Low Basal Expression Research Articles

Synaptic activity-responsive element in the Arc / Arg3.1 promoter essential for synapse-to-nucleus signaling in activated neurons

The neuronal immediate early gene Arc/Arg-3.1 is widely used as one of the most reliable molecular markers for intense synaptic activity in vivo. However, the cis-acting elements responsible for such stringent activity dependence have not been firmly identified. Here we combined luciferase reporter assays in cultured cortical neurons and comparative genome mapping to identify the critical synaptic activity-responsive elements (SARE) of the Arc/Arg-3.1 gene. A major SARE was found as a unique approximately 100-bp element located at >5 kb upstream of the Arc/Arg-3.1 transcription initiation site in the mouse genome. This single element, when positioned immediately upstream of a minimal promoter, was necessary and sufficient to replicate crucial properties of endogenous Arc/Arg-3.1's transcriptional regulation, including rapid onset of transcription triggered by synaptic activity and low basal expression during synaptic inactivity. We identified the major determinants of SARE as a unique cluster of neuronal activity-dependent cis-regulatory elements consisting of closely localized binding sites for CREB, MEF2, and SRF. Consistently, a SARE reporter could readily trace and mark an ensemble of cells that have experienced intense activity in the recent past in vivo. Taken together, our work uncovers a novel transcriptional mechanism by which a critical 100-bp element, SARE, mediates a predominant component of the synapse-to-nucleus signaling in ensembles of Arc/Arg-3.1-positive activated neurons.

Combination of hyperthermia and bortezomib results in additive killing in mantle cell lymphoma cells

The proteasome inhibitor bortezomib exhibits antitumor activity in many malignancies including mantle cell lymphoma (MCL). Unfortunately, many patients fail to respond to treatment or become refractory. Hyperthermia is an effective chemosensitizer that in combination with some chemotherapeutic agents has shown clinical activity in phase II and III studies. The aim of this study was to use MCL cell lines to investigate the potential benefit of combining clinically relevant doses of bortezomib with two different thermal doses (41.8°C/120 min and 44°C/30 min) that mimic the heterogeneity of the temperature distributions achieved within tumors during hyperthermia. Treated tumor cells were assessed for proliferation using the WST-1 assay and for apoptosis by annexin V staining, while heat shock protein (HSP) levels were determined following western blot analysis. Our results demonstrated that MCL cell lines that are sensitive to bortezomib are also thermosensitive and have low basal expression of hsp27, whereas the bortezomib-resistant MCL cell line strongly expresses hsp27 and is thermoresistant. Interestingly, pre-treatment of MCL cell lines with heat at the two different thermal doses, and the transient elevation of hsp27 and hsp70, do not impair their primary sensitivity to bortezomib. Finally, we show that the concurrent treatment of heat and bortezomib results in additive killing in MCL cell lines.In conclusion, these results suggest that the application of bortezomib, under thermal conditions, in mantle cell lymphoma cells may be beneficial and warrants further investigation.

Defining the chromatin signature of inducible genes in T cells

Inducible genes in T cells show the chromatin characteristics of active genes, suggesting they are primed for transcription.

The presence and activity of SP-D in porcine coronary endothelial cells depend on Akt/PI3K, Erk and nitric oxide and decrease after multiple passaging

Surfactant protein D (SP-D) mediates clearance of microorganisms and modulates inflammation in response to cytotoxic stimulation. It is present in various epithelia, but also in vascular smooth muscle and endothelial cells. Experiments were designed to determine whether or not SP-D is present in porcine coronary arterial endothelial cells and if so, to investigate the molecular mechanisms underlying this presence. The expression of SP-D, NO synthase, Akt 1/2 and Erk 1/2 proteins was determined in cultures at passages 1 (#1) and 4 (#4). SP-D in primary cells existed in three isoforms (37–38kDa and 50kDa). The 37–38kDa SP-D forms were the dominant isoforms in the porcine endothelium and were prominent at #1 but partially lost at #4. Tumor necrosis factor-α (TNF-α) significantly augmented the level of SP-D expression at #1 but not at #4. The basal level of 37–38kDa SP-D isoforms at #1 was reduced by L-NAME, wortmannin and PD 98059. The low basal expression at #4 could be increased by DETA NONOate (donor of NO) or insulin (activator of PI3K/Akt). The presence of nitric oxide synthase was reduced while that of Akt 1/2 and Erk 1/2 was increased at #4. In cells both at passages 1 and 4, TNF-α downregulated NO synthase and up-regulated p-Erk 1/2 protein. The present findings demonstrate the presence of SP-D in endothelial cells which is NO-, PI3K/Akt- and Erk-dependent. They suggest a protective role of SP-D in these cells.

Peripheral formalin injection induces unique spinal cord microglial phenotypic changes

Microglia are resident immune cells of brain and activated by peripheral tissue injury. In the present study, we investigated the possible induction of several microglial surface immunomolecules in the spinal cord, including leukocyte common antigen (LCA/CD45), MHC class I antigen, MHC class II antigen, Fc receptor, and CD11c following formalin injection into the rat's hind paw. CD45 and MHC class I were upregulated in the activated microglia, which was evident on day 3 with the peak expression on day 7 following peripheral formalin injection. There was a very low basal expression of MHC class II, CD11c, and the Fc receptor, which did not change after the formalin injection. These results, for the first time, indicate that peripheral formalin injection can induce phenotypic changes of microglia with distinct upregulation of CD45 and MHC class I antigen. The data suggest that phenotypic changes of the activated microglia may be a unique pattern of central changes following peripheral tissue injury.

Architectural changes to CA1 pyramidal neurons in adult and aged mice after peripheral immune stimulation

The expression of several inflammatory cytokines that inhibit synaptic plasticity and hippocampal-dependent learning and memory is higher in the brains of aged mice compared to young adults after peripheral injection of lipopolysaccharide (LPS). In this study we investigated whether the exaggerated inflammatory cytokine response in the hippocampus of aged mice after IP injection of LPS is associated with architectural changes to dendrites of pyramidal neurons in the dorsal CA1 hippocampus. Compared to young adults, aged mice had higher basal expression of MHC class II, lower basal expression of two neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and a decrease in total dendritic length in both the basal and apical tree. After IP LPS administration, expression of IL-1beta, IL-6, and TNFalpha mRNA was higher in hippocampus of aged mice compared to young adults whereas NGF and BDNF mRNA was reduced similarly in both age groups. The basal dendritic tree was not affected by LPS in either adult or aged mice 72h after treatment; however, length and branching of the apical tree was reduced by LPS in aged but not adult mice. The present findings indicate that a peripheral infection in the aged can cause a heightened inflammatory cytokine response in the hippocampus and atrophy of hippocampal neurons. Architectural changes to dorsal CA1 hippocampal neurons may contribute to cognitive disorders evident in elderly patients with an infection.

Subcutaneous Abdominal Adipose Tissue Subcompartments: Potential Role in Rosiglitazone Effects

Abdominal visceral tissue (VAT) and subcutaneous adipose tissue (SAT), comprised of superficial-SAT (sSAT) and deep-SAT (dSAT), are metabolically distinct. The antidiabetic agents thiazolidinediones (TZDs), in addition to their insulin-sensitizing effects, redistribute SAT suggesting that TZD action involves adipose tissue depot-specific regulation. We investigated the expression of proteins key to adipocyte metabolism on differentiated first passage (P1) preadipocytes treated with rosiglitazone, to establish a role for the diverse depots of abdominal adipose tissue in the insulin-sensitizing effects of TZDs. Adipocytes and preadipocytes were isolated from sSAT, dSAT, and VAT samples obtained from eight normal subjects. Preadipocytes (P1) left untreated (U) or treated with a classic differentiation cocktail (DI) including rosiglitazone (DIR) for 9 days were evaluated for strata-specific differences in differentiation including peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and lipoprotein lipase (LPL) expression, insulin sensitivity via adiponectin and glucose transport-4 (GLUT4), glucocorticoid metabolism with 11 beta-hydroxysteroid dehydrogenase type-1 (11 beta HSD1), and alterations in the adipokine leptin. While depot-specific differences were absent with the classic differentiation cocktail, with rosiglitazone sSAT had the most potent response followed by dSAT, whereas VAT was resistant to differentiation. With rosiglitazone, universal strata effects were observed for PPAR-gamma, LPL, and leptin, with VAT in all cases expressing significantly lower basal expression levels. Clear dSAT-specific changes were observed with decreased intracellular GLUT4. Specific sSAT alterations included decreased 11 beta HSD1 whereas secreted adiponectin was potently upregulated in sSAT with respect to dSAT and VAT. Overall, the subcompartments of SAT, sSAT, and dSAT, appear to participate in the metabolic changes that arise with rosiglitazone administration.

Selenoprotein P expression is controlled through interaction of the coactivator PGC-1α with FoxO1a and hepatocyte nuclear factor 4α transcription factors

Selenoprotein P (SeP), the major selenoprotein in plasma, is produced mainly by the liver, although SeP expression is detected in many organs. Recently, we reported stimulation of SeP promoter activity by the forkhead box transcription factor FoxO1a in hepatoma cells and its attenuation by insulin. Here, we demonstrate that this translates into fine-tuning of SeP production and secretion by insulin. Overexpression of peroxisomal proliferator activated receptor-gamma coactivator 1alpha (PGC-1alpha) enhanced the stimulatory effect of FoxO1a on SeP promoter activity. We identified a novel functional binding site for hepatocyte nuclear factor (HNF)-4alpha, termed hepatocyte nuclear factor binding element 1, in the human SeP promoter directly upstream of the FoxO-responsive element daf16-binding element 2 (DBE2). Point mutations in hepatocyte nuclear factor binding element 1 alone or together with DBE2 decreased basal activity and responsiveness of the SeP promoter to PGC-1alpha. Moreover, the PGC-1alpha-inducing glucocorticoid dexamethasone strongly enhanced SeP messenger RNA levels and protein secretion in cultured rat hepatocytes, whereas insulin suppressed the stimulation of both PGC-1alpha and SeP caused by dexamethasone treatment. In a brain-derived neuroblastoma cell line with low basal SeP expression, SeP transcription was stimulated by PGC-1alpha together with FoxO1a, and overexpression of HNF-4alpha potentiated this effect. High-level expression of SeP in liver is ensured by concerted action of the coactivator PGC-1alpha and the transcription factors FoxO1a and HNF-4alpha. Hence, the production of SeP is regulated similarly to that of the gluconeogenic enzyme glucose-6-phosphatase. As hepatic SeP production is crucial for selenium distribution throughout the body, the present study establishes PGC-1alpha as a key regulator of selenium homeostasis.

Evidence for an association of prion protein and sphingolipid‐mediated signaling

The physiological function of the cellular prion protein (PrP(c)) is unclear. PrP(c) associates with lipid rafts, highly glycolipid-rich membrane domains containing a large variety of signaling molecules, e.g., sphingolipids (SL). In this study, we investigated possible connections between PrP(c) and sphingolipid-associated signaling pathways. Using PrP(c)-wt and PrP(c)-k.o. hippocampal cell lines and mouse brains we showed higher activity of neutral and acid sphingomyelinase (SMase) in PrP(c)-k.o.-groups, while ceramide and sphingomyelin-levels were unchanged. Furthermore, despite lower basal expression levels of sphingosine kinase (SphK) in PrP(c)-k.o.-groups, the levels of its metabolite sphingosine-1-phosphate were increased, whereas S1P(3)-receptor expression was higher in PrP(c)-wt-groups again. In addition, we detected enhanced activity of phospholipase D1, an enzyme that seems to be suitable to act as a connector between the S1P(3) receptor and continuative signaling. Finally, evidence for an impact on downstream signaling cascades, especially activation of the PI3K/Akt pathway, was found. In summary, our data suggest that PrP(c) is involved in sphingolipid-associated signaling, modulating pathways that exert anti-apoptotic functions, hence indicating that PrP(c) plays a role in neuroprotection.

Why has sleepwalking research been “sleepwalking”?

Interactions between pattern-recognition receptors shape innate immune responses to pathogens. NOD1 and TLR4 are synergistically interacting receptors playing a pivotal role in the recognition of Gram-negative bacteria. However, mechanisms of their cooperation are poorly understood. It is unclear whether synergy is produced at the level of signaling pathways downstream of NOD1 and TLR4 or at more distal levels such as gene transcription. We analyzed sequential stages of human macrophage activation by a combination of NOD1 and TLR4 agonists (<i>N</i>-acetyl-d-muramyl-l-alanyl-d-isoglutamyl-meso-diaminopimelic acid [M-triDAP] and LPS, respectively). We show that events preceding or not requiring activation of transcription, such as activation of signaling kinases, rapid boost of glycolysis, and most importantly, nuclear translocation of NF-κB, are regulated nonsynergistically. However, at the output of the nucleus, the combination of M-triDAP and LPS synergistically induces expression of a subset of M-triDAP– and LPS-inducible genes, particularly those encoding proinflammatory cytokines (<i>TNF</i>, <i>IL1B</i>, <i>IL6</i>, <i>IL12B</i>, and <i>IL23A</i>). This synergistic response develops between 1 and 4 h of agonist treatment and requires continuous signaling through NOD1. The synergistically regulated genes have a lower basal expression and higher inducibility at 4 h than those regulated nonsynergistically. Both gene subsets include NF-κB–inducible genes. Therefore, activation of the NF-κB pathway does not explain synergistic gene induction, implying involvement of other transcription factors. Inhibition of IKKβ or p38 MAPK lowers agonist-induced <i>TNF</i> mRNA expression but does not abolish synergy. Thus, nonsynergistic activation of NOD1- and TLR4-dependent signaling pathways results in the synergistic induction of a proinflammatory transcriptional program.

An Inducible and Reversible Mouse Genetic Rescue System

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications.

Epigenetic and HIF-1 regulation of stanniocalcin-2 expression in human cancer cells

Mammalian stanniocalcin-2 (STC2) is a secreted glycoprotein hormone with a putative role in unfolded protein response and apoptosis. Here we reported that STC2 expression was sporadically abrogated in human cancer cells by transcriptional silencing associated with CpG island promoter hypermethylation. Direct sequencing of bisulfite-modified DNA from a panel of seven human cancer cell lines revealed that CpG dinucleotides in STC2 promoter was methylated in human ovarian epithelial cancer (SKOV3, OVCAR3 and CaOV3), pancreatic cancer (BxP3), colon adenoma (HT29), and leukemia (Jurkat cells). STC2 CpG island hypermethylation was accompanied with a low basal STC2 expression level. Treatment of these cancer cells with 5-aza-2′-deoxycytidine (5-aza-CdR), an inhibitor of DNA methylation significantly induced STC2 expression. Using SKOV3 cells as a model, the link between DNA demethylation and STC2 expression was consistently demonstrated with hydralazine treatment, which was shown to reduce the protein level of DNA methyltransferase 1 (DNMT1) but stimulated STC2 expression. Two human normal surface ovarian cell-lines (i.e. IOSE 29 and 398) showed no methylation at CpG dinucleotides in the examined promoter region and were accompanied with high basal STC2 levels. Hypoxia stimulated STC2 expression in SKOV3 cells was markedly increased in 5-aza-CdR pretreated cells, showing that DNA methylation may hinder the HIF-1 mediated activation. To elucidate this possibility, RNA interference studies confirmed that endogenous HIF-1α was a key factor for STC2 gene activation as well as in the synergistic induction of STC2 expression in 5-aza-CdR pretreated cells. Chromatin immunoprecipitation (ChIP) assay demonstrated the binding of HIF-1α to STC2 promoter. The binding was increased in 5-aza-CdR pretreated cells. Collectively, this is the first report to show that STC2 was aberrantly hypermethylated in human cancer cells. The findings demonstrated that STC2 epigenetic inactivation may interfere with HIF-1 mediated activation of STC2 expression.

Genetic determinants of basal C-reactive protein expression in Filipino systemic lupus erythematosus families

Basal C-reactive protein (CRP) is a heritable trait associated with long-term cardiovascular disease risk. Existing studies leave ambiguity over the key functional polymorphisms and fail to adjust for trans-acting effects. In a novel cohort of 285 Filipino systemic lupus erythematosus probands and their first-degree relatives, we quantified serum CRP and typed a dense map of CRP single-nucleotide polymorphisms (SNPs), along with SNPs in the interleukin-1 beta, interleukin-6 and apolipoprotein E genes. Ten CRP SNPs demonstrated association with basal CRP in a regression model (P=0.011-0.002). These delineated two haplotypes associated with high and low basal CRP expression (P=0.002). Differences in allele frequency were seen compared with Caucasian populations, enabling us to argue for an independent genetic effect from a phylogenetically distinct haplotype tagged by SNP rs1800947. We demonstrated an association between Apo epsilon 2 and higher basal CRP. Interleukin-6 genotype was associated with basal CRP, highlighting a role for acute-phase cytokines even in basal expression. Identifying these trans-acting variants may improve the use of basal CRP as a predictor cardiovascular risk, and increase our power to detect associations between CRP and disease.

The low density lipoprotein receptor is not necessary for maintaining mouse brain polyunsaturated fatty acid concentrations

The brain cannot synthesize n-6 or n-3 PUFAs de novo and requires their transport from the blood. Two models of brain fatty acid uptake have been proposed. One requires the passive diffusion of unesterified fatty acids through endothelial cells of the blood-brain barrier, and the other requires the uptake of lipoproteins via a lipoprotein receptor on the luminal membrane of endothelial cells. This study tested whether the low density lipoprotein receptor (LDLr) is necessary for maintaining brain PUFA concentrations. Because the cortex has a low basal expression of LDLr and the anterior brain stem has a relatively high expression, we analyzed these regions separately. LDLr knockout (LDLr(-/-)) and wild-type mice consumed an AIN-93G diet ad libitum until 7 weeks of age. After microwaving, the cortex and anterior brain stem (pons and medulla) were isolated for phospholipid fatty acid analyses. There were no differences in phosphatidylserine, phosphatidylinositol, ethanolamine, or choline glycerophospholipid esterified PUFA or saturated or monounsaturated fatty acid concentrations in the cortex or brain stem between LDLr(-/-) and wild-type mice. These findings demonstrate that the LDLr is not necessary for maintaining brain PUFA concentrations and suggest that other mechanisms to transport PUFAs into the brain must exist.

Exploration of the β2-adrenergic receptor regulatory regions: the next step in the holy grail of asthma pharmacogenetics research

exploration of the β2-adrenergic receptor (ADRB2) has been the holy grail of asthma pharmacogenetics research since the gene was cloned two decades ago. The evolution in our understanding of genetic variants in the ADRB2 reflects both the progress and the limitations in exploring the contributions

Immuno‐qPCR detection of the tandem affinity purification (TAP)‐tag as a sensitive and accurate tool suitable for large‐scale protein quantification

The tandem affinity purification (TAP)-tag has rapidly gained a wide popularity, mostly in studies on protein interactions, but lately also in large-scale protein quantification studies. We have developed an immuno-quantitative real-time PCR (qPCR) method to achieve rapid, sensitive and accurate quantification of TAP-tagged (and protein A-tagged) proteins in yeast with a detection range between 10(7) and 10(10) molecules. The immuno-qPCR protein quantification showed an excellent correlation to the published in vivo fluorescent protein (GFP)-based large-scale protein quantifications, but allowed for a much higher sensitivity. The correlation with published data from the large-scale Western blotting-based quantification of the TAP-tag was lower, but the sensitivity of detection was on roughly the same level. The practical use of the immuno-qPCR approach was demonstrated by analysis of osmo-regulated proteins, where the 2000-fold increase in expression of Catalase (Ctt1p), from an extremely low basal expression, could be accurately quantified. All steps of the method, from cell growth, to protein extraction and determination and the immuno-qPCR reaction itself are potentially amenable to automatization. Therefore, since the TAP-tag and protein A are useful in most model organisms, the immuno-qPCR method is both generic and suitable for large-scale studies.

Altered levels of STAT1 and STAT3 influence the neuronal response to interferon gamma

As immune responses in the CNS are highly regulated, cell-specific differences in IFNγ signaling may be integral in dictating the outcome of host cell responses. In comparing the response of IFNγ-treated primary neurons to control MEF, we observed that neurons demonstrated lower basal expression of both STAT1 and STAT3, the primary signal transducers responsible for IFNγ signaling. Following IFNγ treatment of these cell populations, we noted muted and delayed STAT1 phosphorylation, no detectable STAT3 phosphorylation, and a 3–10-fold lower level of representative IFNγ-responsive gene transcripts. Moreover, in response to a brief pulse of IFNγ, a steady increase in STAT1 phosphorylation and IFNγ gene expression over 48 h was observed in neurons, as compared to rapid attenuation in MEF. These distinct response kinetics in IFNγ-stimulated neurons may reflect modifications in the IFNγ negative feedback loop, which may provide a mechanism for the cell-specific heterogeneity of responses to IFNγ.

Characterisation of Nilotinib Transport in Chronic Myeloid Leukaemia Cells.

We have previously shown that imatinib is a substrate for both the efflux transporter P-glycoprotein (ABCB1; MDR-1 gene product) and the influx transporter human Organic Cation Transporter 1 (hOCT1; SLCA22). High hOCT1 and low ABCB1 expression levels in chronic myeloid leukaemia (CML) patients correlate with improved clinical outcome (Wang L. et al. Clinical Pharmacology and Therapeutics; epub June 13th 2007). The second generation tyrosine kinase inhibitor nilotinib is 30-fold more potent than imatinib and is effective in imatinib-resistant patients. However, its cellular transport has not been characterised. The CML cell line KCL22 was used as it has low basal hOCT1 expression. Drug transport studies of 14C-radiolabelled nilotinib (kind gift from Novartis) were performed using high expressing hOCT1 transfected KCL22 cells, with or without OCT1 and ABCB1 inhibitors. Further vectorial ABCB1 efflux studies were performed on confluent monolayers of ABCB1 transfected Type II Madin Darby canine kidney (MDCKII) cells. Nilotinib efflux was not modulated in the presence of the ABCB1 inhibitors PSC-833 (10μM), tariquidar (500nM) and verapamil (500μM), and no evidence of transport via ABCB1 was seen in the transfected ABCB1 MDCKII cells. Nilotinib influx was not modulated in hOCT1 over-expressing cells, or in the presence of OCT1 inhibitors prazosin (100μM) and amantadine (500μM). Nilotinib uptake was also not modulated by the hOCT1 substrate tetraethylamine (TEA) (5μM), however nilotinib inhibited TEA uptake. The distribution coefficient (logD) of nilotinib was 2.1, compared with 0.81 for imatinib, demonstrating high lipophilicity for nilotinib. Flow cytometry revealed greater suppression of phospho-CrKL (a surrogate marker for BCR-ABL blockade) with nilotinib after 1 hour when compared with imatinib, regardless of hOCT1 expression, indicating that nilotinib is effective at BCR-ABL suppression even at low hOCT1 levels, in contrast to imatinib. Nilotinib was not a substrate for the ABCB1 and hOCT1 transporters; however it may be a potential inhibitor of hOCT1. Therefore the level of expression of ABCB1 and hOCT1 in CML is unlikely to be an indicator of the clinical outcome for patients receiving nilotinib, in contrast to what we have shown for imatinib. Nilotinib may therefore be effective in patients who have low hOCT1 expression before start of therapy.

Alterations in immune response and PPAR/LXR regulation in cystic fibrosis macrophages

Background Cystic fibrosis (CF) is characterized by an excessive inflammatory response in epithelial cells and macrophages. In CF mice, lung inflammation can be abrogated by oral treatment with docosahexaenoic acid (DHA). Since PPARs and LXRs are important regulators of inflammation and fatty acid metabolism in macrophages, we hypothesized that these pathways are dysregulated in CF macrophages and are corrected with DHA treatment. Methods Peritoneal macrophages were obtained from wild type and cftr −/− mice. LPS induced cytokine secretion and NFκB activity were analyzed with and without oral DHA treatment. The expression and activity of PPARα,γ, δ and LXRα were analyzed by RT-PCR and EMSA. Results LPS induced TNFα and IL-6 secretion and NFκB p65 activity were increased in CF macrophages. This was associated with low basal PPARγ expression and attenuated LPS induced induction of PPARδ, LXRα and ABCA1. DHA pretreatment in vivo decreased TNFα secretion and p65 activity, and increased PPARα and γ expression and function. The effects of DHA could be reproduced by PPAR agonists and blocked by a PPARα antagonist. Conclusion Impaired regulation of nuclear receptors may contribute to the abnormal LPS induced signaling in CF macrophages and is reversed by DHA.

Reduction of GRP78 expression with siRNA activates unfolded protein response leading to apoptosis in HeLa cells

The 78-kDa glucose-regulated protein GRP78 is a central regulator of endoplasmic reticulum (ER) homeostasis, functioning in protein folding, ER calcium binding and modulation of transmembrane ER stress sensor activity. ER stress uncouples the interaction between GRP78 and ER stress sensors, leading to activation of the unfolded protein response (UPR), including upregulation of ER chaperone proteins. In the present study, we observed unexpected and remarkable induction of glucose-regulated protein 94 (GRP94) in HeLa cells following their transfection with 2′-O-methyl-modified siRNA specific to GRP78 mRNA. Additionally, we found that this siRNA also increased the expression of other UPR-induced genes, such as CHOP, ERdj4 and P5. Activation of UPR-dependent transcription and induction of apoptosis were also observed in cells transfected with GRP78 siRNA. Induction of apoptosis by GRP78 siRNA was also observed in PC-3 cells, which expressed high basal levels of GRP78 protein similar to that observed in HeLa cells. By contrast, five other human cell lines with lower basal expression of GRP78 protein did not undergo apoptosis when treated with GRP78 siRNA. Possible reasons for the strong activation of the UPR and apoptosis induced by GRP78 knockdown in HeLa cells, and the therapeutic utility of 2′-O-methyl-modified GRP78 siRNAs in anticancer treatment, are discussed.