 expression in pancreatic ß cells. MATERIAL/SUBJECTS AND METHODS: PREB mRNA and protein expression were evaluated in pancreatic ß cell line (INS-1 cells) treated with Ex-4 (10 nM). RESULTS: Ex-4 stimulated PREB protein and mRNA expression in INS-1 cells. 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 small interfering RNA to inhibit PREB expression in the cells and demonstrated that the knockdown of PREB expression attenuated the effects of Ex-4 on ABCA1 expression. CONCLUSION: PREB mediates Ex-4-stimulated transcription of the ABCA1 gene in pancreatic ß cells.

Suppression of prolactin expression by cabergoline requires prolactin regulatory element-binding protein (PREB) in GH3 cells.

The prolactin regulatory element-binding protein (PREB) is a transcriptional factor that regulates prolactin (PRL) promoter activity in the anterior pituitary. Prolactinomas are the most common pituitary tumors. Administration of cabergoline, a selective dopamine D2-receptor agonist, has become the initial therapy of choice for most patients with prolactinomas. Although activation of the D2 receptor results in the inhibition of PRL synthesis, the details of the underlying mechanisms remain unknown. Samples of ten prolactinomas and ten nonfunctioning pituitary adenomas were analyzed by immunohistochemistry to detect the expression of PREB. The effect of cabergoline on PREB expression was assessed by western blotting and real-time polymerase chain reaction (PCR) analysis. Reporter gene analysis of PRL was employed to examine the role of PREB on cabergoline-induced suppression of PRL transcription. Immunohistochemical analysis revealed strong positive PREB expression in the prolactinoma tissue, but extremely weak or undetected expression in the nonfunctioning pituitary tumor tissue. Western blots probed with a PREB-specific antiserum revealed that the relative abundance of the PREB protein in the GH3 cells decreased in a dose-dependent manner in response to cabergoline treatment, as did the relative abundance of PREB mRNA. Although cabergoline inhibited the activity of the PRL promoter, mutation of PREB-binding site within the promoter abrogated the ability of cabergoline to inhibit the PRL promoter activity. We have demonstrated that PREB is expressed in prolactinomas and that the suppression of PRL expression by cabergoline requires the transcriptional factor PREB.

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.

Exendin-4 regulates glucokinase expression by CaMKK/CaMKIV pathway in pancreatic beta-cell line.

AIM: Glucokinase (GK) in pancreatic beta cells is thought to be involved in insulin secretion and glucose homeostasis. This study investigates whether the long-acting agonist of the glucagon-like peptide 1, namely exendin-4, mediates stimulatory effects on GK gene expression through the Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) cascade. METHODS: GK expression was examined by real-time PCR, western blot analysis and reporter gene assay in rat insulin-secreting INS-1 cells incubated with exendin-4. CaMKIV activity was assessed by detection of activation loop phosphorylation (Thr(196)) of CaMKIV. We investigated the effect of the constitutively active form (CaMKIVc) of CaMKIV on GK promoter activity. RESULTS: Increased expression level of GK protein was noted in response to rising concentrations of exendin-4 with maximum induction at 10 nM. Real-time PCR analysis showed a significant increase in the amount of GK mRNA in response to rising concentrations of exendin-4. Exendin-4 also stimulated GK promoter activity but failed to do so in the presence of STO-609, a CaMKK inhibitor. This result is consistent with the observations that the upregulation of CaMKIV phosphorylation (at Thr(196)) peaked after 15 min of exposure to exendin-4 and that CaMKIVc enhanced or upregulated GK promoter activity in INS-1 cells. Furthermore, STO-609 significantly suppressed the exendin-4 - upregulated the expression of the GK protein. CONCLUSION: Activation of the CaMKK/CaMKIV cascade might be required for exendin-4-induced GK gene transcription, indicating that exendin-4 plays an important role in insulin secretion in pancreatic beta cells.

Interferon alpha decreases expression of human scavenger receptor class BI, a possible HCV receptor in hepatocytes.

BACKGROUND: Infection with the hepatitis C virus (HCV) causes acute hepatitis. This disease has a high probability of becoming chronic and leading to cirrhosis, but a more deadly consequence is hepatocellular carcinoma. Interferon alpha (IFN alpha)-based treatment combined with ribavirin is the major therapeutic choice available for the treatment of chronic HCV infection. AIMS: The scavenger receptor class B type I (SR-BI) or its human homologue CD36 and LIMPII Analogous-1 (hSR-BI/CLA-1) has recently been shown to interact with HCV envelope glycoprotein E2, thus suggesting that it might participate in entry of the virus into host cells. This rationale underlies current interest in the potential role of IFN alpha in hSR-BI/CLA-1 expression in HepG2 cells. RESULTS: It was shown that endogenous hepatocyte expression of hSR-BI/CLA-1 was suppressed by exposure to IFN alpha. Decreased hSR-BI/CLA-1 expression in IFN alpha-treated cells was due to lower transcriptional activity of the promoter. A potential pathway for the effect of IFN alpha on hSR-BI/CLA-1 promoter activity was identified when the inhibitory action of IFN was abrogated in signal transducer and activator of transcription 1 (STAT1)/STAT2 knocked-down cells. Exposure of HepG2 cells to IFN alpha elicited a rapid phosphorylation of STAT1/STAT2, a known target of IFN alpha signalling. In addition, the mutagenesis of a STAT1/STAT2 response element in the hSR-BI/CLA-1 promoter abolished the ability of IFN alpha to suppress promoter activity. CONCLUSIONS: Together, these results indicate that the STAT1/STAT2 pathway participates in IFN alpha inhibition of hSR-BI/CLA-1 expression, and raise the possibility that lowering the expression of this gene may be of therapeutic value for treating HCV infections.

Phosphatidylinositol 3-OH kinase-Akt/protein kinase B pathway mediates Gas6 induction of scavenger receptor a in immortalized human vascular smooth muscle cell line.

The growth arrest-specific gene 6 encodes a secreted protein, Gas6, which was originally identified as the ligand of a receptor, Axl, with tyrosine kinase activity. The class A scavenger receptor (SRA) mediates lipid uptake into cells, leading to the formation of foam cells, an important step in atherogenesis. Although Gas6 induces SRA expression, the underlying mechanism is not clear. In this report, we show that the Gas6-induced expression of SRA was mediated by the phosphatidylinositol 3-OH kinase (PI3-kinase)-serine/threonine kinase (Akt/protein kinase B [PKB]) pathway involving Akt phosphorylation. This pathway was activated by exposure to Gas6. Furthermore, the effect of Gas6 was abrogated by wortmannin, a specific inhibitor of PI3-kinase. We also demonstrated that the constitutively active form of Akt enhanced activity of the SRA promoter but that the dominant-negative mutant of Akt completely abolished the expression of SRA after treatment with Gas6. These results show that the PI3-kinase-Akt/PKB pathway participates in Gas6-induced SRA expression and suggests that the activation of Akt/PKB plays an important role in Gas6-induced atherosclerosis and foam cell formation in human vascular smooth muscle cells.

Hydrolytic activity of alpha-amylase in anaerobic digested sludge.

Hydrolysis is usually considered to be a rate-limiting step in anaerobic digestion. For improving anaerobic solid waste treatments, it is essential to elucidate the mechanism of hydrolysis. In this study, alpha-amylase, one of the hydrolytic enzymes, was investigated for the elucidation of more precise mechanism of hydrolysis. Alpha-amylase activity of solid starch-degrading bacteria (SDB) was estimated through batch experiments with several different substrates and with distinction between cell-bound and cell-free alpha-amylase. Monitoring of newly isolated strains of SDB was done by fluorescence in situ hybridization. Results indicated that cell-bound alpha-amylase is chiefly responsible for the hydrolysis in the digested sludge, providing very useful information that the contact between microbial cells and solids is significantly important. The activity of alpha-amylase of the digested sludge remained quite low when not required, but increased as they recognized appropriate substrates. Several-fold higher activity was obtained for starch or maltose as compared to glucose only.

DEEP BIOSPHERE. Exploring deep microbial life in coal-bearing sediment down to ~2.5 km below the ocean floor.

Microbial life inhabits deeply buried marine sediments, but the extent of this vast ecosystem remains poorly constrained. Here we provide evidence for the existence of microbial communities in ~40° to 60°C sediment associated with lignite coal beds at ~1.5 to 2.5 km below the seafloor in the Pacific Ocean off Japan. Microbial methanogenesis was indicated by the isotopic compositions of methane and carbon dioxide, biomarkers, cultivation data, and gas compositions. Concentrations of indigenous microbial cells below 1.5 km ranged from <10 to ~10(4) cells cm(-3). Peak concentrations occurred in lignite layers, where communities differed markedly from shallower subseafloor communities and instead resembled organotrophic communities in forest soils. This suggests that terrigenous sediments retain indigenous community members tens of millions of years after burial in the seabed.

Expression of menin in parathyroid tumors.

The multiple endocrine neoplasia type 1 (MEN1) gene seems to be a tumor suppressor that encodes a 610-amino acid protein termed menin and that plays an important role in the development of MEN1 syndrome. Recent reports indicate that heterozygous germline mutations of this gene are responsible for the disease onset of MEN1. In this study we examined the expression of menin in parathyroid tumors from primary hyperparathyroidism (PHP), secondary hyperparathyroidism (SHP), and MEN1 and thyroid tumors including Basedow's disease, thyroid cancer, and adrenocortical tumors. Both ribonucleic acid and protein from these tumors were applied to RT-PCR and Western blotting, respectively. Primers for RT-PCR were designed to amplify the sequence between exons 2 and 3 of the MEN1 gene. Specific antibody against menin was generated in guinea pigs immunized with the recombinant peptide from amino acid residues 443-535 of menin made by using glutathione-S-transferase (GST) gene fusion. Menin messenger ribonucleic acid was strongly expressed on RT-PCR analysis in the parathyroid tumors from both PHP and SHP. Western blotting revealed a specific band of approximately 67 kDa in parathyroid tumors from PHP and SHP, with a much weaker such band detected in thyroid tumors. Menin expression was down-regulated in MEN1 samples, including nonsense mutation and deletion mutant. These findings suggest that menin is predominantly synthesized and stored in parathyroid tumors resulting from PHP and SHP.

Thiazolidinedione inhibits the production of monocyte chemoattractant protein-1 in cytokine-treated human vascular endothelial cells.

The chemokine monocyte chemoattractant protein-1 is a potent chemoattractant for monocytes. Monocyte chemoattractant protein-1 is produced by vascular endothelial cells during inflammatory diseases such as atherosclerosis. In this study, we examined the effects of a thiazolidinedione on monocyte chemoattractant protein-1 expression in human vascular endothelial cells. In human vascular endothelial cells, interleukin-1beta and tumor necrosis factor-alpha induced endogenous monocyte chemoattractant protein-1 protein secretion, mRNA expression and promoter activity. The thiazolidinedione inhibited these effects. In summary, our results indicated that the suppression of the expression of monocyte chemoattractant protein-1 can be accomplished by thiazolidinedione treatment, raising the possibility that thiazolidinedione may be of therapeutic value in the treatment of diseases such as atherosclerosis.

A product of growth arrest-specific gene 6 modulates scavenger receptor expression in human vascular smooth muscle cells.

Although Gas6 is identified as a growth factor for vascular smooth muscle cells (VSMCs), its roles in these cells have not been clearly elucidated. To examine the role of Gas6 in atherosclerosis, we examined the effects of Gas6 on scavenger receptor family expression in VSMCs. Scavenger receptor class A, one of the scavenger receptor family members, was upregulated in VSMCs by Gas6. Furthermore, the atherogenic lipoprotein, oxidized LDL, induced Gas6 production in these cells. These results indicate that Gas6 plays an important role in foam cell formation in human VSMCs.

Thiazolidinedione inhibits production of RANTES in a cytokine-treated human lung epithelial cell line.

The chemokine RANTES is a potent chemoattractant for eosinophils. RANTES is produced by lung epithelial cells during eosinophil-rich inflammatory diseases such as asthma. In this study, we examined the effects of thiazolidinediones (TZD) on RANTES expression in a human lung epithelial cell line, A549. In A549 cells, interleukin-1beta and tumor necrosis factor-alpha induced endogenous RANTES protein secretion, mRNA expression, and promoter activity. The TZD inhibited these effects. Our data indicate that the suppression of the expression of RANTES can be accomplished by TZD treatment, raising the possibility that TZD might be of therapeutic value in diseases such as asthma.

Phosphatidylserine receptor cooperates with high-density lipoprotein receptor in recognition of apoptotic cells by thymic nurse cells.

The thymus contains many apoptotic cells that arise from the process of positive and negative selection. Both thymic macrophages and thymic nurse cells/nursing thymic epithelial cells (nursing TECs), non-professional phagocytes, recognize and ingest apoptotic cells without inflammation or tissue damage. Previously we reported that human scavenger receptor class B (SR-B1) is involved in recognition of apoptotic thymocytes by nursing TECs. In this study, we examined the expression and role of a phosphatidylserine receptor (PSR). This receptor is believed to participate in the clearance of apoptotic cells. PSR was strongly expressed in nursing TECs. Transforming growth factor-beta augmented the expression of PSR leading to enhanced binding of apoptotic cells to nursing TECs. In nursing TECs, suppressed expression of human SR-B1 with anti-PSR antibody decreased binding of apoptotic thymocytes to nursing TECs. Our results suggest that both PSR and SR-B1 are expressed in nursing TECs and these receptors appear to play a major role in the clearance of apoptotic cells from the thymus.

The multiple endocrine neoplasia type 1 gene product, menin, inhibits the human prolactin promoter activity.

Menin is a protein encoded by the gene mutated in multiple endocrine neoplasia type 1 (MEN1) characterized by multiple endocrine tumors of the parathyroid glands, pancreatic islets and the anterior pituitary, especially prolactinoma. In this study, we examined the effects of menin on human prolactin (hPRL) expression. In rat pituitary GH3 cells stably expressing menin, both PRL gene expression/secretion and thymidine incorporation into DNA were inhibited as compared with mock-transfected cells. The transcriptional activity of PRL promoter in GH3 cells co-transfected with menin was significantly decreased. A deletion mutation (569 delC), which we identified in a Japanese MEN1 family, was introduced into menin. When GH3 cells were transfected with a mutant menin expression vector, inhibition of hPRL promoter activity was partially reversed. These observations suggest that menin inhibits hPRL promoter activity and cell proliferation, raising the possibility that menin might play an important role in the tumorigenesis of prolactinoma.

Inhibition of monocyte chemoattractant protein-1 expression in cytokine-treated human lung epithelial cells by thiazolidinedione.

STUDY OBJECTIVE: Several lung diseases are characterized by the presence of increased numbers of activated macrophages. The recruitment and activation of peripheral blood monocytes are potentially critical regulatory events for the control of pulmonary inflammation. The chemokine monocyte chemoattractant protein (MCP)-1 is a potent chemoattractant for monocytes. MCP-1 is produced by lung epithelial cells during the course of inflammatory lung diseases. In the present study, we examined the effects of a thiazolidinedione (TZD), which is used to improve the insulin resistance of individuals with diabetes mellitus, on MCP-1 expression in a human lung epithelial cell line, A549. MEASUREMENTS AND RESULTS: In A549 cells, interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha induced endogenous MCP-1 protein secretion and messenger RNA expression. The TZD inhibited the increase of MCP-1 secretion by IL-1beta and TNF-alpha treatment. The TZD inhibited the expression of MCP-1 messenger RNA with IL-1beta treatment, but not with TNF-alpha treatment. This observation was confirmed by the results of a monocyte chemotactic assay. The transcriptional activity of human MCP-1 promoter in A549 cells paralleled the endogenous messenger RNA expression by cytokines and TZD treatment. CONCLUSIONS: Our findings indicated that the suppression of the expression of MCP-1 could be accomplished by TZD treatment, raising the possibility that TZD may be of therapeutic value in several lung diseases in which MCP-1 plays an important role.

CD36 LIMPII analogous-1, a human homolog of the rodent scavenger receptor B1, provides the cholesterol ester for steroidogenesis in adrenocortical cells.

CD36 and LIMPII analogous-1 (CLA-1), a human homolog of the rodent scavenger receptor B1 (SR-B1), binds high-density lipoprotein (HDL) and mediates the selective uptake of HDL cholesterol ester (CE) by cultured transfected cells. CLA-1 is strongly expressed in steroidogenic tissues, including the adrenal gland, suggesting that CLA-1 plays a role in providing substrates for steroidogenesis. To address this, we established an adrenocortical cell line that highly expresses CLA-1. These cells increased CE uptake from HDL to 140.5% of the level in mock-transfected cells. After incubation of the transfected cells with HDL, corticosterone secretion from CLA-1-transfected cells increased to about two times the level in mock-transfected cells. These results indicate the possibility that CLA-1 (a close structural homolog of SR-B1)-mediated uptake of HDL CE may be a significant source of precursor cholesterol for steroidogenesis in humans as it is in mice.