A TEAD1/p65 complex regulates the eutherian-conserved MnSOD intronic enhancer, eRNA transcription and the innate immune response.

Manganese superoxide dismutase (MnSOD), a critical anti-oxidant enzyme, detoxifies the mitochondrial-derived reactive oxygen species, superoxide, elicited through normal respiration or the inflammatory response. Proinflammatory stimuli induce MnSOD gene expression through a eutherian-conserved, intronic enhancer element. We identified two prototypic enhancer binding proteins, TEAD1 and p65, that when co-expressed induce MnSOD expression comparable to pro-inflammatory stimuli. TEAD1 causes the nuclear sequestration of p65 leading to a novel TEAD1/p65 complex that associates with the intronic enhancer and is necessary for cytokine induction of MnSOD. Unlike typical NF-κB-responsive genes, the induction of MnSOD does not involve p50. Beyond MnSOD, the TEAD1/p65 complex regulates a subset of genes controlling the innate immune response that were previously viewed as solely NF-κB-dependent. We also identified an enhancer-derived RNA (eRNA) that is induced by either proinflammatory stimuli or the TEAD1/p65 complex, potentially linking the intronic enhancer to intra- and interchromosomal gene regulation through the inducible eRNA.

KCNC3(R420H), a K(+) channel mutation causative in spinocerebellar ataxia 13 displays aberrant intracellular trafficking.

Spinocerebellar ataxia 13 (SCA13) is an autosomal dominant disease resulting from mutations in KCNC3 (Kv3.3), a voltage-gated potassium channel. The KCNC3(R420H) mutation was first identified as causative for SCA13 in a four-generation Filipino kindred with over 20 affected individuals. Electrophysiological analyses in oocytes previously showed that this mutation did not lead to a functional channel and displayed a dominant negative phenotype. In an effort to identify the molecular basis of this allelic form of SCA13, we first determined that human KCNC3(WT) and KCNC3(R420H) display disparate post-translational modifications, and the mutant protein has reduced complex glycan adducts. Immunohistochemical analyses demonstrated that KCNC3(R420H) was not properly trafficking to the plasma membrane and surface biotinylation demonstrated that KCNC3(R420H) exhibited only 24% as much surface expression as KCNC3(WT). KCNC3(R420H) trafficked through the ER but was retained in the Golgi. KCNC3(R420H) expression results in altered Golgi and cellular morphology. Electron microscopy of KCNC3(R420H) localization further supports retention in the Golgi. These results are specific to the KCNC3(R420H) allele and provide new insight into the molecular basis of disease manifestation in SCA13.

A distal enhancer controls cytokine-dependent human cPLA2α gene expression.

Specific control of group IVA cytosolic phospholipase A2 (cPLA2α or PLA2G4A) expression modulates arachidonic acid production, thus tightly regulating the downstream effects of pro- and anti-inflammatory eicosanoids. The significance of this pathway in human disease is apparent in a range of pathologies from inflammation to tumorigenesis. While much of the regulation of cPLA2α has focused on posttranslational phosphorylation of the protein, studies on transcriptional regulation of this gene have focused only on proximal promoter regions. We have identified a DNase I hypersensitive site encompassing a 5' distal enhancer element containing a highly conserved consensus AP-1 site involved in transcriptional activation of cPLA2α by interleukin (IL)-1ß. Chromatin immunoprecipitation (ChIP), knockdown, knockout, and overexpression analyses have shown that c-Jun acts both in a negative and positive regulatory role. Transcriptional activation of cPLA2α occurs through the phosphorylation of c-Jun in conjunction with increased association of C/EBPß with the distal novel enhancer. The association of C/EBPß with the transcriptional activation complex does not require an obvious DNA binding site. These data provide new and important contributions to the understanding of cPLA2α regulation at the transcriptional level, with implications for eicosanoid metabolism, cellular signaling, and disease pathogenesis.

Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP.

Isoprenoid lipid carriers are essential in protein glycosylation and bacterial cell envelope biosynthesis. The enzymes involved in their metabolism (synthases, kinases and phosphatases) are therefore critical to cell viability. In this review, we focus on two broad groups of isoprenoid pyrophosphate phosphatases. One group, containing phosphatidic acid phosphatase motifs, includes the eukaryotic dolichyl pyrophosphate phosphatases and proposed recycling bacterial undecaprenol pyrophosphate phosphatases, PgpB, YbjB and YeiU/LpxT. The second group comprises the bacterial undecaprenol pyrophosphate phosphatase, BacA/UppP, responsible for initial formation of undecaprenyl phosphate, which we predict contains a tyrosine phosphate phosphatase motif resembling that of the tumour suppressor, phosphatase and tensin homologue (PTEN). Based on protein sequence alignments across species and 2D structure predictions, we propose catalytic and lipid recognition motifs unique to BacA/UppP enzymes. The verification of our proposed active-site residues would provide new strategies for the development of substrate-specific inhibitors which mimic both the lipid and pyrophosphate moieties, leading to the development of novel antimicrobial agents.

Regulation of human microsomal prostaglandin E synthase-1 by IL-1ß requires a distal enhancer element with a unique role for C/EBPß.

The studies of PGE2 (prostaglandin E2) biosynthesis have focused primarily on the role of cyclo-oxygenases. Efforts have shifted towards the specific PGE2 terminal synthases, particularly mPGES-1 (microsomal PGE synthase 1), which has emerged as the crucial inducible synthase with roles in pain, cancer and inflammation. mPGES-1 is induced by pro-inflammatory cytokines with studies focusing on the proximal promoter, mediated specifically through Egr-1 (early growth-response factor 1). Numerous studies demonstrate that the mPGES-1 promoter (PTGES) alone cannot account for the level of IL-1ß (interleukin 1ß) induction. We identified two DNase I-hypersensitive sites within the proximal promoter near the Egr-1 element and a novel distal site near -8.6 kb. Functional analysis of the distal site revealed two elements that co-operate with basal promoter expression and a stimulus-dependent enhancer. A specific binding site for C/EBPß (CCAAT/enhancer-binding protein ß) in the enhancer was directly responsible for inducible enhancer activity. ChIP (chromatin immunoprecipitation) analysis demonstrated constitutive Egr-1 binding to the promoter and induced RNA polymerase II and C/EBPß binding to the promoter and enhancer respectively. Knockout/knockdown studies established a functional role for C/EBPß in mPGES-1 gene regulation and the documented interaction between Egr-1 and C/EBPß highlights the proximal promoter co-operation with a novel distal enhancer element in regulating inducible mPGES-1 expression.

Induction of group IVC phospholipase A2 in allergic asthma: transcriptional regulation by TNFα in bronchoepithelial cells.

Airway inflammation in allergen-induced asthma is associated with eicosanoid release. These bioactive lipids exhibit anti- and pro-inflammatory activities with relevance to pulmonary pathophysiology. We hypothesized that sensitization/challenge using an extract from the ubiquitous fungus Aspergillus fumigatus in a mouse model of allergic asthma would result in altered phospholipase gene expression, thus modulating the downstream eicosanoid pathway. We observed the most significant induction in the group IVC PLA2 (phospholipase A2) [also known as cPLA2γ (cytosolic PLA2γ) or PLA2G4C]. Our results infer that A. fumigatus extract can induce cPLA2γ levels directly in eosinophils, whereas induction in lung epithelial cells is most likely to be a consequence of TNFα (tumour necrosis factor α) secretion by A. fumigatus-activated macrophages. The mechanism of TNFα-dependent induction of cPLA2γ gene expression was elucidated through a combination of promoter deletions, ChIP (chromatin immunoprecipitation) and overexpression studies in human bronchoepithelial cells, leading to the identification of functionally relevant CRE (cAMP-response element), NF-κB (nuclear factor κB) and E-box promoter elements. ChIP analysis demonstrated that RNA polymerase II, ATF-2 (activating transcription factor 2)-c-Jun, p65-p65 and USF (upstream stimulating factor) 1-USF2 complexes are recruited to the cPLA2γ enhancer/promoter in response to TNFα, with overexpression and dominant-negative studies implying a strong level of co-operation and interplay between these factors. Overall, our results link cytokine-mediated alterations in cPLA2γ gene expression with allergic asthma and outline a complex regulatory mechanism.

Endothelin-1-mediated vasoconstriction alters cerebral gene expression in iron homeostasis and eicosanoid metabolism.

Endothelins are potent vasoconstrictors and signaling molecules. Their effects are broad, impacting processes ranging from neurovascular and cardiovascular health to cell migration and survival. In stroke, traumatic brain injury or subarachnoid hemorrhage, endothelin-1 (ET-1) is induced resulting in cerebral vasospasm, ischemia, reperfusion and the activation of various pathways. Given the central role that ET-1 plays in these patients and to identify the downstream molecular events specific to transient vasoconstriction, we studied the consequences of ET-1-mediated vasoconstriction of the middle cerebral artery in a rat model. Our observations demonstrate that ET-1 can lead to increases in gene expression, including genes associated with the inflammatory response (Ifnb, Il6, Tnf) and oxidative stress (Hif1a, Myc, Sod2). We also observed inductions (>2 fold) of genes involved in eicosanoid biosynthesis (Pla2g4a, Pla2g4b, Ptgs2, Ptgis, Alox12, Alox15), heme metabolism (Hpx, Hmox1, Prdx1) and iron homeostasis (Hamp, Tf). Our findings demonstrate that mRNA levels for the hormone hepcidin (Hamp) are induced in the brain in response to ET-1, providing a novel target in the treatment of multiple conditions. These changes on the ipsilateral side were also accompanied by corresponding changes in a subset of genes in the contralateral hemisphere. Understanding ET-1-mediated events at the molecular level may lead to better treatments for neurological diseases and provide significant impact on neurological function, morbidity and mortality.

Characterization of a mechanism to inhibit ovarian follicle activation.

OBJECTIVE: To demonstrate that a small molecule can induce the transcription factor Foxo3 in the ovary and lead to inhibition of follicle activation. DESIGN: Cell culture, organ culture, and animal studies. SETTING: University-based laboratory. ANIMAL(S): 23 female C57BL/6 mice. INTERVENTION(S): Human ovary cells and mouse ovaries in culture treated with 2-deoxyglucose (2-DG) to mimic glucose deprivation, and mice intraperitoneally injected with 100 mg/kg, 300 mg/kg, or 600 mg/kg 2-DG daily for 2 weeks. MAIN OUTCOME MEASURE(S): In cell and organ culture, Foxo3 expression analyzed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR); in treated animals, expression of genes regulated by nutrient deprivation (Foxo3, ATF4, GRP78, CHOP, ASNS, c-Myc) measured in brain, kidney, and ovary by qRT-PCR; and ovarian follicles histologically classified and counted. RESULT(S): Foxo3 expression is induced by 2-DG at both the mRNA and protein level in human ovarian cell culture, possibly through ATF4-dependent gene regulation. Foxo3 expression is also induced by 2-DG in ovarian organ culture. Treatment of mice with 100 mg/kg 2-DG resulted in a 2.6 fold induction of Foxo3 in the ovary and a 58% decrease in type 3a primary follicles. CONCLUSION(S): Expression of Foxo3 is induced by nutrient deprivation in cell culture, organ culture, and in vivo. In mice, 2-DG treatment results in an inhibition of primordial follicle activation. These data indicate that Foxo3 induction by 2-DG may be useful for fertility preservation.

Effect of allergy and inflammation on eicosanoid gene expression in CFTR deficiency.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is a complicating factor in cystic fibrosis (CF), affecting 2-15% of patients. We hypothesized that sensitization/challenge of CFTR(-/-) mice with an Aspergillus fumigatus (Af) extract will affect eicosanoid pathway gene expression, impacting ABPA and CF. METHODS: FABP-hCFTR(+/-)-CFTR(-/-) mice were sensitized/challenged with an Af extract and gene expression of lung mRNA was evaluated for >40 genes, with correlative data in human CF (IB3.1) and CFTR-corrected (S9) bronchoepithelial cell lines. RESULTS: Pla2g4c, Pla2g2c, Pla2g2d and Pla2g5 were induced in response to Af in CFTR(-/-) mice. Interestingly, PLA2G2D was induced by LPS, IL-2, IL-6, IL-13, and Af only in CFTR-deficient human IB3.1 cells. Prostanoid gene expression was relatively constant, however, several 12/15-lipoxygenase genes were induced in response to Af. Numerous cytokines also caused differential expression of ALOX15 only in IB3.1 cells. CONCLUSIONS: The distinct regulation of PLA2G4C, PLA2G2D and ALOX15 genes in Aspergillus sensitization and/or cystic fibrosis could provide new insights into diagnosis and treatment of ABPA and CF.

cPLA(2)α gene activation by IL-1ß is dependent on an upstream kinase pathway, enzymatic activation and downstream 15-lipoxygenase activity: a positive feedback loop.

Cytosolic phospholipase A(2)α (cPLA(2)α) is the most widely studied member of the Group IV PLA(2) family. The enzyme is Ca(2+)-dependent with specificity for phospholipid substrates containing arachidonic acid. As the pinnacle of the arachidonic acid pathway, cPLA(2)α has a primary role in the biosynthesis of a diverse family of eicosanoid metabolites, with potent physiological, inflammatory and pathological consequences. cPLA(2)α activity is regulated by pro-inflammatory stimuli through pathways involving increased intracellular Ca(2+) levels, phosphorylation coupled to increased enzymatic activity and de novo gene transcription. This study addresses the signal transduction pathways for protein phosphorylation and gene induction following IL-1ß stimulation in human fetal lung fibroblasts. Our results utilizing both inhibitors and kinase-deficient cells demonstrate that cPLA(2)α is phosphorylated within 10min of IL-1ß treatment, an event requiring p38 MAPK as well as the upstream kinase, MKK3/MKK6. Inhibition of p38 MAPK also blocks the phosphorylation of a downstream, nuclear kinase, MSK-1. Our results further demonstrate that the activities of both cPLA(2)α and a downstream lipoxygenase (15-LOX2) are required for IL-1ß-dependent induction of cPLA(2)α mRNA expression. Overall, these data support an MKK3/MKK6→p38 MAPK→MSK-1→cPLA(2)α→15-LOX2-dependent, positive feedback loop where a protein's enzymatic activity is required to regulate its own gene induction by a pro-inflammatory stimulus.

Metabolic regulation of manganese superoxide dismutase expression via essential amino acid deprivation.

Organisms respond to available nutrient levels by rapidly adjusting metabolic flux, in part through changes in gene expression. A consequence of adaptations in metabolic rate is the production of mitochondria-derived reactive oxygen species. Therefore, we hypothesized that nutrient sensing could regulate the synthesis of the primary defense of the cell against superoxide radicals, manganese superoxide dismutase. Our data establish a novel nutrient-sensing pathway for manganese superoxide dismutase expression mediated through essential amino acid depletion concurrent with an increase in cellular viability. Most relevantly, our results are divergent from current mechanisms governing amino acid-dependent gene regulation. This pathway requires the presence of glutamine, signaling via the tricarboxylic acid cycle/electron transport chain, an intact mitochondrial membrane potential, and the activity of both the MEK/ERK and mammalian target of rapamycin kinases. Our results provide evidence for convergence of metabolic cues with nutrient control of antioxidant gene regulation, revealing a potential signaling strategy that impacts free radical-mediated mutations with implications in cancer and aging.