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Specific ligands of the peripheral benzodiazepine receptor (PBR) activate pro-apoptotic and anti-proliferative signaling pathways. Previously, we found that PBR ligands activated the p38 mitogen-activated protein kinase (MAPK) pathway in esophageal cancer cells, and that the activation of p38MAPK contributed to tumor cell apoptosis and cell cycle arrest. Here, we report that PBR ligands also activate the pro-survival MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway in esophageal cancer cells, which might compromise the efficacy of PBR ligands. Hence, a combination treatment of PBR ligands and MEK inhibitors, which are emerging as promising anticancer agents, was pursued to determine whether this treatment could lead to enhanced apoptosis and cell cycle arrest. Using Western blotting we demonstrated a time- and dose-dependent phosphorylation of ERK1/2 in response to PBR ligands. Apoptosis was investigated by assessment of mitochondrial alterations and caspase-3 activity. Cell cycle arrest was measured by flow cytometric analysis of stained isolated nuclei. The inhibition of MEK/ERK with a pharmacologic inhibitor, 2'-amino-3'-methoxyflavone (PD 98059), resulted in a synergistic enhancement of PBR-ligand-induced growth inhibition, apoptosis and cell cycle arrest. Specifity of the pharmacologic inhibitor was confirmed by the use of 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U 0126), a second MEK/ERK inhibitor, and 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U 0124), a structural analogue of it which does not display any affinity to MEK. Enhanced pro-apoptotic and anti-proliferative effects were observed both in KYSE-140 esophageal squamous cancer and OE-33 adenocarcinoma cells, suggesting that this effect was not cell-type specific. In addition, the PBR-mediated overexpression of the stress response gene (growth arrest and DNA-damage-inducible gene gadd153) was synergistically enhanced by MEK inhibition. This is the first report of enhanced PBR-ligand-mediated apoptosis and cell cycle arrest by simultaneous MEK inhibition, suggesting a new anticancer strategy.

Activation of vascular smooth muscle cells (VSMCs) by proinflammatory cytokines is a key feature of atherosclerotic lesion formation. Transforming growth factor (TGF)-beta1 is a pleiotropic growth factor that can modulate the inflammatory response in diverse cell types including VSMCs. However, the mechanisms by which TGF-beta1 is able to mediate these effects remains incompletely understood. We demonstrate here that the ability of TGF-beta1 to inhibit markers of VSMC activation, inducible nitric-oxide synthase (iNOS) and interleukin (IL)-6, is mediated through its downstream effector Smad3. In reporter gene transfection studies, we found that among a panel of Smads, Smad3 could inhibit iNOS induction in an analogous manner as exogenous TGF-beta1. Adenoviral overexpression of Smad3 potently repressed inducible expression of endogenous iNOS and IL-6. Conversely, TGF-beta1 inhibition of cytokine-mediated induction of iNOS and IL-6 expression was completely blocked in Smad3-deficient VSMCs. Previous studies demonstrate that CCAAT/enhancer-binding protein (C/EBP) and NF-kappaB sites are critical for cytokine induction of both the iNOS and IL-6 promoters. We demonstrate that the inhibitory effect of Smad3 occurs via a novel antagonistic effect of Smad3 on C/EBP DNA-protein binding and activity. Smad3 mediates this effect in part by inhibiting C/EBP-beta and C/EBP-delta through distinct mechanisms. Furthermore, we find that Smad3 prevents the cooperative induction of the iNOS promoter by C/EBP and NF-kappaB. These data demonstrate that Smad3 plays an essential role in mediating TGF-beta1 anti-inflammatory response in VSMCs.

Depriving cells of iron likely stresses them and can result in cell death. To examine the potential relationship between this form of stress and cell death, Jurkat T-lymphocytes were made iron-deficient by exposing them to the iron chelator, deferoxamine (DFO). Such treatment produced evidence of apoptosis, including cell shrinkage, membrane blebbing, chromatin condensation and fragmentation, and also formation of apoptotic bodies. Additionally, proteolytic cleavage of poly(ADP-ribose)polymerase was detected, suggesting involvement of caspases in initiating apoptosis. Indeed, a selective caspase-3 inhibitor prevented the effects of DFO. During the early induction period of apoptosis, GRP78 and HSP70 mRNA expression was not affected. In contrast, there was mainly increased mRNA expression of Growth Arrest and DNA Damage-inducible gene 153 (GADD153), which seemed to be at the level of transcription rather than mRNA stability. Furthermore, fortifying cells with antioxidants did not prevent the increased GADD153 mRNA expression, and no evidence of single-strand breaks in DNA was found, suggesting that neither reactive oxygen species nor DNA damage was involved in triggering GADD153 gene activation. DFO also caused GADD153 protein to be expressed. Because GADD153 is recognized as a pro-apoptotic gene, these findings generate the notion that GADD153 might help mediate apoptosis in iron-deficient cells.

The CHOP gene is transcriptionally induced by amino acid starvation. We have previously identified a genomic cis-acting element (amino acid response element (AARE)) involved in the transcriptional activation of the human CHOP gene by leucine starvation and shown that it binds the activating transcription factor 2 (ATF2). The present study was designed to identify other transcription factors capable of binding to the CHOP AARE and to establish their role with regard to induction of the gene by amino acid deprivation. Electrophoretic mobility shift assay and transient transfection experiments show that several transcription factors that belong to the C/EBP or ATF families bind the AARE sequence and activate transcription. Among all these transcription factors, only ATF4 and ATF2 are involved in the amino acid control of CHOP expression. We show that inhibition of ATF2 or ATF4 expression impairs the transcriptional activation of CHOP by amino acid starvation. The transacting capacity of ATF4 depends on its expression level and that of ATF2 on its phosphorylation state. In response to leucine starvation, ATF4 expression and ATF2 phosphorylation are increased. However, induction of ATF4 expression by the endoplasmic reticulum stress pathway does not fully activate the AARE-dependent transcription. Taken together our results demonstrate that at least two pathways, one leading to ATF4 induction and one leading to ATF2 phosphorylation, are necessary to induce CHOP expression by amino acid starvation. This work was extended to the regulation of other amino acid regulated genes and suggests that ATF4 and ATF2 are key components of the amino acid control of gene expression.

The human trophoblast has the capacity to invade maternal tissue in a controlled fashion and to produce a wide range of hormones. The transcription factors belonging to the CCAAT/enhancer-binding protein (C/EBP) family are regulators of intracellular processes and mediators of hormone action. C/EBP binding sites have been described in the promoters of several placenta-expressed target genes. In the present study, we used immunohistochemistry and Western-blot analysis to investigate the expression pattern of the three most important members of this family, C/EBP-alpha, -beta, and -delta, in the normal human placenta as well as in isolated trophoblast cell populations. We found C/EBP-alpha and C/EBP-beta expression in the villous syncytiotrophophoblast (ST) and the extravillous (intermediate) trophoblast (EVT), but it was absent from the villous cytotrophoblast (CT). Interestingly, expression of C/EBP-beta continued to be very strong up to the third trimester of pregnancy, especially in the ST. C/EBP-delta showed overall lower expression levels, stronger only in the EVT, while CT/ST showed very low/negative expression. These data show for the first time the expression pattern and tissue localization of C/EBP factors in the human placenta, indicating that these factors (especially C/EBP-beta) may play important roles in the regulation of placenta-specific genes and processes.

The eukaryotic Y-box binding protein YB-1 is involved in various biological processes, including DNA repair, cell proliferation and the regulation of transcription and translation. YB-1 protein is abundant and expressed ubiquitously in human cells, functioning in cell proliferation and transformation. Its concentration is thought to be highly regulated at both the levels of transcription and translation. Therefore, we investigated whether or not the 5'-UTR of YB-1 mRNA affects the translation of YB-1 protein, thus influencing expression levels. Luciferase mRNA ligated to the YB-1 mRNA 5'-UTR was used as a reporter construct. Ligation of the full-length YB-1 5'-UTR (331 bases) enhanced translation as assessed by in vitro and in vivo translation assays. Deletion constructs of the YB-1 5'-UTR also resulted in a higher efficiency of translation, especially in the region mapped to +197 to +331 from the major transcription start site. RNA gel shift assays revealed that the affinity of YB-1 for various 5'-UTR probe sequences was higher for the full-length 5'-UTR than for deleted 5'-UTR sequences. An in vitro translation assay was used to demonstrate that recombinant YB-1 protein inhibited translation of the full-length 5'-UTR of YB-1 mRNA. Thus, our findings provide evidence for the autoregulation of YB-1 mRNA translation via the 5'-UTR.

Background: The expression of pro-angiogenic cytokines, such as vascular endothelial growth factor (VEGF) and interleukin-8/CXCL8 (IL-8), plays an important role in tumor growth and metastasis. Low oxygen tension within poorly-vascularized tumors is thought to be the prime stimulus causing the secretion of VEGF. The expression of IL-8 by solid tumors is thought to be primarily due to intrinsic influences, such as constitutive activation of nuclear factor kappa B (NF-kappaB). However, VEGF expression is responsive to glucose deprivation, suggesting that low concentrations of nutrients other than oxygen may play a role in triggering the pro-angiogenic phenotype. Glucose deprivation causes endoplasmic reticulum (ER) stress and alters gene expression through the unfolded protein response (UPR) signaling pathway. A branch of the UPR, known as the ER overload response (EOR), can cause NF-kappaB activation. Thus, we hypothesized that treatments that cause ER stress and deprivation of other nutrients, such as amino acids, would trigger the expression of angiogenic cytokines by breast cancer cell lines., Results: We found that glutamine deprivation and treatment with a chemical inducer of ER stress (tunicamycin) caused a marked induction of the secretion of both VEGF and IL-8 protein by a human breast adenocarcinoma cell line (TSE cells). Glutamine deprivation, glucose deprivation and several chemical inducers of ER stress increased VEGF and IL-8 mRNA expression in TSE and other breast cancer cell lines cultured under both normoxic and hypoxic conditions, though hypoxia generally diminished the effects of glucose deprivation. Of all amino acids tested, ambient glutamine availability had the largest effect on VEGF and IL-8 mRNA expression. The induction of VEGF mRNA expression, but not IL-8, was sustained and closely corresponded with the upregulated expression of the ER stress-responsive genes glucose-regulated protein 78 (GRP78) and growth arrest and DNA damage inducible gene 153 (GADD153)., Conclusion: These results suggest that nutrient deprivation within the solid tumor microenvironment might contribute to the activation of a pro-angiogenic phenotype. The angiogenic switch may act to increase blood supply in response to nutrient deprivation as well as hypoxia.

Background and Objectives: C/EBP homologous protein (CHOP) is an endoplasmic reticulum (ER) stress inducible transcription factor involved in the development of apoptosis, growth arrest, and differentiation. CHOP deficient (chop - / - ) mouse embryonic fibroblasts (MEFs) exposed to ER stresses such as tunicamycin exhibit decreased apoptosis and reduced toxicity when compared to chop + / + control cells. Overexpression of the 70 kDa heat shock stress protein (HSP-70) can inhibit apoptotic pathways. The biological significance of photodynamic therapy (PDT) protocols that induce cellular damage resulting in differential CHOP and stress protein expression patterns was examined., Study Design/materials and Methods: Wild type mouse radiation induced fibrosarcoma (RIF) cells as well as MEFs with chop + / + and chop - / - genotypes were used with either a mitochondrial and ER localizing porphyrin (PH) photosensitizer or a lysosomal localizing chlorin (NPe6) photosensitizer. PDT induced cytotoxicity, apoptosis, and stress protein expression patterns were determined as a function of cell type and photosensitizer., Results: PH mediated PDT induced expression of CHOP and 78 kDa glucose regulated protein (GRP-78), but not HSP-70 while NPe6 mediated PDT induced protein expression of HSP-70 but did not activate CHOP or GRP-78 expression. Enhanced apoptosis and toxicity were observed in chop + / + cells following exposure to tunicamycin or PH mediated PDT when compared to identical treatments in chop - / - cells. NPe6 mediated PDT induced minimally detectable apoptosis in both chop + / + and chop - / - cells and only a modest increase in survival for chop - / - cells., Conclusions: These results demonstrate that PDT activation of CHOP, GRP-78, and HSP-70 varied as a function of photosensitizer subcellular localization and that a single oxidative stress response was not observed following PDT. We also show that CHOP expression increased apoptosis following PH mediated PDT and that increased CHOP expression is associated with enhanced PDT photosensitization., ((c) 2004 Wiley-Liss, Inc.)

Arsenic trioxide is valuable for treatment of promyelocytic leukemia, but less attention has been paid to its therapeutic potential for other cancers. In this study, the effects of arsenic trioxide were tested in human pancreatic (AsPC-1), colonic (HT-29), and breast (MCF-7) cancer cells. In all three cancer cell lines, arsenic trioxide inhibited proliferation in a concentration and time-dependent manner, as measured by 3H-methyl thymidine incorporation and cell counting. Coincident with inhibition of growth, arsenic trioxide induced marked morphologic changes, including reduced cytoplasmic volume, membrane blebbing, and nuclear condensation consistent with apoptosis. Propidium iodide DNA staining at 24 hours revealed cell cycle arrest in the G0/G1 phase and an increase in the S phase, while at 72 hr there was G2/M phase arrest with a marked increase in the sub-G0/G1, apoptotic cell population. The DNA fragmentation induced by arsenic trioxide was confirmed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay in all cell lines. Western blot analysis revealed activation of caspase -3, -7, and -9 by arsenic trioxide. Caspase-3 activity was confirmed by demonstrating cleavage of its downstream target, poly ADP-ribose polymerase (PARP). Expression of the antiapoptosis protein, Bcl-2, was time-dependently decreased. In contrast, arsenic trioxide markedly enhanced the expression of the p21 protein, GADD45 and GADD153, in a time-dependent manner. These findings suggest that arsenic trioxide has potential as a therapeutic agent for these cancers.

The fluorescence-based real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantify mRNA level in cells and tissues and is now a crucial tool for basic biological researches and biotechnology. In the present study, on the basis of the real-time quantitative RT-RCR, we detected and quantified mRNA copies of the transcription factor, CCAAT/enhancer binding protein (C/EBP: an immediate-early gene that is involved in synaptic plasticity and learning and memory) in the central nervous system of the pond snail Lymnaea stagnalis. We designed the primer set and the probe in the specific insert for the detection of Lymnaea C/EBP (LymC/EBP) clone 1. This insert is not contained in LymC/EBP clone 2 by alternative splicing. The copy number of LymC/EBP clone 1 was linearly decreased relative to the dilution of cDNA, and it was estimated 30 copies/microl in test sample. The availability of the present study showed that the real-time quantitative RT-PCR technique is more accurate and more specific for the detection and quantification of the mRNA level of genes in L. stagnalis than the other PCR methods.

Previous work showed that acute stimulation of a conditionally active protein kinase B (PKB or cAKT) was sufficient to elicit insulin-like induction of GCK (glucokinase) and SREBP1 (sterol regulatory element-binding protein 1) in hepatocytes [Iynedjian, Roth, Fleischmann and Gjinovci (2000) Biochem. J. 351, 621-627; Fleischmann and Iynedjian (2000) Biochem. J. 349, 13-17]. The objective of the present study was to determine whether activation of PKB during insulin stimulation of hepatocytes was a necessary condition for the induction of the two genes. Activation of PKB by insulin was inhibited by pretreatment of the hepatocytes with C2 ceramide. This resulted in the inhibition of insulin-dependent increases in GCK and SREBP1 mRNAs. A triple mutant of PKB failed to interfere with insulin activation of PKB in hepatocytes even at high overexpression levels achieved after adenovirus transduction. A PKB-CaaX fusion protein, which can act as a dominant-negative inhibitor of PKB activation in other cells, was shown to be constitutively activated in hepatocytes and to trigger insulin-like induction of GCK and SREBP1. In addition, constitutive PKB-CaaX activity caused refractoriness of the hepatocytes to insulin signalling at an upstream step resulting in the inhibition of both extracellular-signal-regulated kinase 1/2 and endogenous PKB activation. The stimulation of gene expression by constitutively active PKB-CaaX and inhibition of the insulin effect by ceramide are compatible with a role for PKB in the insulin-dependent induction of GCK and SREBP1.

Hepatic genes crucial for carbohydrate and lipid homeostasis are regulated by insulin and glucose metabolism. However, the relative contributions of insulin and glucose to the regulation of metabolic gene expression are poorly defined in vivo. To address this issue, adenovirus-mediated hepatic overexpression of glucokinase was used to determine the effects of increased hepatic glucose metabolism on gene expression in fasted or ad libitum fed rats. In the fasted state, a 3 fold glucokinase overexpression was sufficient to mimic feeding-induced increases in pyruvate kinase and acetyl CoA carboxylase mRNA levels, demonstrating a primary role for glucose metabolism in the regulation of these genes in vivo. Conversely, glucokinase overexpression was unable to mimic feeding-induced alterations of fatty acid synthase, glucose-6-phosphate dehydrogenase, carnitine palmitoyl transferase I or PEPCK mRNAs, indicating insulin as the primary regulator of these genes. Interestingly, glucose-6-phosphatase mRNA was increased by glucokinase overexpression in both the fasted and fed states, providing evidence, under these conditions, for the dominance of glucose over insulin signaling for this gene in vivo. Importantly, glucokinase overexpression did not alter sterol regulatory element binding protein 1-c mRNA levels in vivo and glucose signaling did not alter the expression of this gene in primary hepatocytes. We conclude that a modest hepatic overexpression of glucokinase is sufficient to alter expression of metabolic genes without changing the expression of SREBP-1c.

Fenretinide induces apoptosis of neuroblastoma cells in vitro and interacts synergistically with the chemotherapeutic drugs cisplatin and etoposide. The stress-inducible transcription factor known as growth and DNA damage (GADD)-inducible transcription factor 153 is induced in response to fenretinide and in other cell types modulates apoptosis via pro- and antiapoptotic members of the BCL2 family. Because BCL2-family proteins are important in apoptosis induced by chemotherapeutic drugs, GADD153 may be a key mediator of synergy between fenretinide and chemotherapeutic drugs. To investigate this, GADD153 cDNA in sense and antisense orientations was stably transfected into SH-SY5Y neuroblastoma cells using a tetracycline-inducible vector. Increased expression of GADD153 raised the background level of apoptosis and increased apoptosis induced by fenretinide or the chemotherapeutic drugs cisplatin and etoposide. However, there was no increase in synergy between fenretinide and chemotherapeutic drugs. Conversely, expression of antisense-GADD153 virtually abolished the induction of apoptosis in response to fenretinide but overall had no significant effect on apoptosis induced by chemotherapeutic drugs. The effect of antisense-GADD153 on synergy between chemotherapeutic drugs and fenretinide varied with the drug used: there was no effect on synergy between fenretinide and cisplatin, but the combination of fenretinide with etoposide became antagonistic. These results suggest that mechanisms mediating synergy between fenretinide and chemotherapeutic drugs lie upstream of GADD153.

Insulin receptor substrate-1 (IRS-1) plays an essential role in mediating the insulin signals that trigger mitogenesis, lipid synthesis, and uncoupling protein-1 gene expression in mouse brown adipocytes. Expression of IRS-3 is restricted mainly to white adipose tissue; expression of this IRS protein is virtually absent in brown adipocytes. We have tested the capacity of IRS-3 to mediate insulin actions in IRS-1-deficient brown adipocytes. Thus, we expressed exogenous IRS-3 in immortalized IRS-1-/- brown adipocytes at a level comparable with that of endogenous IRS-3 in white adipose tissue. Under these conditions, IRS-3 signaling in response to insulin was observed, as revealed by tyrosine phosphorylation of IRS-3, and the activation of phosphatidylinositol (PI) 3-kinase associated with this recombinant protein. However, although insulin promoted the association of Grb-2 with recombinant IRS-3 in IRS-1-/- cells, the exogenous expression of this IRS family member failed to activate p42/44 MAPK and mitogenesis in brown adipocytes lacking IRS-1. Downstream of PI 3-kinase, IRS-3 expression restored insulin-induced Akt phosphorylation, which is impaired by the lack of IRS-1 signaling. Whereas the generation of IRS-3 signals enhanced adipocyte determination and differentiation-dependent factor 1/sterol regulatory element-binding protein (ADD-1/SREBP-1c) and fatty acid synthase mRNA and protein expression, activation of this pathway was unable to reconstitute CCAAT/enhancer-binding protein alpha and uncoupling protein-1 transactivation and gene expression in response to insulin. Similar results were obtained following insulin-like growth factor-I stimulation. In brown adipocytes expressing the IRS-3F4 mutant, the association of the p85alpha regulatory subunit via Src homology 2 binding was lost, but insulin nevertheless induced PI 3-kinase activity and Akt phosphorylation in a wortmannin-dependent manner. In contrast, activation of IRS-3F4 signaling failed to restore the induction of ADD-1/SREBP-1c and fatty acid synthase gene expression in IRS-1-deficient brown adipocytes. These studies demonstrate that recombinant IRS-3 may reconstitute some, but not all, of the signals required for insulin action in brown adipocytes. Thus, our data further implicate a unique role for IRS-1 in triggering insulin action in brown adipocytes.

Adipocyte differentiation of mouse embryonic fibroblasts (MEFs) derived from c-Src wild-type or c-Src-deficient (abbreviated as MEF+/+ and MEF-/- hereafter) C57BL/6 mice was induced by ascorbic acid (A) and beta-glycerophosphate (G). TCDD clearly suppressed differentiation of MEF+/+, but not MEF-/-, as measured by increased accumulation of triglycerides associated with increased expression of adipocyte differentiation-specific genes such as peroxisome proliferators activated receptor (PPAR)gamma, stearoyl-CoA desaturase (SCD-1). Studies on inducibility of TCDD-activated genes such as cytochrome P450 (CYP)1A1 and CYP1B1 revealed a comparable dose response in both MEF+/+ and MEF-/-. Furthermore, the binding activity of AhR complexes to xenobiotic response elements (XREs) was similar in both cell lines. We further studied the effect of TCDD on CCAAT/enhancer binding proteins (C/EBP), which are known to be important regulators of cell differentiation. TCDD induced C/EBPbeta and C/EBPdelta mRNA expression and DNA binding activity in a time- and dose-dependent manner in MEF+/+ but not in MEF-/-. The levels of C/EBPbeta and C/EBPdelta were still elevated in differentiated MEF+/+ after 10 days of treatment with TCDD. In MEF-/-, C/EBPbeta and C/EBPdelta are highly expressed constitutively. In contrast to MEF+/+, TCDD does not cause any significant change of these transcription factors in MEF-/-. These data indicate that suppression of differentiation by TCDD in MEF requires a functional c-Src activity and induced levels of C/EBPbeta and C/EBPdelta, including their maintenance at high levels by TCDD, rather than ultimate high levels of these C/EBP isoforms.

Background: The genes for the sterol regulatory element-binding protein-1a (SREBP-1a), -1c, and -2, the low-density lipoprotein (LDL) receptor, and the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase play a key role in the intracellular cholesterol and lipid metabolism., Methods: To enable the absolute and relative quantitation of the mRNA levels of these genes we developed a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The inclusion of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene for reference and normalization enabled us to accurately discriminate between a twofold variance in the expression levels of these genes. We used this assay to study their expression in mononuclear peripheral blood cells (PBMNC)., Results: We found that the relative expression of SREBP-1a is tenfold higher than that of SREBP-1c, but only half of that of SREBP-2. The level of SREBP-1a transcripts correlated with that of the SREBP-1c, LDL receptor, HMG-CoA reductase, and SREBP-2 genes, whereas the amount of SREBP-1c mRNA did not show a relationship with that of the latter three genes. The most abundant transcript in PBMNC is that of SREBP-2, followed by that of SREBP-1a, whereas SREBP-1c mRNA is only found in smaller amounts., Conclusions: This competitive RT-PCR method is very well suited for the accurate quantitation of the respective mRNAs.

Sterol regulatory element-binding proteins (SREBPs) are a family of membrane-bound transcription factors that regulate cholesterol and fatty acid homeostasis. In mammals, three SREBP isoforms designated SREBP-1a, SREBP-1c, and SREBP-2 have been identified. SREBP-1a and SREBP-1c are derived from the same gene by virtue of alternatively spliced first exons. SREBP-1a has a longer transcriptional activation domain and is a more potent transcriptional activator than SREBP-1c in cultured cells and liver. Here, we describe the physiologic consequences of overexpressing the nuclear form of SREBP-1a (nSREBP-1a) in adipocytes of mice using the adipocyte-specific aP2 promoter (aP2-nSREBP-1a). The transgenic aP2-nSREBP-1a mice developed markedly enlarged white and brown adipocytes that were fully differentiated. Adipocytes isolated from aP2-nSREBP-1a mice had significantly increased rates of fatty acid synthesis and enhanced fatty acid secretion. The increased production and release of fatty acids from adipocytes led, in turn, to a fatty liver. Overexpression of the alternative SREBP-1 isoform, nSREBP-1c, in adipose tissue inhibits adipocyte differentiation; as a result, the transgenic nSREBP-1c mice develop a syndrome resembling human lipodystrophy, which includes a loss of peripheral white adipose tissue, diabetes, and fatty livers (Shimomura, I., Hammer, R. E., Richardson, J. A., Ikemoto, S., Bashmakov, Y., Goldstein, J. L., and Brown, M. S. (1998) Genes Dev. 12, 3182-3194). In striking contrast, nSREBP-1a overexpression in fat resulted in the hypertrophy of fully differentiated adipocytes, no diabetes, and mild hepatic steatosis. These results suggest that nSREBP-1a and nSREBP-1c have distinct roles in adipocyte fat metabolism in vivo.

The aim of this study was to investigate the sterol regulatory element-binding protein 1c (SREBP1c) mRNA muscle expression in morbid obese subjects before and after massive lipid malabsorption due to bariatric surgery (bilio-pancreatic diversion, BPD). We studied 11 obese subjects (BMI 49+/-2 kg/m2) before and 24 months after BPD. Skeletal muscle SREBP1c mRNA expression was determined using RT-competitive PCR. Intramyocytic triglycerides were quantified by HPLC. Insulin sensitivity (M/I) was assessed by euglycemic-hyperinsulinemic clamp. Energy expenditure and respiratory quotient (RQ) were measured over 24 h in a calorimetric chamber. Total cardiovascular risk dropped from 2 before to -2.5 after BPD (P<0.0001). The M/I value was normalized after surgery (0.036+/-0.0148 to 0.095+/-0.0147 micromol kgFFM(-1) min(-1) pmoles(-1) P<0.001). SREBP-1c mRNA levels were decreased (from 4.12+/-2.43 to 2.69+/-1.83% of cyclophilin mRNA, P=0.02) after BPD. In a multiple regression analysis, M/I values (P<0.0001) as well as the intramyocytic triglyceride levels (P=0.039) were the most powerful independent variables for predicting cardiovascular risk. Our results show that the reduction of cardiovascular risk after bariatric massive weight loss is strongly related to the reversion of insulin resistance and to the lowering of intramyocytic triglyceride depots. These two parameters are associated with a significant reduction in SREBP-1c mRNA expression in skeletal muscle, suggesting that this transcription factor might be involved in the accumulation of triglycerides in muscle cells of morbidly obese subjects.

Insulin-like growth factor-1 (IGF-1) promotes the survival of cerebellar granule neurons by enhancing calcium influx through L-type calcium channels, whereas NMDA receptor-mediated calcium influx can lead to excitotoxic death. Here we demonstrate that L and NMDA receptor channel activities differentially regulate the transcription factor C/EBPbeta to control neuronal survival. Specifically, we show that L channel-dependent calcium influx results in increased CaMKIV activity, which acts to decrease nuclear C/EBPbeta levels. Conversely, NMDA receptor-mediated influx rapidly elevates nuclear C/EBPbeta and induces excitotoxic death via activation of the calcium-dependent phosphatase, calcineurin. Moderate levels of AMPA receptor activity stimulate L channels to improve survival, whereas higher levels stimulate NMDA receptors and reduce neuronal survival, suggesting differential synaptic effects. Finally, N-type calcium channel activity reduces survival, potentially by increasing glutamate release. Together, these results show that the L-type calcium channel-dependent survival and NMDA receptor death pathways converge to regulate nuclear C/EBPbeta levels, which appears to be pivotal in these mechanisms.

Specific ligands of the peripheral benzodiazepine receptor (PBR) are known to induce apoptosis and cell cycle arrest in oesophageal cancer cells. However, the underlying mechanisms are still unknown. Here, we investigated the transcriptional alterations and activation of protein kinases in response to PBR-specific ligands. Using cDNA arrays, we examined the transcriptional effects of the PBR-specific ligand FGIN-1-27 in two oesophageal cancer cell lines, KYSE-140 (squamous cell carcinoma) and OE-33 (adenocarcinoma). In oesophageal cancer cells, FGIN-1-27 induced extensive changes in the expression of genes involved in the regulation of apoptosis and cell cycle. Both in oesophageal cancer cell lines (KYSE-140, OE-33) we observed a strong upregulation of the growth arrest and DNA-damage-inducible genes, gadd45 and gadd153, in response to PBR ligands. gadd genes are known to be induced by p38MAPK activation. Using Western blotting we detected a time- and dose-dependent phosphorylation of p38MAPK, which was found to be functionally involved in gadd induction, apoptosis, and cell cycle arrest. In conclusion, our data indicate that PBR-specific ligands cause apoptosis and cell cycle arrest by activation of the p38MAPK pathway and induction of gadd45 and gadd153.

Understanding the strengths and limitations of alternative models, such as the Tg.AC assay, for evaluation of the potential carcinogenicity of pharmaceuticals requires assessment of assay specificity through studies that specifically target biologically active compounds that are known to not be carcinogens in rodents. To identify drugs that might provoke a false positive response in the Tg.AC assay, we screened pharmaceuticals for in vitro induction of the gadd153 promoter and the zeta-globin promoter. We have previously found a high correlation between induction of the gadd153 promoter in HepG2 cells and activity in the Tg.AC assay. The three drugs selected through screening 99 noncarcinogenic pharmaceuticals were amiloride, dipyridamole, and pyrimethamine. A 26-week skin paint study was conducted in hemizygous Tg.AC mice with the three drugs at two doses selected by a 4-week dose range finding study. Evidence of systemic toxicity was observed in animals dosed chronically with pyrimethamine or amiloride, but no skin papillomas were observed in mice treated with amiloride, dipyridamole, or pyrimethamine for 26 weeks. All male mice and 80% of female mice treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) in acetone developed a maximal tumor burden. However, mice treated with TPA in a vehicle containing 2.4% DMSO had greatly reduced incidences of papillomas. In summary, the correct negative response was shown in the Tg.AC assay for three noncarcinogenic pharmaceuticals, which adds further favorable evidence of appropriate specificity of this model system. However, vehicle composition must be carefully selected because the outcome of this assay can be confounded by certain commonly used solvents.

Short-term assays for carcinogenicity testing of chemicals that use transgenic mice designed to have altered expression of genes mechanistically relevant to carcinogenesis are attractive alternatives to two-year dosing studies in rodents. The models that have been the received the greatest level of performance evaluation include p53(+/-), rasH2, Xpa/p53(+/-), and Tg.AC mice. For use of these models in a regulatory setting to evaluate the carcinogenic potential of pharmaceuticals, it is important to establish an assurance of assay specificity and positive predictivity based on studies using drugs with a wide spectrum of pharmacologic activity. For this purpose, 99 noncarcinogenic drugs were prioritized based on their activity in an in vitro induction assay correlative with a positive response in the Tg.AC assay (induction of the gadd153 promoter in HepG2 cells). Activities in two assays less predictive of Tg.AC activity (induction of c-fos and zeta-globin gene promoters) were also measured. Nine percent of the screened drugs induced the gadd153 promoter by at least fourfold. Several criteria were used to select candidates for subsequent in vivo testing in the Tg.AC assay: (1) sufficient drug solubility in appropriate skin paint vehicles to elicit systemic toxicity, (2) the level of induction of the gadd153 promoter by the drug, (3) the in vitro potency of the drug, and (4) the cost of the drug required for a 6-month study. Based on these criteria, amiloride, dipyridamole, and pyrimethamine were selected from 99 rodent noncarcinogens in a drug database for testing the specificity of the Tg.AC assay.

Liver X receptors (LXRs) regulate the expression of genes involved in cholesterol and fatty acid homeostasis, including the genes for ATP-binding cassette transporter A1 (ABCA1) and sterol response element binding protein 1 (SREBP1). Loss of LXR leads to derepression of the ABCA1 gene in macrophages and the intestine, while the SREBP1c gene remains transcriptionally silent. Here we report that high-density-lipoprotein (HDL) cholesterol levels are increased in LXR-deficient mice, suggesting that derepression of ABCA1 and possibly other LXR target genes in selected tissues is sufficient to result in enhanced HDL biogenesis at the whole-body level. We provide several independent lines of evidence indicating that the repressive actions of LXRs are dependent on interactions with the nuclear receptor corepressor (NCoR) and the silencing mediator of retinoic acid and thyroid hormone receptors (SMRT). While dissociation of NCoR and SMRT results in derepression of the ABCA1 gene in macrophages, it is not sufficient for derepression of the SREBP1c gene. These findings reveal differential requirements for corepressors in the regulation of genes involved in cholesterol and fatty acid homeostasis and raise the possibility that these interactions may be exploited to develop synthetic ligands that selectively modulate LXR actions in vivo.

Previous studies have revealed that LPS can activate transcription of the IL-10 gene promoter through an SV40 promoter factor 1 (Sp1) binding site in mouse macrophage RAW264.7. In this study, we determined that, in addition to Sp1, C/EBPbeta and delta were also involved in LPS-induced gene expression of IL-10. By transient transfection with 5'-deletion mutants of the IL-10 promoter, we found that there were two LPS-responsive elements in the promoter of the mouse IL-10 gene. Analysis of these two regions by gel shift assay suggested that Sp1 and C/EBPbeta and delta were bound to these two regions, respectively. By site-directed mutagenesis, we found that disruption at both the Sp1 and C/EBP binding sites almost completely blocked the LPS response. By gel shift assay and Western blotting, we found that the DNA binding complex and protein expression of C/EBPbeta and delta were increased by LPS treatment, but these results were not found for Sp1. Overexpression of C/EBPbeta or C/EBPdelta, respectively, activated the promoter of the IL-10 gene, and they were enhanced by LPS. Coimmunoprecipitation experiments in intact cells indicated that LPS stimulated interaction between Sp1 and C/EBPbeta and delta. These results suggested that the interaction between Sp1 and C/EBPbeta and delta induced by LPS cooperatively activated expression of the IL-10 gene. The increase of C/EBPbeta and delta proteins and the enhancement of transactivation activity of C/EBPbeta and delta by LPS treatment, at least in part, explain the activation of IL-10 gene expression.

ICBP90 (Inverted CCAAT box Binding Protein of 90 kDa) is a recently identified nuclear protein that binds to one of the inverted CCAAT boxes of the topoisomerase IIalpha (TopoIIalpha) gene promoter. Here, we show that ICBP90 shares structural homology with several other proteins, including Np95, the human and mouse NIRF, suggesting the emergence of a new family of nuclear proteins. Towards elucidating the functions of this family, we analysed the expression of ICBP90 in various cancer or noncancer cell lines and in normal or breast carcinoma tissues. We found that cancer cell lines express higher levels of ICBP90 and TopoIIalpha than noncancer cell lines. By using cell-cycle phase-blocking drugs, we show that in primary cultured human lung fibroblasts, ICBP90 expression peaks at late G1 and during G2/M phases. In contrast, cancer cell lines such as HeLa, Jurkat and A549 show constant ICBP90 expression throughout the entire cell cycle. The effect of overexpression of E2F-1 is more efficient on ICBP90 and TopoIIalpha expression in noncancer cells (IMR90, WI38) than in cancer cells (U2OS, SaOs). Together, these results show that ICBP90 expression is altered in cancer cell lines and is upregulated by E2F-1 overexpression with an efficiency depending on the cancer status of the cell line.

Elevated levels of both fibrinogen and cholesterol are risk factors in coronary artery disease. Previously we reported a metabolic link between fibrinogen and lipid metabolism in that HepG2 cells that were programmed by transfection of Bbeta-fibrinogen cDNA to overexpress fibrinogen exhibited increased synthesis of cholesterol and increased secretion of apolipoprotein B. In this study we demonstrate that oxysterols, which participate in maintaining cholesterol homeostasis, also down regulate fibrinogen expression. Treatment of HepG2 cells with 25-hydroxycholesterol lowered fibrinogen Aalpha, Bbeta and gamma mRNA levels and inhibited fibrinogen synthesis and secretion but had no effect on alpha1 -antitrypsin which, like fibrinogen, is an acute-phase protein. The inhibition of fibrinogen synthesis by oxysterols was maintained in interleukin-6 treated cells. Other oxysterols, that inhibit cholesterol synthesis by a feedback mechanism, also diminished fibrinogen expression in HepG2, rat H-4-II-E hepatoma cells and in primary human hepatocytes. Overexpression of SREBP-1 and SREBP-2 by transfection of HepG2 cells, or treatment with a synthetic LXRalpha agonist, which affect cholesterol metabolism, did not affect fibrinogen expression. We conclude that fibrinogen and cholesterol may share a novel common regulatory pathway.

The synthesis of phosphatidylcholine (PtdCho) by the CDP-choline pathway is under the control of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT). Sterol regulatory element binding proteins (SREBPs) have been proposed to regulate CCT at the transcriptional level, or via the synthesis of lipid activators or substrates of the CDP-choline pathway. To assess the contributions of these two mechanisms, we examined CCTalpha expression and PtdCho synthesis by the CDP-choline pathway in cholesterol and fatty acid auxotrophic CHO M19 cells inducibly expressing constitutively active nuclear forms of SREBP1a or SREBP2. Induction of either SREBP resulted in increased expression of mRNAs for sterol-regulated genes, elevated fatty acid and cholesterol synthesis (>10-50-fold) and increased PtdCho synthesis (2-fold). CCTalpha mRNA was increased 2-fold by enforced expression of SREBP1a or SREBP2. The resultant increase in CCTalpha protein and activity (2-fold) was restricted primarily to the soluble fraction of cells, and increased CCTalpha activity in vivo was not detected. Inhibition of the synthesis of fatty acids or their CoA esters by cerulenin or triacsin C respectively following SREBP induction effectively blocked the accompanying elevation in PtdCho synthesis. Thus PtdCho synthesis was driven by increased synthesis of fatty acids or a product thereof. These data show that transcriptional activation of CCTalpha is modest relative to that of other SREBP-regulated genes, and that stimulation of PtdCho synthesis by SREBPs in CHO cells is due primarily to increased fatty acid synthesis.

Hamsters were fed a control diet or diets containing palm, olive, safflower, or fish oil for 2 weeks. In villus cell populations from duodenum, jejunum, and ileum, rates of intestinal fatty acid and cholesterol synthesis were estimated, as were sterol regulatory element-binding protein (SREBP)-1a, SREBP-1c, SREBP-2, HMG-CoA synthase, fatty acid synthase, ATP citrate lyase, acetyl-CoA carboxylase mRNA levels, and SREBP-1 and SREBP-2 mass. Plasma cholesterol and triacylglcerol levels were increased in animals ingesting palm oil and decreased in animals ingesting fish oil. Fatty acid synthesis and fatty acid synthase activity were decreased in the proximal intestine of animals ingesting all the fat-containing diets. Intestinal cholesterol synthesis was unaltered. In animals fed fat, SREBP-1c gene expression was modestly increased in the duodenum of hamsters fed palm oil or olive oil, and decreased in animals ingesting safflower oil or fish oil. Fatty acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, SREBP-2, and HMG-CoA synthase mRNA levels were not altered, nor were SREBP-1 or SREBP-2 mass. In the intestine, dietary polyunsaturated fatty acids suppress SREBP-1c mRNA without altering expression of its target genes, fatty acid synthase, acetyl-CoA carboxylase, or ATP citrate lyase. Fatty acid influx decreases intestinal fatty acid synthesis by a posttranscriptional mechanism independent of the SREBP pathway.

Accumulation of lipids in non-adipose tissues is often associated with Type 2 diabetes and its complications. Elevated expression of the lipogenic transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), has been demonstrated in islets and liver of diabetic animals. To elucidate the molecular mechanisms underlying SREBP-1c-induced beta-cell dysfunction, we employed the Tet-On inducible system to achieve tightly controlled and conditional expression of the nuclear active form of SREBP-1c (naSREBP-1c) in INS-1 cells. Controlled expression of naSREBP-1c induced massive accumulation of lipid droplets and blunted nutrient-stimulated insulin secretion in INS-1 cells. K(+)-evoked insulin exocytosis was unaltered. Quantification of the gene expression profile in this INS-1 stable clone revealed that naSREBP-1c induced beta-cell dysfunction by targeting multiple genes dedicated to carbohydrate metabolism, lipid biosynthesis, cell growth, and apoptosis. naSREBP-1c elicits cell growth-arrest and eventually apoptosis. We also found that the SREBP-1c processing in beta-cells was irresponsive to acute stimulation of glucose and insulin, which was distinct from that in lipogenic tissues. However, 2-day exposure to these agents promoted SREBP-1c processing. Therefore, the SREBP-1c maturation could be implicated in the pathogenesis of beta-cell glucolipotoxicity.

Aging is generally accompanied by reduced tolerance to oxidative stress and altered responsiveness to proliferative signals. We have shown that hepatocytes derived from aged rats (24-26 months) exhibit greater sensitivity to H(2)O(2) treatment and reduced proliferation following epidermal growth factor (EGF) treatment than cells of young adult rats (5-6 months). Here we examined the effects of aging and calorie restriction (CR) on expression of the oxidative stress-inducible and pro-apoptotic gene gadd153 (chop) in these hepatocytes, and we investigated its influence on sensitivity to oxidants. We show that aging was associated with elevated expression of gadd153, both basally and in response to H(2)O(2) treatment. CR, which attenuates age-associated declines in stress tolerance, prevented the age-related increase in gadd153 expression. EGF treatment also resulted in gadd153 induction in old cells. This effect was absent in young cells and in old cells of CR rats. gadd153 induction by EGF was reactive oxygen species-dependent and correlated with heightened sensitivity to subsequent H(2)O(2) treatment, suggesting that elevated Gadd153 contributes to the greater sensitivity of EGF-pretreated old cells to oxidative stress. Additional support for this hypothesis was provided by experiments with Rat1 fibroblasts in which conditional expression of Gadd153 conferred increased sensitivity to H(2)O(2). We propose a model whereby the diminished ability of old hepatocytes to overcome an EGF-triggered reactive oxygen species load leads to induction of the proapoptotic gene gadd153, which, in turn, sensitizes the cells to oxidant injury. Our findings point to gadd153 expression levels as an important factor in liver aging.

The unfolded protein response, which is activated in response to the loss of endoplasmic reticulum (ER) Ca(2+) homeostasis and/or the accumulation of misfolded, unassembled, or aggregated proteins in the ER lumen, involves both transcriptional and translational regulation. In the current studies we sought to identify novel ER stress-induced genes by conducting microarray analysis on tunicamycin-treated cells. We identified P58(IPK), an inhibitor of the interferon-induced double-stranded RNA-activated protein kinase, as induced during ER stress. Additional studies suggested that p58(IPK) induction was mediated via ATF6 and that P58(IPK) played a role in down-regulating the activity of the pancreatic eIF2 kinase/eukaryotic initiation factor 2alpha (eIF2alpha)-like ER kinase/activation transcription factor (ATF) 4 pathway. Modulation of P58(IPK) levels altered the phosphorylation status of eIF2alpha, and thereby affected expression of its downstream targets, ATF4 and Gadd153. Overexpression of P58(IPK) inhibited eIF2alpha phosphorylation and reduced ATF4 and Gadd153 protein accumulation, whereas silencing of P58(IPK) expression enhanced pancreatic eIF2alpha-like ER kinase and eIF2alpha phosphorylation and increased ATF4 and Gadd153 accumulation. These findings implicate P58(IPK) as an important component of a negative feedback loop used by the cell to inhibit eIF2alpha signaling, and thus attenuate the unfolded protein response.

The expression of acetyl-CoA carboxylase (ACC) in mouse peripheral nervous system (PNS) was investigated. Both ACC 265 and ACC 280 isoforms were expressed in the sciatic nerve, although ACC 265 was predominant. ACC 265 transcripts originating from promoters P1 and P2 could be detected in the developing nerve, as well as the two splice products, which are characterized by the presence or the absence of a 24-base sequence before the codon serine-1200. The mRNA levels for ACC 265 parallel those of other lipogenic genes whose expression is linked to the myelination process. In addition, ACC 265 mRNA and protein levels in the nerves of the trembler mutant, which is a mouse model of PNS dysmyelination, represented around 30% of the normal values. The expression of the sterol regulatory element-binding proteins (SREBPs) was also studied. SREBP 1 mRNAs were expressed at a constant level during nerve development, and their quantities were normal in trembler. On the contrary, SREBP 2 mRNA quantities varied during the myelination period similarly to the lipogenic gene mRNAs, and the levels measured in trembler represented only 10% of the normal values. Taken together, these results suggest that the coordinate expression of several lipogenic genes, which occurs during PNS myelination, could possibly be regulated by SREBP 2.

Activation of fatty acid synthase (FAS) expression and fatty acid synthesis is a common event in human breast cancer. Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate genes involved in lipid metabolism, including FAS. SREBP-1c expression is induced in liver and adipose tissue by insulin and by fasting/refeeding and is critical for nutritional regulation of lipogenic gene expression. In contrast, upregulation of fatty acid metabolism during in vitro transformation of human mammary epithelial cells and in breast cancer cells was driven by increased MAP kinase and PI 3-kinase signaling, which increased SREBP-1 levels. SREBP-1a was more abundant than SREBP-1c in many proliferative tissues and cultured cells and was thus a candidate to regulate lipogenesis for support of membrane synthesis during cell growth. We now show that SREBP-1c and FAS mRNA were both increased by H-ras transformation of MCF-10a breast epithelial cells and were both reduced by exposure of MCF-7 breast cancer cells to the MAP kinase inhibitor, PD98059, or the PI 3-kinase inhibitor, wortmannin, while SREBP-1a and SREBP-2 showed less variation. Similarly, the mRNA levels for FAS and SREBP-1c in a panel of primary human breast cancer samples showed much greater increases than did those for SREBP-1a and SREBP-2 and were significantly correlated with each other, suggesting coordinate regulation of SREBP-1c and FAS in clinical breast cancer. We conclude that regulation of FAS expression in breast cancer is achieved through modulation of SREBP-1c, similar to the regulation in liver and adipose tissue, although the upstream regulation of liopgenesis differs in these tissues.

Regulation of sterol regulatory element-binding proteins (SREBPs) by fatty acid flux was investigated in CaCo-2 cells. Cells were incubated with 1 mM taurocholate with or without 250 microM 18:0, 18:1, 18:2, 20:4, 20:5 or 22:6 fatty acids. Fatty acid synthase (FAS) and acetyl-CoA carboxylase mRNA levels and gene and protein expression of SREBPs were estimated. 18:2, 20:4, 20:5 and 22:6 fatty acids decreased the amount of mature SREBP-1 and mRNA levels of SREBP-1c, SREBP-1a, FAS and acetyl-CoA carboxylase. SREBP-2 gene or mature protein expression was not altered. Liver X receptor (LXR) activation by T0901317 increased gene expression of SREBP-1c, SREBP-1a, FAS and acetyl-CoA carboxylase without altering SREBP-2. 20:5, but not 18:1, prevented the full expression of SREBP-1c mRNA by T0901317. T0901317 increased SREBP-1 mass without altering the mass of mature SREBP-2. Although only 18:2, 20:4, 20:5 and 22:6 suppressed SREBP-1, acetyl-CoA carboxylase and FAS expression, all fatty acids decreased the rate of fatty acid synthesis. T0901317 increased endogenous fatty acid synthesis yet did not increase secretion of triacylglycerol-rich lipoproteins. In CaCo-2 cells, polyunsaturated fatty acids decrease gene and protein expression of SREBP-1 and FAS mRNA, probably through interference with LXR activity. Since all fatty acids decreased fatty acid synthesis, mechanisms other than changes in SREBP-1c expression must be entertained. Increased endogenous fatty acid synthesis does not promote triacylglycerol-rich lipoprotein secretion.

P58(IPK) is an Hsp40 family member known to inhibit the interferon (IFN)-induced, double-stranded RNA-activated, eukaryotic initiation factor 2alpha (eIF2alpha) protein kinase R (PKR) by binding to its kinase domain. We find that the stress of unfolded proteins in the endoplasmic reticulum (ER) activates P58(IPK) gene transcription through an ER stress-response element in its promoter region. P58(IPK) interacts with and inhibits the PKR-like ER-localized eIF2alpha kinase PERK, which is normally activated during the ER-stress response to protect cells from ER stress by attenuating protein synthesis and reducing ER client protein load. Levels of phosphorylated eIF2alpha were lower in ER-stressed P58(IPK)-overexpressing cells and were enhanced in P58(IPK) mutant cells. In the ER-stress response, PKR-like ER kinase (PERK)-mediated translational repression is transient and is followed by translational recovery and enhanced expression of genes that increase the capacity of the ER to process client proteins. The absence of P58(IPK) resulted in increased expression levels of two ER stress-inducible genes, BiP and Chop, consistent with the enhanced eIF2alpha phosphorylation in the P58(IPK) deletion cells. Our studies suggest that P58(IPK) induction during the ER-stress response represses PERK activity and plays a functional role in the expression of downstream markers of PERK activity in the later phase of the ER-stress response.

Dopamine (DA) metabolism and oxidation produce both reactive oxygen species (ROS) and reactive quinones. These chemical species are implicated in dopamine neurotoxicity and neurodegeneration. In the present studies, human neuroblastoma (SK-N-SH) cells were exposed to toxic concentrations of dopamine (333 microM) in order to investigate molecular pathways involved in dopamine toxicity. cDNA hybridization array (microarray) technology demonstrated that GADD45 and GADD153 (growth arrest and DNA-damage inducible) gene expression was increased in dopamine-treated cells (333 microM for 18 h). Subsequent Northern and Western blot analysis confirmed these changes in GADD45 and GADD153 gene expression. The antioxidant, ascorbic acid, significantly reduced the increase in GADD45 gene expression but did not significantly reduce GADD153 gene expression. Currently, the precise function of the GADD gene products is not known. It is known, however, that these genes are upregulated in response to stress to allow cells time to repair macromolecular damage. In the present case, GADD gene expression (manifested as increased mRNA and protein levels) preceded dopamine-induced cytotoxicity. It appears that dopamine, through the formation of reactive oxygen species and quinones, may damage cellular macromolecules to the point of inducing GADD gene expression. Other genes that displayed changes, but that have not been subjected to post-hoc confirmation, include clusterin (increased), ubiquitin (increased), CD27 ligand (increased), CD27BP (increased), and rac-PK-beta (decreased).

We have recently identified ICBP90 as being a protein able to bind in vitro a CCAAT box of the topoisomerase II alpha gene promoter. The aim of the present work was to check whether ICBP90 is able to regulate in vivo topoisomerase II alpha expression in human lung fibroblasts under various proliferating conditions. Transient transfection experiments performed on moderately growing human lung fibroblasts (50% of confluence) showed that overexpression of ICBP90 is associated with an elevation of topoisomerase II alpha expression and an increase of the cell proliferation rate. In highly proliferating human lung fibroblasts (20% confluence) overexpression of ICBP90 had no effect. In contrast, in non-proliferating fibroblasts (100% confluence) overexpression of ICBP90 allowed recovery of topoisomerase II alpha expression levels with a concomitant overgrowth of confluent cell cultures. Our results show that ICBP90 regulates topoisomerase II alpha expression and is able to overcome cell contact inhibition signaling, suggesting that increased ICBP90 expression may be involved in carcinogenesis.

Stearoyl-CoA desaturase (SCD) catalyzes the rate-limiting step in the cellular synthesis of monounsaturated fatty acids, mainly oleate (18:1) and palmitoleate (16:1), which are the major monounsaturated fatty acids of membrane phospholipids, cholesteryl esters, waxes, and triglycerides. The mouse expresses three well-characterized SCD genes (SCD1, 2, and 3). SCD1 is the main isoform expressed in the liver of mice. Previous in vivo studies have shown that the transcriptional repression by n-3 and n-6 polyunsaturated fatty acids (PUFAs) and the induction by cholesterol of the SCD1 gene are dependent on the maturation of the sterol regulatory element-binding protein-1c (SREBP-1c). We studied the regulation of SREBP-1, SCD1, and other SREBP-1 target genes when a high cholesterol diet is combined with PUFA as n-6 PUFA rich soybean oil (SO), or n-3 PUFA rich fish oil (FO). While the PUFA/cholesterol (PUFA/CH) diets repressed the maturation of the SREBP-1, the SCD1 mRNA levels, and protein and enzyme activity were induced. Compared with PUFA diets, hepatic cholesterol ester and triglyceride were enriched with 16:1 and 18:1 monounsaturated fatty acids in mice fed PUFA/CH diets. Total plasma cholesterol levels were not altered but plasma triglycerides were reduced in SO/CH-fed mice compared with SO-fed mice. The mRNA for SREBP-1 was increased by the PUFA/CH diet but the mRNA levels of SREBP-1 target genes such as fatty acid synthase and LDL receptor were decreased, indicating that the main control of PUFA-mediated suppression of SREBP-1 target genes is the maturation of SREBP-1. This study demonstrates that cholesterol overrides the PUFA-mediated repression of the SCD1 gene and regulates SCD1 gene expression through a mechanism independent of SREBP-1 maturation.

Endoplasmic reticulum (ER) stress signaling is an adaptive cellular response to the loss of ER Ca(2+) homeostasis and/or the accumulation of misfolded, unassembled, or aggregated proteins in the ER lumen. ER stress-activated signaling pathways regulate protein synthesis initiation and can also trigger apoptosis through the ER-associated caspase 12. Viruses that utilize the host cell ER as an integral part of their life cycle would be predicted to cause some level of ER stress. Bovine viral diarrhea virus (BVDV) is a positive-stranded RNA virus of the Flaviviridae family. BVDV and related flaviviruses use the host ER as the primary site of envelope glycoprotein biogenesis, genomic replication, and particle assembly. We are using a cytopathic strain of BVDV (cpBVDV) that causes cellular apoptosis as a model system to determine how virus-induced ER stress contributes to pathogenesis. We show that, in a natural infection of MDBK cells, cpBVDV activates the ER transmembrane kinase PERK (PKR-like ER kinase) and causes hyperphosphorylation of the translation initiation factor eIF2 alpha, consistent with the induction of an ER stress response. Additionally, we show that initiation of cellular apoptosis correlates with downregulation of the antiapoptotic Bcl-2 protein, induced expression of caspase 12, and a decrease in intracellular glutathione levels. Defining the molecular stress pathways leading to cpBVDV-induced apoptosis provides the basis to study how other ER-tropic viruses, such as hepatitis C and B viruses, modulate the host cell ER stress response during the course of persistent infection.

Metallothionein (MT) promoter was methylated in rat hepatoma and in mouse lymphosarcoma cells by methylation of cytosine within the CpG dinucleotide region. After demethylation of MT-I promoter in mouse lymphosarcoma cells or in the transplanted rat hepatoma with 5-azacytidine, a potent inhibitor of DNA methyltransferase, the promoter was activated in response to heavy metal treatment. MT-I promoter was also suppressed in human prostate cancer lines PC3 and DU145, probably by promoter methylation, whereas cadmium induced MT-I in the human prostate cancer line LNCaP. In the prostate cancer lines where MT-I was suppressed, glutathione-S-transferase-pi (GST-pi) was expressed. On the contrary, GST-pi gene was repressed in the cell line where MT-I was induced, which suggests an inverse relationship between MT-I induction and GST-pi expression in some prostate cancer lines. The expressions of GST-pi and gamma-glutamyl cysteine synthase were also significantly higher (5- to 12-fold) in the lymphosarcoma cells and the hepatoma relative to the parental tissues. The higher expressions of these two genes suggest a compensatory mechanism in the cells where the gene for the antioxidant MT-I/II is not induced. MT-I/II may function as a growth suppressor either alone or in concert with other factor(s), and consequently their lack of expression could facilitate the tumor growth. In addition to suppression of MT-I/II expression by promoter methylation, the lack of MT induction could also be brought about by nuclear factor I (NFI), probably by interaction with the metal transcription factor MTF-1. An inverse relationship was observed between the level of NFI and MT-I expression in some cells, which suggests a role for NFI in the relatively low constitutive levels of MT-I expression in these cells.

Up-regulation of several stress proteins such as heat-shock proteins and glucose-regulated proteins participate in tolerance against environmental stress. Previously, we found that protein-disulfide isomerase (PDI) is specifically up-regulated in response to hypoxia/brain ischemia in astrocytes. In addition, the overexpression of this gene into neurons protects against apoptotic cell death induced by hypoxia/brain ischemia. To address the detailed function of PDI, we screened for proteins that interact with PDI using the yeast two-hybrid system. We report here that PDI interacts with ubiquilin, which has a ubiquitin-like domain and a ubiquitin-associated domain. Interestingly, ubiquilin is also up-regulated in response to hypoxia in glial cells with a time course similar to that of PDI induction. In hypoxia-treated glial cells, the endogenous ubiquilin and PDI were almost completely co-localized, suggesting that ubiquilin is an endoplasmic reticulum-associated protein. Overexpression of this gene in neuronal cells resulted in significant inhibition of the DNA fragmentation triggered by hypoxia, but not that induced by nitric oxide or staurosporine. Moreover, ubiquilin has the ability to attenuate CHOP induction by hypoxia. These observations suggested that ubiquilin together with PDI have critical functions as regulatory proteins for CHOP-mediated cell death, and therefore up-regulation of these proteins may result in acquisition of tolerance against ischemic stress in glial cells.

The synthetic retinoid fenretinide induces apoptosis of neuroblastoma cells and in vitro acts synergistically with chemotherapeutic drugs used to treat neuroblastoma. The mechanisms of fenretinide-induced cell death of neuroblastoma cells are complex, involving cellular signaling pathways as yet incompletely defined but, in part, involving the generation of reactive oxygen species (ROS). In an attempt to characterize the mechanism of action of fenretinide, cDNA array filters were screened to identify apoptotic genes regulated in response to treatment of SH-SY5Y cells with fenretinide. Expression of the stress-induced transcription factor, GADD153, was up-regulated at both the protein and mRNA levels in response to fenretinide. Overexpression of GADD153 increased apoptosis in the presence and absence of fenretinide, whereas reduced expression of GADD153 by expression of antisense DNA abrogated the response to fenretinide. Although fenretinide is a partial retinoic acid receptor (RAR)-beta/gamma agonist, RARbeta/gamma antagonists did not block the induction of GADD153 by fenretinide; conversely, the induction of GADD153 was blocked by antioxidants. Enzyme inhibitors were used to identify pathways mediating the ROS-dependent effects of fenretinide: inhibitors of phospholipase A(2) and lypoxygenases (LOX), and specific inhibitors of 12-LOX, but not 5-LOX or 15-LOX, inhibited the induction of ROS, apoptosis, and GADD153 in response to fenretinide. The inhibition of ROS and apoptosis was reversed by the addition of the 12-LOX products, 12 (S)-hydroperoxyeicosatetraenoic acid (12-HpETE) and 12 (S)-hydroxyeicosatetraenoic acid (12-HETE). Fenretinide did not increase free arachidonic acid levels, but increased LOX activity without a detectable increase in 12-LOX protein. These results suggest that fenretinide induces apoptosis via RAR-dependent and -independent pathways in which the RAR-independent pathway is characterized by a fenretinide-dependent increase in 12-LOX activity, leading to the induction of GADD153. The targeting of 12-LOX and/or GADD153 in neuroblastoma cells may thus present a novel pathway for the development of drugs inducing apoptosis of neuroblastoma with improved tumor specificity.

Activation of fatty acid synthase (FAS) expression and fatty acid synthesis is a common event in tumor cells from a variety of human cancers and is closely linked to malignant transformation and to tumor virulence in population studies of human cancer. We now show that, in contrast to nutritional regulation of lipogenesis in liver or adipose tissue, changes in fatty acid metabolism during in vitro transformation of the human mammary epithelial cell line MCF-10a are driven by increases in epidermal growth factor signaling, acting in major part through the mitogen-activated protein (MAP) kinase and phosphatidylinositol (PI) 3-kinase signaling cascades. H-ras transformation of MCF-10a cells resulted in upregulation of MAP kinase and PI 3-kinase signals, upregulation of sterol regulatory element binding protein 1 (SREBP-1) transcription factor levels, and upregulation of FAS expression and FA synthesis. Deletion of the major SREBP binding site from the FAS promoter abrogated transcription in transformed MCF-10a cells. Inhibitors of MAP and PI 3-kinases downregulated SREBP-1 levels and decreased transcription from the FAS promoter, reducing FAS expression and fatty acid synthesis in transformed MCF-10a cells and in MCF-7 and HCT116 carcinoma cells. H-ras transformation sensitized MCF-10a cells to the FAS inhibitors cerulenin and C-75. These results confirm an important role for SREBP-1 in neoplastic lipogenesis, and provide a likely basis for the linkage of upregulated fatty acid metabolism with neoplastic transformation and with tumor virulence, since MAP and PI 3-kinase signaling contributes to both.

Nephrotic syndrome (NS) is characterized by the presence of proteinuria and hyperlipidemia. However, ingestion of soy protein has a hypolipidemic effect. The present study was designed to determine whether the ingestion of a 20% soy protein diet regulates the expression of hepatic sterol regulatory element binding protein (SREBP)-1, fatty acid synthase (FAS), malic enzyme, beta-hydroxy-beta-methylglutaryl-CoA (HMG-CoA) reductase (r) and synthase (s), and LDL receptor (r), and to assess whether soy protein improves lipid and renal abnormalities in rats with chronic NS. Male Wistar rats were injected with vehicle or with puromycin aminonucleoside to induce NS and were fed either 20% casein or soy protein diets for 64 d. NS rats fed 20% soy protein had improved creatinine clearance and reduced proteinuria, hypercholesterolemia, hypertriglyceridemia, as well as VLDL-triglycerides and LDL cholesterol compared with NS rats fed the 20% casein diet. In addition, the soy protein diet decreased the incidence of glomerular sclerosis, and proinflammatory cytokines in kidney. Ingestion of the soy protein diet by control rats reduced the gene expression of SREBP-1, malic enzyme, FAS and increased HMG-CoAr, HMG-CoAs and LDLr. However, NS rats fed either casein or soy protein diets had low insulin concentrations with reductions in SREBP-1, FAS and malic enzyme expression compared with control rats fed the casein diet. NS rats fed the soy diet also had lower HMG-CoAr and LDLr mRNA levels than NS rats fed casein. In conclusion, the beneficial effects of soy protein on lipid metabolism are modulated in part by SREBP-1. However, in NS rats, the benefit may be through a direct effect of this protein on kidney rather than mediated by changes in expression of hepatic lipid metabolism genes.

Molecules differentially expressed or overexpressed by malignant cells can serve in detecting and tracking of tumor. Additionally, they potentially can be applied in histologic-specific antitumor therapy. Few breast cancer-associated candidate molecules have been identified. Here we describe the use of combinatorial immunoglobulin [antigen-binding fragment of immunoglobulin molecule (Fab) fragment] phage libraries generated from patients with breast carcinoma to identify cancer-associated gene expression. The libraries were enriched for tumor-binding Fab by 3 logs and yielded a group of antibodies against DNA-binding protein B (DbpB), a 35-kDa thrombin-inducible nuclear factor and member of the Y-box family of proteins, which are known to act both negatively in selective gene suppression and positively as promoters of gene transcription. Sequencing of the anti-DbpB showed a degree of heterogeneity and bp substitutions suggesting that the Fabs selected from the combinatorial library represented a varied anti-DbpB immune response and did not simply arise from in vitro amplification by PCR of a single or limited numbers of immunoglobulin genes. Sequencing of the DbpB molecule expressed in malignant breast cancer showed no evidence of tumor-specific mutations. Evaluation of levels of DbpB gene product expression however showed the molecule to be constitutively expressed in normal nonmalignant breast tissues but to have consistently differentially higher expression in breast cancer. Immunohistological staining revealed DbpB to be present both intracellularly and on the cell surface, which suggests it may be a means whereby malignant cells repair and replicate DNA in a selectively advantageous manner as compared with nonmalignant cells. DbpB expression in breast cancer may advance the basic understanding of the role of Y-box binding proteins as regulatory agents, and in defining malignant cell phenotypes. In addition, DbpB and the antibodies generated against it may have direct application in tumor detection and in molecule-targeted immunotherapy.

Downstream of the gene for the liposarcoma-associated fusion oncoprotein 54 (DOL54) is a target gene of the myxoid liposarcoma and round cell liposarcoma (M-LPS/RC-LPS) oncogene, TLS/FUS-CHOP. The DOL54 gene product is closely associated with adipogenic differentiation. DOL54 overexpression resulted in tumorigenicity when Chinese Hamster Ovary (CHO) cells were injected subcutaneously into nude mice. The biological significance of DOL54 expression for human malignant soft tissue tumors, however, has not yet been investigated. We examined TLS-CHOP and DOL54 expression in M-LPS/RC-LPS, well-differentiated liposarcoma and malignant fibrous histiocytoma (MFH), a tumor whose cellular origin has not been determined. We observed DOL54 expression in 50% of M-LPS/RC-LPS cases (in which TLS-CHOP was also expressed) and 33% of MFH cases, suggesting that a portion of MFH lesions may either derive from adipocytic precursor cells or have the potential to undergo adipogenic differentiation. In this manner, M-LPS/RC-LPS and MFH lesions may share tumorigenic characteristics, resulting from the unscheduled expression of DOL54.

Effects of lead nitrate (LN), a hepatic mitogen, on hepatic gene expressions of lanosterol 14alpha-demethylase (CYP51) and the sterol regulatory element binding proteins (SREBP-1a, SREBP-1c and SREBP-2), which are thought to be transcription factors for hepatic CYP51 gene, were examined by the methods of Northern blot and/or real time reverse transcriptase-polymerase chain reaction (RT-PCR). In both immature (4-week-old) and mature (7-week-old) rats, LN treatment resulted in definite increases in hepatic gene expression of CYP51 at 12 h and in the liver weight at 48 h. As for transcription factors for the CYP51 gene, enhanced gene expression of SREBP-2 was observed 6-12 h after LN treatment, whereas no enhanced gene expression of other SREBPs, SREBP-1a and SREBP-1c, was observed at any time after the treatment; for SREBP-1a, there was no significant change; for SREPB-1c, there was a drastic decrease. In addition, the serum total cholesterol level was increased 12 h after LN treatment to 7-week-old rats, and the increased level was maintained at least up to 48 h later. In the present study, we demonstrate for the first time that LN, a heavy-metal ion, activates the expression of the SREBP-2 and CYP51 genes without decreasing the serum total cholesterol level and further suggest that only SREBP-2 among SREBPs might play an important role in the LN-enhanced CYP51 gene expression.

Heregulin-beta1 (HRG), a combinatorial ligand for human epidermal growth factor receptor 3 (HER3) and HER4, is a regulatory polypeptide having distinct biological effects, such as growth stimulation, differentiation, invasiveness, and migration in mammary epithelial cells. The mechanism underlying the diverse functions of HRG is not well established but is believed to depend on induced changes in the expression of specific cellular gene products, their modification, or both. Here, we identified the basic leucine zipper transcription factor, the growth-arrest and DNA-damage 153 (GADD153)/CCAAT-enhancer binding protein (C/EBP) homologous protein (CHOP) as one of the HRG-inducible genes. We demonstrated that HRG stimulation of mammary epithelial cells induces the expression of GADD153 mRNA and protein and transcription of GADD153 promoter. The transcriptional activity of the GADD153 promoter as well as transcription from the C/EBP-activating transcription factor (ATF) composite motif in the GADD153 promoter was also stimulated by HRG-inducible ATF-4 and activated HER2 but not wild-type HER2. GADD153 expression was upregulated by the lactogenic hormones insulin and progesterone and associated with differentiation of normal mammary epithelial cells. Consistent with its role as transcriptional modifier, GADD153 stimulated transcription of beta-casein promoter activity in a STAT5a-sensitive manner in mammary epithelial cells. Analysis of mouse mammary gland development revealed that GADD153 expression was predominantly restricted in the early lactating stages. Because cyclic AMP responsive element and ATF binding sites are present in a variety of growth-regulating cellular genes, these findings suggest that stimulation of GADD153 expression and its transactivating functions may constitute an important mechanism of regulation of putative genes having diverse functions during cell growth and differentiation.

The findings of this study represent the first report, to the authors' knowledge, of CCAAT/enhancer binding protein (C/EBP) cDNA sequence in a fish species. C/EBP epsilon of Japanese flounder was 1861 bp in length (ORF of 822 bp) encoding for 274 amino acids, with a calculated molecular weight of 30 kDa. Japanese flounder C/EBP beta was found to be 1561 bp in length (ORF of 1041 bp), encoding for 347 amino acids and a calculated molecular weight of 39 kDa. These genes were expressed in various fish organs, tissues and secretions. C/EBP epsilon was detected by Northern blot from total RNA of head and posterior kidney, heart and spleen. However, RT-PCR also detected C/EBP epsilon in brain, spleen and peritoneal cavity fluid and peripheral blood leucocyte cDNA. C/EBP beta was detected by Northern blot analysis in the head and posterior kidney, spleen, intestine, liver, brain, heart, gill and testis and further found by RT-PCR to be detected in mucus, peritoneal cavity fluid, peripheral blood leucocytes and eye cDNA. Phylogenetic analysis placed the Japanese flounder C/EBP beta within the same cluster as previously reported C/EBP beta sequences. However, Japanese flounder C/EBP epsilon sequence data were not found to cluster with the three reported mammalian C/EBP epsilon sequences currently available. Understanding C/EBP transcriptional gene control in commercially important fish species may lead to a better control of disease.