@article{vonLoeffelholzLieskeNeuschaeferRubeetal.2017,
  author    = {von Loeffelholz, Christian and Lieske, Stefanie and Neuschaefer-Rube, Frank and Willmes, Diana M. and Raschzok, Nathanael and Sauer, Igor M. and K{\"o}nig, J{\"o}rg and Fromm, Martin F. and Horn, Paul and Chatzigeorgiou, Antonios and Pathe-Neuschaefer-Rube, Andrea and Jordan, Jens and Pfeiffer, Andreas F. H. and Mingrone, Geltrude and Bornstein, Stefan R. and Stroehle, Peter and Harms, Christoph and Wunderlich, F. Thomas and Helfand, Stephen L. and Bernier, Michel and de Cabo, Rafael and Shulman, Gerald I. and Chavakis, Triantafyllos and P{\"u}schel, Gerhard Paul and Birkenfeld, Andreas L.},
  title     = {The human longevity gene homolog INDY and interleukin-6 interact in hepatic lipid metabolism},
  series = {Hepatology},
  volume    = {66},
  journal   = {Hepatology},
  number    = {2},
  publisher = {Wiley},
  address   = {Hoboken},
  issn      = {0270-9139},
  doi       = {10.1002/hep.29089},
  pages     = {616 -- 630},
  year      = {2017},
  abstract  = {Reduced expression of the Indy ("I am Not Dead, Yet") gene in lower organisms promotes longevity in a manner akin to caloric restriction. Deletion of the mammalian homolog of Indy (mIndy, Slc13a5) encoding for a plasma membrane-associated citrate transporter expressed highly in the liver, protects mice from high-fat diet-induced and aging-induced obesity and hepatic fat accumulation through a mechanism resembling caloric restriction. We studied a possible role of mIndy in human hepatic fat metabolism. In obese, insulin-resistant patients with nonalcoholic fatty liver disease, hepatic mIndy expression was increased and mIndy expression was also independently associated with hepatic steatosis. In nonhuman primates, a 2-year high-fat, high-sucrose diet increased hepatic mIndy expression. Liver microarray analysis showed that high mIndy expression was associated with pathways involved in hepatic lipid metabolism and immunological processes. Interleukin-6 (IL-6) was identified as a regulator of mIndy by binding to its cognate receptor. Studies in human primary hepatocytes confirmed that IL-6 markedly induced mIndy transcription through the IL-6 receptor and activation of the transcription factor signal transducer and activator of transcription 3, and a putative start site of the human mIndy promoter was determined. Activation of the IL-6-signal transducer and activator of transcription 3 pathway stimulated mIndy expression, enhanced cytoplasmic citrate influx, and augmented hepatic lipogenesis in vivo. In contrast, deletion of mIndy completely prevented the stimulating effect of IL-6 on citrate uptake and reduced hepatic lipogenesis. These data show that mIndy is increased in liver of obese humans and nonhuman primates with NALFD. Moreover, our data identify mIndy as a target gene of IL-6 and determine novel functions of IL-6 through mINDY. Conclusion: Targeting human mINDY may have therapeutic potential in obese patients with nonalcoholic fatty liver disease. German Clinical Trials Register: DRKS00005450.},
  language  = {en}
}
@unpublished{SeelaenderLavianoBusquetsetal.2015,
  author    = {Seelaender, Marilia and Laviano, A. and Busquets, S. and P{\"u}schel, Gerhard Paul and Margaria, T. and Batista Jr., Miguel Luiz},
  title     = {Inflammation in Cachexia},
  series = {Mediators of inflammation},
  journal   = {Mediators of inflammation},
  publisher = {Hindawi Publishing Corp.},
  address   = {New York},
  issn      = {0962-9351},
  doi       = {10.1155/2015/536954},
  pages     = {2},
  year      = {2015},
  language  = {en}
}
@misc{SchenkeSchjeidePuescheletal.2020,
  author    = {Schenke, Maren and Schjeide, Brit-Maren and P{\"u}schel, Gerhard Paul and Seeger, Bettina},
  title     = {Analysis of motor neurons differentiated from human induced pluripotent stem cells for the use in cell-based Botulinum neurotoxin activity assays},
  series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe},
  journal   = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe},
  number    = {1083},
  issn      = {1866-8372},
  doi       = {10.25932/publishup-47207},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-472071},
  pages     = {22},
  year      = {2020},
  abstract  = {Botulinum neurotoxins (BoNTs) are potent neurotoxins produced by bacteria, which inhibit neurotransmitter release, specifically in their physiological target known as motor neurons (MNs). For the potency assessment of BoNTs produced for treatment in traditional and aesthetic medicine, the mouse lethality assay is still used by the majority of manufacturers, which is ethically questionable in terms of the 3Rs principle. In this study, MNs were differentiated from human induced pluripotent stem cells based on three published protocols. The resulting cell populations were analyzed for their MN yield and their suitability for the potency assessment of BoNTs. MNs produce specific gangliosides and synaptic proteins, which are bound by BoNTs in order to be taken up by receptor-mediated endocytosis, which is followed by cleavage of specific soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins required for neurotransmitter release. The presence of receptors and substrates for all BoNT serotypes was demonstrated in MNs generated in vitro. In particular, the MN differentiation protocol based on Du et al. yielded high numbers of MNs in a short amount of time with high expression of BoNT receptors and targets. The resulting cells are more sensitive to BoNT/A1 than the commonly used neuroblastoma cell line SiMa. MNs are, therefore, an ideal tool for being combined with already established detection methods.},
  language  = {en}
}
@misc{CamargoRiccardiRibeiroetal.2017,
  author    = {Camargo, Rodolfo Gonzalez and Riccardi, Daniela Mendes dos Reis and Ribeiro, Henrique Quintas Teixeira and Carnevali Junior, Luiz Carlos and Matos-Neto, Emidio Marques de and Enjiu, Lucas and Neves, Rodrigo Xavier and Lima, Joanna Darck Carola Correia and Figuer{\^e}do, Raquel Galv{\~a}o and Alc{\^a}ntara, Paulo S{\´e}rgio Martins de and Maximiano, Linda and Otoch, Jos{\´e} and Batista Jr., Miguel Luiz and P{\"u}schel, Gerhard Paul and Seelaender, Marilia},
  title     = {NF-kappa Bp65 and expression of its pro-inflammatory target genes are upregulated in the subcutaneous adipose tissue of cachectic cancer patients},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-400163},
  pages     = {15},
  year      = {2017},
  abstract  = {Cancer cachexia, of which the most notable symptom is severe and rapid weight loss, is present in the majority of patients with advanced cancer. Inflammatory mediators play an important role in the development of cachexia, envisaged as a chronic inflammatory syndrome. The white adipose tissue (WAT) is one of the first compartments affected in cancer cachexia and suffers a high rate of lipolysis. It secretes several cytokines capable of directly regulating intermediate metabolism. A common pathway in the regulation of the expression of pro-inflammatory cytokines in WAT is the activation of the nuclear transcription factor kappa-B (NF-κB). We have examined the gene expression of the subunits NF-κBp65 and NF-κBp50, as well as NF-κBp65 and NF-κBp50 binding, the gene expression of pro-inflammatory mediators under NF-κB control (IL-1β, IL-6, INF-γ, TNF-α, MCP-1), and its inhibitory protein, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α). The observational study involved 35 patients (control group, n = 12 and cancer group, n = 23, further divided into cachectic and non-cachectic). NF-κBp65 and its target genes expression (TNF-α, IL-1β, MCP-1 and IκB-α) were significantly higher in cachectic cancer patients. Moreover, NF-κBp65 gene expression correlated positively with the expression of its target genes. The results strongly suggest that the NF-κB pathway plays a role in the promotion of WAT inflammation during cachexia.},
  language  = {en}
}
@misc{PatheNeuschaeferRubeNeuschaeferRubeHaasetal.2018,
  author    = {Pathe-Neusch{\"a}fer-Rube, Andrea and Neusch{\"a}fer-Rube, Frank and Haas, Gerald and Langoth-Fehringer, Nina and P{\"u}schel, Gerhard Paul},
  title     = {Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins},
  series = {Toxins},
  journal   = {Toxins},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-418141},
  pages     = {10},
  year      = {2018},
  abstract  = {Despite the implementation of cell-based replacement methods, the mouse lethality assay is still frequently used to determine the activity of botulinum toxin (BoNT) for medical use. One explanation is that due to the use of neoepitope-specific antibodies to detect the cleaved BoNT substrate, the currently devised assays can detect only one specific serotype of the toxin. Recently, we developed a cell-based functional assay, in which BoNT activity is determined by inhibiting the release of a reporter enzyme that is liberated concomitantly with the neurotransmitter from neurosecretory vesicles. In theory, this assay should be suitable to detect the activity of any BoNT serotype. Consistent with this assumption, the current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) was inhibited by BoNT-A and-C. Furthermore, this was also inhibited by BoNT-B and tetanus toxin to a lesser extent and at higher concentrations. In order to provide support for the suitability of this technique in practical applications, a dose-response curve obtained with a pharmaceutical preparation of BoNT-A closely mirrored the activity determined in the mouse lethality assay. In summary, the newly established cell-based assay may represent a versatile and specific alternative to the mouse lethality assay and other currently established cell-based assays.},
  language  = {en}
}
@article{HenkelNeuschaeferRubePatheNeuschaeferRubeetal.2009,
  author    = {Henkel, Janin and Neuschaefer-Rube, Frank and Pathe-Neuschaefer-Rube, Andrea and P{\"u}schel, Gerhard Paul},
  title     = {Aggravation by prostaglandin e-2 of interleukin-6-dependent insulin resistance in hepatocytes},
  issn      = {0270-9139},
  doi       = {10.1002/Hep.23064},
  year      = {2009},
  abstract  = {Hepatic insulin resistance is a major contributor to fasting hyperglycemia in patients with metabolic syndrome and type 2 diabetes. Circumstantial evidence suggests that cyclooxygenase products in addition to cytokines might contribute to insulin resistance. However, direct evidence for a role of prostaglandins in the development of hepatic insulin resistance is lacking. Therefore, the impact of prostaglandin E-2 (PGE(2)) alone and in combination with interleukin-6 (IL-6) on insulin signaling was studied in primary hepatocyte cultures. Rat hepatocytes were incubated with IL-6 and/or PGE(2) and subsequently with insulin. Glycogen synthesis was monitored by radiochemical analysis; the activation state of proteins of the insulin receptor signal chain was analyzed by western blot with phosphospecific antibodies. In hepatocytes, insulin-stimulated glycogen synthesis and insulin-dependent phosphorylation of Akt-kinase were attenuated synergistically by prior incubation with IL-6 and/or PGE(2) while insulin receptor autophosphorylation was barely affected. IL-6 but not PGE(2) induced suppressors of cytokine signaling (SOCS3). PGE(2) but not IL-6 activated extracellular signal-regulated kinase 1/2 (ERK1/2) persistently. Inhibition of ERK1/2 activation by PD98059 abolished the PGE(2)-dependent but not the IL-6-dependent attenuation of insulin signaling. In HepG2 cells expressing a recombinant EP3-receptor, PGE(2) pre-incubation activated ERK1/2, caused a serine phosphorylation of insulin receptor substrate 1 (IRS1), and reduced the insulin-dependent Akt-phosphorylation. Conclusion: PGE(2) might contribute to hepatic insulin resistance via an EP3-receptor-dependent ERK1/2 activation resulting in a serine phosphorylation of insulin receptor substrate, thereby preventing an insulin-dependent activation of Akt and glycogen synthesis. Since different molecular mechanisms appear to be employed, PGE(2) may synergize with IL-6, which interrupted the insulin receptor signal chain, principally by an induction of SOCS, namely SOCS3.},
  language  = {en}
}
@article{NeuschaeferRubeOppermannMoelleretal.1999,
  author    = {Neusch{\"a}fer-Rube, Frank and Oppermann, Martin and M{\"o}ller, Ulrike and B{\"o}er, Ulrike and P{\"u}schel, Gerhard Paul},
  title     = {Agonist-induced phosphorylation by G protein-coupled receptor kinases of the EP4 receptor carboxyl-terminal domain in an EP3/EP4 prostaglandin E(2) receptor hybrid},
  issn      = {1521-0111},
  year      = {1999},
  abstract  = {Prostaglandin E(2) receptors (EP-Rs) belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains. They can be subdivided into four subtypes according to their ligand-binding and G protein-coupling specificity: EP1 couple to G(q), EP2 and EP4 to G(s), and EP3 to G(i). The EP4-R, in contrast to the EP3beta-R, shows rapid agonist-induced desensitization. The agonist-induced desensitization depends on the presence of the EP4-R carboxyl-terminal domain, which also confers desensitization in a G(i)-coupled rEP3hEP4 carboxyl-terminal domain receptor hybrid (rEP3hEP4-Ct-R). To elucidate the possible mechanism of this desensitization, in vivo phosphorylation stimulated by activators of second messenger kinases, by prostaglandin E(2), or by the EP3-R agonist M\&B28767 was investigated in COS-7 cells expressing FLAG-epitope-tagged rat EP3beta-R (rEP3beta-R), hEP4-R, or rEP3hEP4- Ct-R. Stimulation of protein kinase C with phorbol-12-myristate-13-acetate led to a slight phosphorylation of the FLAG- rEP3beta-R but to a strong phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R, which was suppressed by the protein kinase A and protein kinase C inhibitor staurosporine. Prostaglandin E(2) stimulated phosphorylation of the FLAG- hEP4-R in its carboxyl-terminal receptor domain. The EP3-R agonist M\&B28767 induced a time- and dose-dependent phosphorylation of the FLAG-rEP3hEP4-Ct-R but not of the FLAG-rEP3beta-R. Agonist-induced phosphorylation of the FLAG- hEP4-R and the FLAG-rEP3hEP4-Ct-R were not inhibited by staurosporine, which implies a role of G protein-coupled receptor kinases (GRKs) in agonist-induced receptor phosphorylation. Overexpression of GRKs in FLAG-rEP3hEP4-Ct-R- expressing COS-7 cells augmented the M\&B28767-induced receptor phosphorylation and receptor sequestration. These findings indicate that phosphorylation of the carboxyl-terminal hEP4-R domain possibly by GRKs but not by second messenger kinases may be involved in rapid agonist-induced desensitization of the hEP4-R and the rEP3hEP4-Ct-R.},
  language  = {en}
}
@article{WienekeNeuschaeferRubeBodeetal.2009,
  author    = {Wieneke, Nadine and Neuschaefer-Rube, Frank and Bode, L. M. and Kuna, Manuela and Andres, Jesus and Carnevali Junior, Luiz Carlos and Hirsch-Ernst, Karen I. and P{\"u}schel, Gerhard Paul},
  title     = {Synergistic acceleration of thyroid hormone degradation by phenobarbital and the PPAR alpha agonist WY14643 in rat hepatocytes},
  issn      = {0041-008X},
  doi       = {10.1016/j.taap.2009.07.014},
  year      = {2009},
  abstract  = {Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feed back-control led regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase 11 enzymes of xenobiotic metabolism. We have recently shown, that PPAR alpha agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR alpha agonist could enhance the phenobarbital-dependent acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPARa agonist WY14643 to a larger extent than after induction with either Compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UCT1A10. The PPAR alpha-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting- incluced reduction in energy expenditure by fatty acids as natural PPARa ligands. The synergism of the PPAR alpha agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics.},
  language  = {en}
}
@article{NeuschaeferRubeHermosillaRehwaldetal.2004,
  author    = {Neusch{\"a}fer-Rube, Frank and Hermosilla, Ricardo and Rehwald, Matthias and Ronnstrand, Lars and Sch{\"u}lein, Ralf and Wernstedt, Christer and P{\"u}schel, Gerhard Paul},
  title     = {Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site},
  year      = {2004},
  abstract  = {hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a G(s)-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C- terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced beta-arrestin complex formation, which is stabilized by phosphorylation. Subsequently beta-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist- induced phosphorylation, interaction with beta-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370-382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389-Ser-390-Thr-391-Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of beta-arrestin1. However, phosphorylation greatly enhanced the stability of the beta-arrestin1-receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both beta-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary.},
  language  = {en}
}
@article{RehwaldNeuschaeferRubeDeVriesetal.1999,
  author    = {Rehwald, Matthias and Neusch{\"a}fer-Rube, Frank and DeVries, Christa and P{\"u}schel, Gerhard Paul},
  title     = {Possible role for ligand binding of histidine 81 in the second transmembrane domain of the rat prostaglandin F2alpha receptor},
  year      = {1999},
  abstract  = {For the five principal prostanoids PGD2, PGE2, PGF2alpha, prostacyclin and thromboxane A2 eight receptors have been identified that belong to the family of G-protein-coupled receptors. They display an overall homology of merely 30\%. However, single amino acids in the transmembrane domains such as an Arg in the seventh transmembrane domain are highly conserved. This Arg has been identified as part of the ligand binding pocket. It interacts with the carboxyl group of the prostanoid. The aim of the current study was to analyze the potential role in ligand binding of His-81 in the second transmembrane domain of the rat PGF2alpha receptor, which is conserved among all PGF2alpha receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand binding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exchanged by site-directed mutagenesis to Gln (Q), Asp (D), Arg (R), Ala (A) and Gly (G). The receptor molecules were N-terminally extended by a Flag epitope for immunological detection. All mutant proteins were expressed at levels between 50\% and 80\% of the wild type construct. The H81Q and H81D receptor bound PGF2alpha with 2-fold and 25-fold lower affinity, respectively, than the wild type receptor. Membranes of cells expressing the H81R, H81A or H81G mutants did not bind significant amounts of PGF2alpha. Wild type receptor and H81Q showed a shallow pH optimum for PGF2alpha binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF2alpha with a sharp optimum at pH 4.5, a pH at which the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pH values. The data indicate that the His-81 in the second transmembrane domain of the PGF2alpha receptor in concert with Arg-291 in the seventh transmembrane domain may be involved in ligand binding, most likely not by ionic interaction with the prostaglandin's carboxyl group but rather as a hydrogen bond donor.},
  language  = {en}
}
@article{NeuschaeferRubeMoellerPueschel2000,
  author    = {Neusch{\"a}fer-Rube, Frank and M{\"o}ller, Ulrike and P{\"u}schel, Gerhard Paul},
  title     = {Structure of the 5'-flanking region of the rat prostaglandin f(2alpha) receptor},
  year      = {2000},
  abstract  = {Prostaglandin F(2alpha) (PGF(2alpha)), modulates hepatocyte functions via a heptahelical G(q)-coupled PGF(2alpha)-receptor (FP-R) which in liver is expressed exclusively in hepatocytes. The aim of the present study was to isolate the 5'-flanking region of the rat FP-R gene and to elucidate its basal and IL-6-modulated transcription control function in rat hepatocytes. The 5'-non-translated region of the rat hepatocyte FP-R mRNA differed from the corresponding region in rat fetal astrocyte or corpus luteum. It was encoded by exons 1a and 2 which were separated by a 1. 4 kb intron containing the exons 1b and 1c coding for the 5'-untranslated region of rat fetal astrocyte and corpus luteum FP-R mRNA, respectively. The transcription initiation site in hepatocytes was localized 263 bp upstream of the start ATG by 5'-RACE. A DNA-fragment covering the 5'-flanking region of the rFP-R gene from - 1 of the transcription initiation site to -2590 bp was cloned and sequenced. Its 3'-two thirds had a 65\% sequence identity to the mouse FP-R promoter however no homology to the bovine FP-R promoter. In the overlapping sequence most of the putative transcription factor binding sites were conserved between mouse and rat. The rat promoter contained no classical TATA- or CAAT-boxes but putative binding sites for the transcription factors C/EBP, GATA-1, HNF-1, HNF-3beta, SP-1, and USF. Luciferase reporter gene constructs containing portions of the 5'-flanking region were transfected into rat hepatocytes. Luciferase expression ranked -181 >/= -608 < -1418 > -1821 >/= -2590. The strongest transcriptional activity was conferred by the region between -608 and -1418 containing a cluster of potential HNF-1 and HNF-3beta binding sites that might allow the exclusive expression of FP-R mRNA in hepatocytes. The amount of FP-R mRNA and the luciferase expression under control of the -2590 promoter fragment were reduced by IL-6 in hepatocytes. Copyright 2000 Academic Press.},
  language  = {en}
}
@article{NeuschaeferRubePatheNeuschaeferRubeHippenstieletal.2013,
  author    = {Neusch{\"a}fer-Rube, Frank and Pathe-Neusch{\"a}fer-Rube, A. and Hippenstiel, S. and Kracht, M. and P{\"u}schel, Gerhard Paul},
  title     = {NF-kB-dependent IL-8 induction by prostaglandin EP2 receptors EP1 and EP4},
  series = {British journal of pharmacology : journal of The British Pharmacological Society},
  volume    = {168},
  journal   = {British journal of pharmacology : journal of The British Pharmacological Society},
  number    = {3},
  publisher = {Wiley-Blackwell},
  address   = {Hoboken},
  issn      = {0007-1188},
  doi       = {10.1111/j.1476-5381.2012.02182.x},
  pages     = {704 -- 717},
  year      = {2013},
  abstract  = {Background and Purpose Recent studies suggested a role for PGE2 in the expression of the chemokine IL-8. PGE2 signals via four different GPCRs, EP1-EP4. The role of EP1 and EP4 receptors for IL-8 induction was studied in HEK293 cells, overexpressing EP1 (HEK-EP1), EP4 (HEK-EP4) or both receptors (HEK-EP1 + EP4). Experimental Approach IL-8 mRNA and protein induction and IL-8 promoter and NF-?B activation were assessed in EP expressing HEK cells. Key Results In HEK-EP1 and HEK-EP1 + EP4 but not HEK or HEK-EP4 cells, PGE2 activated the IL-8 promoter and induced IL-8 mRNA and protein synthesis. Stimulation of HEK-EP1 + EP4 cells with an EP1-specific agonist activated IL-8 promoter and induced IL-8 mRNA and protein, whereas a specific EP4 agonist neither activated the IL-8 promoter nor induced IL-8 mRNA and protein synthesis. Simultaneous stimulation of HEK- EP1 + EP4 cells with both agonists activated IL-8 promoter and induced IL-8 mRNA to the same extent as PGE2. In HEK-EP1 + EP4 cells, PGE2-mediated IL-8 promoter activation and IL-8 mRNA induction were blunted by inhibition of I?B kinase. PGE2 activated NF-?B in HEK-EP1, HEK-EP4 and HEK-EP1 + EP4 cells. In HEK-EP1 + EP4 cells, simultaneous activation of both receptors was needed for maximal PGE2-induced NF-?B activation. PGE2-stimulated NF-?B activation by EP1 was blocked by inhibitors of PLC, calcium-signalling and Src-kinase, whereas that induced by EP4 was only blunted by Src-kinase inhibition. Conclusions and Implications These findings suggest that PGE2-mediated NF-?B activation by simultaneous stimulation of EP1 and EP4 receptors induces maximal IL-8 promoter activation and IL-8 mRNA and protein induction.},
  language  = {en}
}
@article{FennekohlSchieferdeckerJungermannetal.1999,
  author    = {Fennekohl, Alexandra and Schieferdecker, Henrike L. and Jungermann, Kurt and P{\"u}schel, Gerhard Paul},
  title     = {Differential expression of prostanoid receptors in hepatocytes, Kupffer cells, sinusoidal endothelial cells and stellate cells of rat liver},
  issn      = {0168-8278},
  year      = {1999},
  abstract  = {BACKGROUND/AIMS: Prostanoids produced by nonparenchymal cells modulate the function of parenchymal and nonparenchymal liver cells during homeostasis and inflammation via eight classes of prostanoid receptors coupled to different G-proteins. Prostanoid receptor expression in parenchymal and nonparenchymal cells was studied in order to get a better insight into the complex prostanoid-mediated intrahepatic signaling network. METHODS: RNA was isolated from freshly purified parenchymal and nonparenchymal rat liver cells and the mRNA level of all eight prostanoid receptor classes was determined by newly developed semiquantitative reverse transcription-polymerase chain reaction protocols. RESULTS: The mRNAs for the prostanoid receptors were differentially expressed. Hepatocytes were the only cell type which contained the mRNA of the Gq-linked prostaglandin F2alpha receptor; they were devoid of any mRNA for the Gs-linked prostanoid receptors. Kupffer cells possessed the largest amount of mRNA for the Gs-linked prostaglandin E2 receptor subtype 2. Endothelial cells expressed high levels of mRNA for the Gq-linked thromboxane receptor and medium levels of mRNA for the Gs-linked prostacyclin receptor, while stellate cells had the highest levels of mRNA for the prostacyclin receptor. The mRNAs for the Gq-linked prostaglandin E2 receptor subtype 1 and the Gi-linked prostaglandin E2 receptor subtype 3 were expressed in hepatocytes and all nonparenchymal cell types at similar high levels, whereas the mRNA of the Gs-linked prostaglandin D2 receptor was expressed in all nonparenchymal cells at very low levels. CONCLUSIONS: In hepatocytes the prostaglandin F2alpha receptor can mediate an increase in glucose output via an increase of intracellular InsP3 while cAMP-dependent glucose output can be inhibited via the subtype 3 prostaglandin E2 receptor. The subtype 2 prostaglandin E2 receptor can restrain the inflammatory response of Kupffer cells via an increase in intracellular cAMP The thromboxane receptor and the prostacyclin receptor in sinusoidal endothelial and the prostacyclin receptor in stellate cells may be involved in the regulation of sinusoidal blood flow and filtration.},
  language  = {en}
}
@article{SchieferdeckerPestelPuescheletal.1999,
  author    = {Schieferdecker, Henrike L. and Pestel, Sabine and P{\"u}schel, Gerhard Paul and G{\"o}tze, Otto},
  title     = {Increase by anaphylatoxin C5a of glucose output in perfused rat liver via prostanoids derived from nonparenchymal cells : direct action of prostaglandins and indirect action of thromboxane A(2) on hepatocytes},
  year      = {1999},
  abstract  = {In the perfused rat liver the anaphylatoxin C5a enhanced glucose output, reduced flow, and elevated prostanoid overflow. Because hepatocytes (HCs) do not express C5a receptors, the metabolic C5a actions must be indirect, mediated by e.g. prostanoids from Kupffer cells (KCs) and hepatic stellate cells (HSCs), which possess C5a receptors. Surprisingly, the metabolic C5a effects were not only impaired by the prostanoid synthesis inhibitor, indomethacin, but also by the thromboxane A(2) (TXA(2)) receptor antagonist, daltroban, even though HCs do not express TXA(2) receptors. TXA(2) did not induce prostaglandin (PG) or an unknown factor release from KCs or sinusoidal endothelial cells (SECs), which express TXA(2) receptors, because (1) daltroban did neither influence the C5a-induced release of prostanoids from cultured KCs nor the C5a-dependent activation of glycogen phosphorylase in KC/HC cocultures and because (2) the TXA(2) analog, U46619, failed to stimulate prostanoid release from cultured KCs or SECs or to activate glycogen phosphorylase in KC/HC or SEC/HC cocultures. In the perfused liver, Ca(2+)-deprivation inhibited not only flow reduction but also glucose output elicited by C5a to similar extents as daltroban. Similarly, in the absence of extracellular Ca(2+), flow reduction and glucose output induced by U46619 were almost completely prevented, whereas glucose output induced by the directly acting PGF(2alpha) was only slightly lowered. Thus, in the perfused rat liver PGs released after C5a- stimulation from KCs and HSCs directly activated glycogen phosphorylase in HCs, and TXA(2) enhanced glucose output indirectly mainly by causing hypoxia as a result of flow reduction.},
  language  = {en}
}
@article{FennekohlLucasPueschel2000,
  author    = {Fennekohl, Alexandra and Lucas, Maria and P{\"u}schel, Gerhard Paul},
  title     = {Induction by interleukin 6 of G(s)-coupled prostaglandin E(2) receptors in rat hepatocytes mediating a prostaglandin e(2)-dependent inhibition of the hepatocyte's acute phase response},
  year      = {2000},
  abstract  = {Prostanoids, that are released from nonparenchymal liver cells in response to proinflammatory stimuli, are involved in the regulation of hepatic functions during inflammation. They exert their effects on their target cells via heptahelical receptors in the plasma membrane. For the 5 prostanoids prostaglandin E(2) (PGE(2)), prostaglandin F(2alpha), prostaglandin D(2) (PGD(2)), prostacyclin, and thromboxane A(2) there exist 8 receptors that are coupled to different heterotrimeric G proteins. These receptors are expressed differentially in the 4 principal liver cell types, i.e., hepatocytes, Kupffer cells, sinusoidal endothelial cells, and hepatic stellate cells. It was intriguing, that the messenger RNA (mRNA) of none of the G(s)-coupled prostanoid receptors (DP-R, EP2-R, EP4-R, and IP-R) that can attenuate the inflammatory reaction were present in hepatocytes. The current study shows that the expression of the G(s)-coupled prostanoid receptors EP2-R, EP4-R, and DP-R, but not the IP-R, was efficiently and rapidly up-regulated by treatment of hepatocytes in vitro or rats in vivo with the key acute phase cytokine interleukin 6 (IL-6). In IL-6-treated hepatocytes PGE(2) in turn attenuated the IL-6-induced alpha(2)-macroglobulin formation via a cyclic adenosine monophosphate (cAMP)- dependent signal chain. The data indicate that an IL-6-mediated induction of the previously not expressed EP2-R and EP4- R on hepatocytes might establish a prostanoid-mediated feedback inhibition loop for the attenuation of the acute phase response.},
  language  = {en}
}
@article{PatheNeuschaeferRubeNeuschaeferRubeGenzetal.2015,
  author    = {Pathe-Neuschaefer-Rube, Andrea and Neuschaefer-Rube, Frank and Genz, Lara and P{\"u}schel, Gerhard Paul},
  title     = {Botulinum Neurotoxin Dose-Dependently Inhibits Release of Neurosecretory Vesicle-Targeted Luciferase from Neuronal Cells},
  series = {Alternatives to animal experimentation : ALTEX ; a journal for new paths in biomedical science},
  volume    = {32},
  journal   = {Alternatives to animal experimentation : ALTEX ; a journal for new paths in biomedical science},
  number    = {4},
  publisher = {Springer},
  address   = {Heidelberg},
  issn      = {1868-596X},
  pages     = {297 -- 306},
  year      = {2015},
  abstract  = {Botulinum toxin is a bacterial toxin that inhibits neurotransmitter release from neurons and thereby causes a flaccid paralysis. It is used as drug to treat a number of serious ailments and, more frequently, for aesthetic medical interventions. Botulinum toxin for pharmacological applications is isolated from bacterial cultures. Due to partial denaturation of the protein, the specific activity of these preparations shows large variations. Because of its extreme potential toxicity, pharmacological preparations must be carefully tested for their activity. For the current gold standard, the mouse lethality assay, several hundred thousand mice are killed per year. Alternative methods have been developed that suffer from one or more of the following deficits: In vitro enzyme assays test only the activity of the catalytic subunit of the toxin. Enzymatic and cell based immunological assays are specific for just one of the different serotypes. The current study takes a completely different approach that overcomes these limitations: Neuronal cell lines were stably transfected with plasmids coding for luciferases of different species, which were N-terminally tagged with leader sequences that redirect the luciferase into neuro-secretory vesicles. From these vesicles, luciferases were released upon depolarization of the cells. The depolarization-dependent release was efficiently inhibited by botulinum toxin in a concentration range (1 to 100 pM) that is used in pharmacological preparations. The new assay might thus be an alternative to the mouse lethality assay and the immunological assays already in use.},
  language  = {en}
}
@article{PatheNeuschaeferRubeNeuschaeferRubeHaasetal.2018,
  author    = {Pathe-Neusch{\"a}fer-Rube, Andrea and Neusch{\"a}fer-Rube, Frank and Haas, Gerald and Langoth-Fehringer, Nina and P{\"u}schel, Gerhard Paul},
  title     = {Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins},
  series = {Toxins},
  volume    = {10},
  journal   = {Toxins},
  number    = {9},
  publisher = {Molecular Diversity Preservation International (MDPI)},
  address   = {Basel},
  issn      = {2072-6651},
  doi       = {10.3390/toxins10090360},
  pages     = {1 -- 10},
  year      = {2018},
  abstract  = {Despite the implementation of cell-based replacement methods, the mouse lethality assay is still frequently used to determine the activity of botulinum toxin (BoNT) for medical use. One explanation is that due to the use of neoepitope-specific antibodies to detect the cleaved BoNT substrate, the currently devised assays can detect only one specific serotype of the toxin. Recently, we developed a cell-based functional assay, in which BoNT activity is determined by inhibiting the release of a reporter enzyme that is liberated concomitantly with the neurotransmitter from neurosecretory vesicles. In theory, this assay should be suitable to detect the activity of any BoNT serotype. Consistent with this assumption, the current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) was inhibited by BoNT-A and-C. Furthermore, this was also inhibited by BoNT-B and tetanus toxin to a lesser extent and at higher concentrations. In order to provide support for the suitability of this technique in practical applications, a dose-response curve obtained with a pharmaceutical preparation of BoNT-A closely mirrored the activity determined in the mouse lethality assay. In summary, the newly established cell-based assay may represent a versatile and specific alternative to the mouse lethality assay and other currently established cell-based assays.},
  language  = {en}
}
@article{SchraplauScheweNeuschaeferRubeetal.2015,
  author    = {Schraplau, Anne and Schewe, Bettina and Neusch{\"a}fer-Rube, Frank and Ringel, Sebastian and Neuber, Corinna and Kleuser, Burkhard and P{\"u}schel, Gerhard Paul},
  title     = {Enhanced thyroid hormone breakdown in hepatocytes by mutual induction of the constitutive androstane receptor (CAR, NR1I3) and arylhydrocarbon receptor by benzo[a]pyrene and phenobarbital},
  series = {Toxicology},
  volume    = {328},
  journal   = {Toxicology},
  publisher = {Elsevier},
  address   = {Clare},
  issn      = {0300-483X},
  doi       = {10.1016/j.tox.2014.12.004},
  pages     = {21 -- 28},
  year      = {2015},
  abstract  = {Xenobiotics may interfere with the hypothalamic-pituitary-thyroid endocrine axis by inducing enzymes that inactivate thyroid hormones and thereby reduce the metabolic rate. This induction results from an activation of xeno-sensing nuclear receptors. The current study shows that benzo[a]pyrene, a frequent contaminant of processed food and activator of the arylhydrocarbon receptor (AhR) activated the promoter and induced the transcription of the nuclear receptor constitutive androstane receptor (CAR, NR1I3) in rat hepatocytes. Likewise, phenobarbital induced the AhR transcription. This mutual induction of the nuclear receptors enhanced the phenobarbital-dependent induction of the prototypic CAR target gene Cyp2b1 as well as the AhR-dependent induction of UDP-glucuronosyltransferases. In both cases, the induction by the combination of both xenobiotics was more than the sum of the induction by either substance alone. By inducing the AhR, phenobarbital enhanced the benzo[a]pyrene-dependent reduction of thyroid hormone half-life and the benzo[a]pyrene-dependent increase in the rate of thyroid hormone glucuronide formation in hepatocyte cultures. CAR ligands might thus augment the endocrine disrupting potential of AhR activators by an induction of the AhR. (C) 2014 Elsevier Ireland Ltd. All rights reserved.},
  language  = {en}
}
@article{PatheNeuschaeferRubeNeuschaeferRubePueschel2005,
  author    = {Pathe-Neusch{\"a}fer-Rube, Andrea and Neusch{\"a}fer-Rube, Frank and P{\"u}schel, Gerhard Paul},
  title     = {Role of the ERC motif in the proximal part of the second intracellular loop and the C-terminal domain of the human prostaglandin F2alpha receptor (hFP-R) in G-protein coupling control},
  year      = {2005},
  abstract  = {The human FP-R (F2alpha prostaglandin receptor) is a Gq-coupled heptahelical ectoreceptor, which is of significant medical interest, since it is a potential target for the treatment of glaucoma and preterm labour. On agonist exposure, it mediates an increase in intracellular inositol phosphate formation. Little is known about the structures that govern the agonist-dependent receptor activation. In other prostanoid receptors, the C-terminal domain has been inferred in the control of agonist-dependent receptor activation. A DRY motif at the beginning of the second intracellular loop is highly conserved throughout the G-protein-coupled receptor family and appears to be crucial for controlling agonist-dependent receptor activation. It is replaced by an ERC motif in the FP-R and no evidence for the relevance of this motif in ligand-dependent activation of prostanoid receptors has been provided so far. The aim of the present study was to elucidate the potential role of the C-terminal domain and the ERC motif in agonist-controlled intracellular signalling in FP-R mutants generated by site-directed mutagenesis. It was found that substitution of the acidic Glu(132) in the ERC motif by a threonine residue led to full constitutive activation, whereas truncation of the receptor's C-terminal domain led to partial constitutive activation of all three intracellular signal pathways that had previously been shown to be activated by the FP-R, i.e. inositol trisphosphate formation, focal adhesion kinase activation and T-cell factor signalling. Inositol trisphosphate formation and focal adhesion kinase phosphorylation were further enhanced by ligand binding in cells expressing the truncation mutant but not the E132T (Glu132-->Thr) mutant. Thus C-terminal truncation appeared to result in a receptor with partial constitutive activation, whereas substitution of Glu132 by threonine apparently resulted in a receptor with full constitutive activity.},
  language  = {en}
}
@article{BoeerFennekohlPueschel2003,
  author    = {B{\"o}er, Ulrike and Fennekohl, Alexandra and P{\"u}schel, Gerhard Paul},
  title     = {Sensitization by interleukin-6 of rat hepatocytes to tumor necrosis factor alpha-induced apoptosis},
  year      = {2003},
  abstract  = {BACKGROUND/AIMS: Tumor necrosis factor (TNF) elicits hepatocyte apoptosis in toxic liver injury and is also central in hepatocyte proliferation after partial hepatectomy. In both circumstances interleukin (IL)-6 levels are also elevated. In mouse liver IL-6 attenuated Fas receptor-mediated apoptosis indicating its interference with pro-apoptotic signal chains. It was, therefore, the aim to examine the modulation by IL-6 of TNFalpha-induced apoptosis in rat hepatocytes. METHODS: Primary rat hepatocytes were treated with IL-6 prior to induction of apoptosis with TNFalpha/ actinomycin D or anti-Fas antibody M-20. Apoptosis was detected by determination of caspase-3 activation and bisbenzimide staining of condensed nuclei. Expression of TNFalpha receptors was analyzed by semi-quantitative polymerase chain reaction and ligand binding studies with [125I]-TNFalpha. RESULTS: IL-6 treatment doubled TNFalpha/actinomycin D- induced caspase-3 activity and significantly enhanced chromatin condensation. By contrast IL-6 inhibited Fas-induced increase in caspase-3 activity by 45\% and significantly reduced chromatin condensation. IL-6 increased the mRNA level of TNF-R1 1.35-fold and augmented cell surface binding of [125I]-TNFalpha 3-fold. The latter and TNFalpha-mediated caspase activation was attenuated by prostaglandin E(2). CONCLUSIONS: IL-6 - in contrast to its anti-apoptotic modulation of the Fas-induced pathway - exerted a pro-apoptotic effect on the TNFalpha/actinomycin D-induced apoptosis by increasing the number of TNF-R on hepatocytes.},
  language  = {en}
}