Regulation of CDKN2B expression by interaction of Arnt with Miz-1--a basis for functional integration between the HIF and Myc gene regulatory pathways.

BACKGROUND: Hypoxia- and Myc-dependent transcriptional regulatory pathways are frequently deregulated in cancer cells. These pathways converge in many cellular responses, but the underlying molecular mechanisms are unclear. METHODS: The ability of Miz-1 and Arnt to interact was identified in a yeast two-hybrid screen. The mode of interaction and the functional consequences of complex formation were analyzed by diverse molecular biology methods, in vitro. Statistical analyses were performed by Student's t-test and ANOVA. RESULTS: In the present study we demonstrate that the aryl hydrocarbon receptor nuclear translocator (Arnt), which is central in hypoxia-induced signaling, forms a complex with Miz-1, an important transcriptional regulator in Myc-mediated transcriptional repression. Overexpression of Arnt induced reporter gene activity driven by the proximal promoter of the cyclin-dependent kinase inhibitor 2B gene (CDKN2B), which is an established target for the Myc/Miz-1 complex. In contrast, mutated forms of Arnt, that were unable to interact with Miz-1, had reduced capability to activate transcription. Moreover, repression of Arnt reduced endogenous CDKN2B expression, and chromatin immunoprecipitation demonstrated that Arnt interacts with the CDKN2B promoter. The transcriptional activity of Arnt was counteracted by Myc, but not by a mutated variant of Myc that is unable to interact with Miz-1, suggesting mutually exclusive interaction of Arnt and Myc with Miz-1. Our results also establish CDKN2B as a hypoxia regulated gene, as endogenous CDKN2B mRNA and protein levels were reduced by hypoxic treatment of U2OS cells. CONCLUSIONS: Our data reveal a novel mode of regulation by protein-protein interaction that directly ties together, at the transcriptional level, the Myc- and hypoxia-dependent signaling pathways and expands our understanding of the roles of hypoxia and cell cycle alterations during tumorigenesis.

Candidate genes for monitoring hydrogen peroxide resistance in the salmon louse, Lepeophtheirus salmonis.

BACKGROUND: Hydrogen peroxide (H2O2) is one of the delousing agents used to control sea lice infestations in salmonid aquaculture. However, some Lepeophtheirus salmonis populations have developed resistance towards H2O2. An increased gene expression and activity of catalase, an enzyme that breaks down H2O2, have been detected in resistant lice, being therefore introduced as a resistance marker in the salmon industry. In the present study the aim was to validate the use of catalase expression as a marker and to identify new candidate genes as additional markers to catalase, related to H2O2 resistance in L. salmonis. METHODS: A sensitive and an H2O2 resistant laboratory strain (P0 generation, not exposed to H2O2 for several years) were batch crossed to generate a cohort with a wide range of H2O2 sensitivities (F2 generation). F2 adult females were then exposed to H2O2 to separate sensitive and resistant individuals. Those F2 lice, the P0 lice and field-collected resistant lice (exposed to H2O2 in the field) were used in an RNA sequencing study. RESULTS: Catalase was upregulated in resistant lice exposed to H2O2 compared to sensitive lice. This was, however, not the case for unexposed resistant P0 lice. Several other genes were found differentially expressed between sensitive and resistant lice, but most of them seemed to be related to H2O2 exposure. However, five genes were consistently up- or downregulated in the resistant lice independent of exposure history. The upregulated genes were: one gene in the DNA polymerase family, one gene encoding a Nesprin-like protein and an unannotated gene encoding a small protein. The downregulated genes encoded endoplasmic reticulum resident protein 29 and an aquaporin (Glp1_v2). CONCLUSIONS: Catalase expression seems to be induced by H2O2 exposure, since it was not upregulated in unexposed resistant lice. This may pose a challenge for its use as a resistance marker. The five new genes associated with resistance are put forward as complementary candidate genes. The most promising was Glp1_v2, an aquaglyceroporin that may serve as a passing channel for H2O2. Lower channel number can reduce the influx or distribution of H2O2 in the salmon louse, being directly involved in the resistance mechanism.

Epac1 null mice have nephrogenic diabetes insipidus with deficient corticopapillary osmotic gradient and weaker collecting duct tight junctions.

AIM: The cAMP-mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1-/- mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium. METHODS: In Epac1-/- and Wt C57BL/6J mice, renal papillae, dissected from snap-frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.-injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [14 C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time-spaced intravenously injected 125 I- and 131 I-labelled albumin. RESULTS: Epac1-/- mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1-/- mice had no increased protein diet-induced urea-dependent osmotic diuresis, and expressed Wt levels of aquaporin-2 (AQP-2) and urea transporter A1/3 (UT-A1/3). Epac1-/- mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide. CONCLUSION: Our results conform to the hypothesis that Epac1-dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs.

Phosphorylation of steroidogenic factor 1 is mediated by cyclin-dependent kinase 7.

The nuclear receptor steroidogenic factor-1 (SF1) is critical for development and function of steroidogenic tissues. Posttranslational modifications are known to influence the transcriptional capacity of SF1, and it was previously demonstrated that serine 203 is phosphorylated. In this paper we report that serine 203 is phosphorylated by a cyclin-dependent kinase 7 (CDK7)-mediated process. As part of the CDK-activating kinase complex, CDK7 is a component of the basal transcription factor TFIIH, and phosphorylation of SF1 as well as SF1-dependent transcription was clearly reduced in cells carrying a mutation that renders the CDK-activating kinase complex unable to interact with the TFIIH core. Coimmunoprecipitation analyses revealed that SF1 and CDK7 reside in the same complex, and kinase assays demonstrated that immunoprecipitated CDK7 and purified TFIIH phosphorylate SF1 in vitro. The CDK inhibitor roscovitine blocked phosphorylation of SF1, and an inactive form of CDK7 repressed the phosphorylation level and the transactivation capacity of SF1. Structural studies have identified phosphoinositides as potential ligands for SF1. Interestingly, we found that mutations designed to block phospholipid binding dramatically decreased the level of SF1 phosphorylation. Together our results suggest a connection between ligand occupation and phosphorylation and association with the basic transcriptional machinery, indicating an intricate regulation of SF1 transactivation.

Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis.

Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- (Rapgef4-/-) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.

Mutations in voltage-gated sodium channels from pyrethroid resistant salmon lice (Lepeophtheirus salmonis).

BACKGROUND: Parasitic salmon lice (Lepeophtheirus salmonis) cause high economic losses in Atlantic salmon farming. Pyrethroids, which block arthropod voltage-gated sodium channels (Nav 1), are used for salmon delousing. However, pyrethroid resistance is common in L. salmonis. The present study characterized Nav 1 homologues in L. salmonis in order to identify channel mutations associated to resistance, called kdr (knockdown) mutations. RESULTS: Genome scans identified three L. salmonis Nav 1 homologues, LsNav 1.1, LsNav 1.2 and LsNav 1.3. Arthropod kdr mutations map to specific Nav 1 regions within domains DI-III, namely segments S5 and S6 and the linker helix connecting S4 and S5. The above channel regions were amplified by RT-PCR and sequenced in deltamethrin-susceptible and deltamethrin-resistant L. salmonis. While LsNav 1.1 and LsNav 1.2 lacked nucleotide polymorphisms showing association to resistance, LsNav 1.3 showed a non-synonymous mutation in S5 of DII occurring in deltamethrin-resistant parasites. The mutation is homologous to a previously described kdr mutation (I936V, numbering according to Musca domestica Vssc1) and was present in two pyrethroid-resistant L. salmonis strains (allele frequencies of 0.800 and 0.357), but absent in two pyrethroid-susceptible strains. CONCLUSIONS: The present study indicates that a kdr-mutation in LsNaV 1.3 may contribute to deltamethrin resistance in L. salmonis. © 2018 Society of Chemical Industry.

Mice depleted for Exchange Proteins Directly Activated by cAMP (Epac) exhibit irregular liver regeneration in response to partial hepatectomy.

The exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2) are expressed in a cell specific manner in the liver, but their biological functions in this tissue are poorly understood. The current study was undertaken to begin to determine the potential roles of Epac1 and Epac2 in liver physiology and disease. Male C57BL/6J mice in which expression of Epac1 and/or Epac2 are deleted, were subjected to partial hepatectomy and the regenerating liver was analyzed with regard to lipid accumulation, cell replication and protein expression. In response to partial hepatectomy, deletion of Epac1 and/or Epac2 led to increased hepatocyte proliferation 36 h post surgery, and the transient steatosis observed in wild type mice was virtually absent in mice lacking both Epac1 and Epac2. The expression of the protein cytochrome P4504a14, which is implicated in hepatic steatosis and fibrosis, was substantially reduced upon deletion of Epac1/2, while a number of factors involved in lipid metabolism were significantly decreased. Moreover, the number of Küpffer cells was affected, and Epac2 expression was increased in the liver of wild type mice in response to partial hepatectomy, further supporting a role for these proteins in liver function. This study establishes hepatic phenotypic abnormalities in mice deleted for Epac1/2 for the first time, and introduces Epac1/2 as regulators of hepatocyte proliferation and lipid accumulation in the regenerative process.

Deoxyribonucleic acid methylation controls cell type-specific expression of steroidogenic factor 1.

Steroidogenic factor 1 (SF1) is expressed in a time- and cell-specific manner in the endocrine system. In this study we present evidence to support that methylation of CpG sites located in the proximal promoter of the gene encoding SF1 contributes to the restricted expression pattern of this nuclear receptor. DNA methylation analyses revealed a nearly perfect correlation between the methylation status of the proximal promoter and protein expression, such that it was hypomethylated in cells that express SF1 but hypermethylated in nonexpressing cells. Moreover, in vitro methylation of this region completely repressed reporter gene activity in transfected steroidogenic cells. Bisulfite sequencing of DNA from embryonic tissue demonstrated that the proximal promoter was unmethylated in the developing testis and ovary, whereas it was hypermethylated in tissues that do not express SF1. Together these results indicate that the DNA methylation pattern is established early in the embryo and stably inherited thereafter throughout development to confine SF1 expression to the appropriate tissues. Chromatin immunoprecipitation analyses revealed that the transcriptional activator upstream stimulatory factor 2 and RNA polymerase II were specifically recruited to this DNA region in cells in which the proximal promoter is hypomethylated, providing functional support for the fact that lack of methylation corresponds to a transcriptionally active gene. In conclusion, we identified a region within the SF1/Sf1 gene that epigenetically directs cell-specific expression of SF1.

Deltamethrin resistance in the salmon louse, Lepeophtheirus salmonis (Krøyer): Maternal inheritance and reduced apoptosis.

Resistance towards deltamethrin (DMT) in the crustacean ectoparasite Lepeophtheirus salmonis (Caligidae) is a problem on fish farms lining the North Atlantic Ocean. Two Norwegian strains with different susceptibility towards DMT were crossed in the parental generation (P0), females from a sensitive strain were crossed with males from a resistant strain and vice versa. Individual susceptibility towards DMT was assessed in the second filial generation (F2). DMT resistance was only found in F2 descendants when the P0 females were from the resistant strain, pointing to maternal inheritance. Since maternal inheritance might be linked to the mitochondrial (mt) genome, the nucleotide sequences and the gene expressions of mt-genes were analysed. Twenty non-synonymous single nucleotide polymorphisms (SNPs) were identified in mt-transcripts from resistant F2 parasites, including SNPs in two cytochrome C oxidase subunits (COX1 and COX3) and two subunits of the NADH dehydrogenase complex (ND1 and ND5) previously linked to DMT resistance in the salmon louse. Differential expression analysis between the sensitive and resistant strain revealed strain effect in seven out of twelve mt-genes. The current study also show that DNA fragmentation (indicating apoptosis) was affected by DMT exposure in skeletal muscle tissue and that resistant parasites undergo less apoptosis than sensitive parasites.

Deletion of exchange proteins directly activated by cAMP (Epac) causes defects in hippocampal signaling in female mice.

Previous studies demonstrate essential roles for the exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2; here collectively referred to as Epac) in the brain. In the hippocampus, Epac contributes to the control of neuronal growth and differentiation and has been implicated in memory and learning as well as in anxiety and depression. In the present study we address the hypothesis that Epac affects hippocampal cellular responses to acute restraint stress. Stress causes activation of the hypothalamus-pituitary-adrenal (HPA)-axis, and glucocorticoid receptor (GR) signaling is essential for proper feedback regulation of the stress response, both in the brain and along the HPA axis. In the hippocampus, GR expression is regulated by cAMP and the brain enriched micro RNA miR-124. Epac has been associated with miR-124 expression in hippocampal neurons, but not in regulation of GR. We report that hippocampal expression of Epac1 and Epac2 increased in response to acute stress in female wild type mice. In female mice genetically deleted for Epac, nuclear translocation of GR in response to restraint stress was significantly delayed, and moreover, miR-124 expression was decreased in these mice. Male mice lacking Epac also showed abnormalities in miR-124 expression, but the phenotype was less profound than in females. Serum corticosterone levels were slightly altered immediately after stress in both male and female mice deleted for Epac. The presented data indicate that Epac1 and Epac2 are involved in controlling cellular responses to acute stress in the mouse hippocampus and provide novel insights into the underlying transcriptional and signaling networks. Interestingly, we observe sex specific differences when Epac is deleted. As the incidence and prevalence of stress-related diseases are higher in women than in men, the Epac knockout models might serve as genetic tools to further elucidate the cellular mechanisms underlying differences between male and female with regard to regulation of stress.

Effects of algal-produced neurotoxins on metabolic activity in telencephalon, optic tectum and cerebellum of Atlantic salmon (Salmo salar).

Neurotoxins from algal blooms have been reported to cause mortality in a variety of species, including sea birds, sea mammals and fish. Farmed fish cannot escape harmful algal blooms and their potential toxins, thus they are more vulnerable for exposure than wild stocks. Sublethal doses of the toxins are likely to affect fish behaviour and may impair cognitive abilities. In the present study, changes in the metabolic activity in different parts of the Atlantic salmon (Salmo salar) brain involved in central integration and cognition were investigated after exposure to sublethal doses of three algal-produced neurotoxins; saxitoxin (STX), brevetoxin (BTX) and domoic acid (DA). Fish were randomly selected to four groups for i.p. injection of saline (control) or one of the neurotoxins STX (10 microg STX/kg bw), BTX (68 microg BTX/kg bw) or DA (6 mg DA/kg bw). In addition, 14C-2-deoxyglucose was i.m. injected to measure brain metabolic activity by autoradiography. The three regions investigated were telencephalon (Tel), optic tectum (OT) and cerebellum (Ce). There were no differences in the metabolic activity after STX and BTX exposure compared to the control in these regions. However, a clear increase was observed after DA exposure. When the subregions with the highest metabolic rate were pseudocoloured in the three brain regions, the three toxins caused distinct differences in the respective patterns of metabolic activation. Fish exposed to STX displayed similar patterns as the control fish, whereas fish exposed to BTX and DA showed highest metabolic activity in subregions different from the control group. All three neurotoxins affected subregions that are believed to be involved in cognitive abilities in fish.

A selection study on a laboratory-designed population of salmon lic (Lepeophtheirus salmonis) using organophosphate and pyrethroid pesticides.

Resistance towards antiparasitic agents in the salmon louse (Lepeophtheirus salmonis) is a widespread problem along the Norwegian coast, reducing treatments efficacies and slowing down the envisioned expansion of Norwegian salmon production. The present study was conducted in order to assess the efficacies of two of the most widely used anti-parasitic substances-azamethiphos and deltamethrin-as well as assessing the benefit of having a resistant genotype compared to being fully sensitive when exposed to one of these substances. Atlantic salmon were exposed to a mix of salmon lice copepodids from a fully sensitive, a double resistant and a multi-resistant strain. Once the lice reached pre-adult stages, one group was exposed to 100 µg/L azamethiphos for 60 minutes, the other to 2 µg/L deltamethrin for 30 minutes, and the last was kept in a seawater control. Detached lice were collected at a series of time points following exposure, and all lice (immobilized and surviving) were analysed for both pyrethroid (sensitive "S" and resistant "R") and azamethiphos (fully sensitive "SS", heterozygous resistant "RS" and fully resistant "RR") resistance markers. We found that the efficacies of deltamethrin on parasites with genotype S and R were 70.3 and 13.2%, respectively. The overall efficacy of the deltamethrin treatment was 32.3%. The efficacies of azamethiphos on parasites with genotype SS, RS and RR were 100, 80 and 19.1%, respectively. The overall efficacy of the azamethiphos treatment was 80.4%. Survival analyses revealed that the median survival time in deltamethrin-sensitive and-resistant parasites were 16.8 and >172 hours, respectively. The differences were even more pronounced in the azamethiphos-treated group, where SS, RS and RR parasites survived for 0.26, 6.6 and >172 hours, respectively. The substantial differences in survival between sensitive and resistant lice following treatment demonstrate the ability of medicinal treatments to drive genetic selection towards a much more resistant salmon lice population within a very short time span if there is no influx of sensitive genotypes.

Epac1-deficient mice have bleeding phenotype and thrombocytes with decreased GPIbß expression.

Epac1 (Exchange protein directly activated by cAMP 1) limits fluid loss from the circulation by tightening the endothelial barrier. We show here that Epac1-/- mice, but not Epac2-/- mice, have prolonged bleeding time, suggesting that Epac1 may limit fluid loss also by restraining bleeding. The Epac1-/- mice had deficient in vitro secondary hemostasis. Quantitative comprehensive proteomics analysis revealed that Epac1-/- mouse platelets (thrombocytes) had unbalanced expression of key components of the glycoprotein Ib-IX-V (GPIb-IX-V) complex, with decrease of GP1bß and no change of GP1bα. This complex is critical for platelet adhesion under arterial shear conditions. Furthermore, Epac1-/- mice have reduced levels of plasma coagulation factors and fibrinogen, increased size of circulating platelets, increased megakaryocytes (the GP1bß level was decreased also in Epac1-/- bone marrow) and higher abundance of reticulated platelets. Viscoelastic measurement of clotting function revealed Epac1-/- mice with a dysfunction in the clotting process, which corresponds to reduced plasma levels of coagulation factors like factor XIII and fibrinogen. We propose that the observed platelet phenotype is due to deficient Epac1 activity during megakaryopoiesis and thrombopoiesis, and that the defects in blood clotting for Epac1-/- is connected to secondary hemostasis.

Role of EPAC in cAMP-Mediated Actions in Adrenocortical Cells.

Adrenocorticotropic hormone regulates adrenal steroidogenesis mainly via the intracellular signaling molecule cAMP. The effects of cAMP are principally relayed by activating protein kinase A (PKA) and the more recently discovered exchange proteins directly activated by cAMP 1 and 2 (EPAC1 and EPAC2). While the intracellular roles of PKA have been extensively studied in steroidogenic tissues, those of EPACs are only emerging. EPAC1 and EPAC2 are encoded by the genes RAPGEF3 and RAPGEF4, respectively. Whereas EPAC1 is ubiquitously expressed, the expression of EPAC2 is more restricted, and typically found in endocrine tissues. Alternative promoter usage of RAPGEF4 gives rise to three different isoforms of EPAC2 that vary in their N-termini (EPAC2A, EPAC2B, and EPAC2C) and that exhibit distinct expression patterns. EPAC2A is expressed in the brain and pancreas, EPAC2B in steroidogenic cells of the adrenal gland and testis, and EPAC2C has until now only been found in the liver. In this review, we discuss current knowledge on EPAC expression and function with focus on the known roles of EPAC in adrenal gland physiology.

The effect of dietary lipid composition on the intestinal uptake and tissue distribution of benzo[a]pyrene and phenanthrene in Atlantic salmon (Salmo salar).

Uptake of polycyclic aromatic hydrocarbons (PAHs) across the intestine is suggested to occur in association with dietary lipids. Partial replacement of fish ingredients by vegetable ingredients in aquafeeds has led to increased levels of PAHs in marine farmed fish. We therefore investigated, intestinal uptake, tissue distribution and PAH metabolism after a single dose of (14)C-benzo[a]pyrene (BaP) or (14)C-phenanthrene (PHE) given to Atlantic salmon (Salmo salar) acclimatized to a fish oil or vegetable oil based diet. Both BaP and PHE were absorbed along the intestine. Fish oil based feed increased BaP concentration in the pyloric caeca and that of PHE in the proximal intestine. In contrast, vegetable oil increased BaP concentrations in the distal intestine. Extraction of whole body autoradiograms removed PHE-associated radiolabeling almost completely from the intestinal mucosa, but not BaP-associated radiolabeling, indicating the presence of BaP metabolites bound to cellular macromolecules. This observation correlates with the increased cyp1a expression in the proximal intestine, distal intestine and liver in the BaP exposed group. Furthermore, BaP-induced cyp1a expression was higher in the distal intestine of salmon fed fish oil compared to the vegetable oil fed group. PHE had no significant effect on cyp1a expression in any of these tissues. We conclude that dietary lipid composition affects intestinal PAH uptake. Fish oil based feed increased intestinal PAH concentrations probably due to an enhanced solubility in micelles composed of fish oil fatty acids. Increased BaP accumulation in the distal intestine of vegetable oil fed fish seems to be associated with a reduced Cyp1a-mediated BaP metabolism.

Cloning and characterization of a novel zinc finger protein that modulates the transcriptional activity of nuclear receptors.

The orphan nuclear receptor steroidogenic factor-1 (SF-1) plays pivotal roles in the development and function of steroidogenic organs. It transcriptionally regulates an array of factors required for biosynthesis of steroid hormones and is also necessary for the expression of genes in the pituitary and the male reproductive tract. Here we describe the identification of a novel zinc finger protein that modifies the transcriptional potential of SF-1. This factor, which we call Zip67 (zinc finger protein 67 kDa), was cloned through a two-hybrid screen of a human testis cDNA library using the C-terminal part of SF-1 as the bait. Transient transfection experiments demonstrated that Zip67 represses SF-1-dependent transcription in the context of both multimerized SF-1-binding sites and natural SF-1-inducible promoters. The interaction between Zip67 and SF-1 was dependent on an intact activation function-2 domain of SF-1, and we propose a mechanism whereby Zip67 represses transcription through competition with p160 coactivators for binding to SF-1. Zip67 was detected in SF-1 expressing tissues such as testis, adrenal, ovary and spleen in addition to other tissues. In line with the broader expression pattern, we found that Zip67 also affected transcription mediated by several other nuclear receptors. In conclusion, we have isolated a novel zinc-finger protein that influences gene activation through interaction with the functionally important activation function-2 domain of nuclear receptors.

Development of a transgenic green fluorescent protein lineage marker for steroidogenic factor 1.

Knockout mice lacking steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a complex phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To explore further how SF-1 regulates endocrine function, we used bacterial artificial chromosome transgenesis to develop a lineage marker for SF-1-expressing cells. A genomic fragment containing 50 kb of the mouse Nr5a1 gene was used to target enhanced green fluorescent protein (eGFP) in transgenic mice. These sequences directed eGFP to multiple cell lineages that express SF-1, including steroidogenic cells of the adrenal cortex, testes, and ovaries, neurons of the ventromedial hypothalamic nucleus, and reticuloendothelial cells of the spleen. Despite the proven role of SF-1 in gonadotrope function, eGFP was not expressed in the anterior pituitary. These experiments show that 50 kb of the mouse Nr5a1 gene can target transgenic expression to multiple cell lineages that normally express SF-1. The SF-1/eGFP transgenic mice will facilitate approaches such as fluorescence-activated cell sorting of eGFP-positive cells and DNA microarray analyses to expand our understanding of the multiple actions of SF-1 in endocrine development and function.

The nuclear receptor coactivators p300/CBP/cointegrator-associated protein (p/CIP) and transcription intermediary factor 2 (TIF2) differentially regulate PKA-stimulated transcriptional activity of steroidogenic factor 1.

Steroidogenic factor-1 (SF-1) is a member of the nuclear receptor superfamily that plays essential roles in the development of endocrine organs. Steroid receptor coactivator 1 and transcription intermediary factor 2 (TIF2) belong to the p160 coactivator family that mediates transcriptional activation by several nuclear receptors, including SF-1. Here, it is reported that another of the p160 coactivators, p/CIP, interacts with SF-1 through the activation function-2 domain. Both p300/CBP/cointegrator-associated protein (p/CIP) and TIF2 potentiated SF-1-mediated transcription from two reporter gene constructs in transfected nonsteroidogenic COS-1 cells and in adrenocortical Y1 cells. PKA was shown to stimulate SF-1 transcriptional activity, and coexpression of p/CIP together with the PKA catalytic subunit stimulated SF-1-mediated transactivation even further. In contrast, PKA catalytic subunit overexpression impaired the ability of TIF2 to potentiate SF-1-dependent transcription. Activation of PKA also inhibited the TIF2-mediated coactivation of other nuclear receptors such as PPAR alpha/-gamma and liver X receptor-alpha. The TIF2 mRNA levels were not affected by PKA, but instead we found that PKA activation led to a decrease in the levels of TIF2 protein. Moreover, the C-terminal activation domain 2 of TIF2 was required for the inhibitory effect of PKA, suggesting that this region is the target for the PKA-mediated down-regulation. Thus, in contrast to the regulation of p/CIP and steroid receptor coactivator 1, we suggest that activation of PKA leads to selective down-regulation of TIF2 and subsequently repression of TIF2 coactivator function.

Identification and Molecular Characterization of Two Acetylcholinesterases from the Salmon Louse, Lepeophtheirus salmonis.

Acetylcholinesterase (AChE) is an important enzyme in cholinergic synapses. Most arthropods have two genes (ace1 and ace2), but only one encodes the predominant synaptic AChE, the main target for organophosphates. Resistance towards organophosphates is widespread in the marine arthropod Lepeophtheirus salmonis. To understand this trait, it is essential to characterize the gene(s) coding for AChE(s). The full length cDNA sequences encoding two AChEs in L. salmonis were molecularly characterized in this study. The two ace genes were highly similar (83.5% similarity at protein level). Alignment to the L. salmonis genome revealed that both genes were located close to each other (separated by just 26.4 kbp on the L. salmonis genome), resulting from a recent gene duplication. Both proteins had all the typical features of functional AChE and clustered together with AChE-type 1 proteins in other species, an observation that has not been described in other arthropods. We therefore concluded the presence of two versions of ace1 gene in L. salmonis, named ace1a and ace1b. Ace1a was predominantly expressed in different developmental stages compared to ace1b and was possibly active in the cephalothorax, indicating that ace1a is more likely to play the major role in cholinergic synaptic transmission. The study is essential to understand the role of AChEs in resistance against organophosphates in L. salmonis.

Mechanism behind Resistance against the Organophosphate Azamethiphos in Salmon Lice (Lepeophtheirus salmonis).

Acetylcholinesterase (AChE) is the primary target for organophosphates (OP). Several mutations have been reported in AChE to be associated with the reduced sensitivity against OP in various arthropods. However, to the best of our knowledge, no such reports are available for Lepeophtheirus salmonis. Hence, in the present study, we aimed to determine the association of AChE(s) gene(s) with resistance against OP. We screened the AChE genes (L. salmonis ace1a and ace1b) in two salmon lice populations: one sensitive (n=5) and the other resistant (n=5) for azamethiphos, a commonly used OP in salmon farming. The screening led to the identification of a missense mutation Phe362Tyr in L. salmonis ace1a, (corresponding to Phe331 in Torpedo californica AChE) in all the samples of the resistant population. We confirmed the potential role of the mutation, with reduced sensitivity against azamethiphos in L. salmonis, by screening for Phe362Tyr in 2 sensitive and 5 resistant strains. The significantly higher frequency of the mutant allele (362Tyr) in the resistant strains clearly indicated the possible association of Phe362Tyr mutation in L. salmonis ace1a with resistance towards azamethiphos. The 3D modelling, short term survival experiments and enzymatic assays further supported the imperative role of Phe362Tyr in reduced sensitivity of L. salmonis for azamethiphos. Based on all these observations, the present study, for the first time, presents the mechanism of resistance in L. salmonis against azamethiphos. In addition, we developed a rapid diagnostic tool for the high throughput screening of Phe362Tyr mutation using High Resolution Melt analysis.