Frank Neuschäfer-Rube

Molecular cloning and characterization of the four rat prostaglandin E2 prostanoid receptor subtypes

We have characterized the rat prostanoid EP1, EP2, EP3alpha and EP4 receptor subtypes cloned from spleen, hepatocyte and/or kidney cDNA libraries. Comparison of the deduced amino acid sequences of the rat EP receptors with their respective homologues from mouse and human showed 91% to 98% and 82% to 89% identity, respectively. Radioreceptor binding assays and functional assays were performed on EP receptor expressing human embryonic kidney (HEK) 293 cells. The KD values obtained with prostaglandin E2 for the prostanoid receptor subtypes EP1, EP2, EP3alpha and EP4 were approximately 24, 5, 1 and 1 nM, respectively. The rank order of affinities for various prostanoids at the prostanoid receptor subtypes EP2, EP3alpha and EP4 receptor subtypes was prostaglandin E2 = prostaglandin E1 > iloprost > prostaglandin F2alpha > prostaglandin D2 > U46619. The rank order at the prostanoid EP1 receptor was essentially the same except that iloprost had the highest affinity of the prostanoids tested. Of the selective ligands, butaprost was selective for prostanoid EP2, M&B28767 and sulprostone were selective for EP3alpha and enprostil displayed dual selectivity, interacting with both prostanoid receptor subtypes EP1 and EP3alpha. All four receptors coupled to their predominant signal transduction pathways in HEK 293 cells. Notably, using a novel aequorin luminescence assay to monitor prostanoid EP1 mediated increases in intracellular calcium, both iloprost and sulprostone were identified as partial agonists. Finally, by Northern blot analysis EP3 transcripts were most abundant in liver and kidney whereas prostanoid EP2 receptor mRNA was expressed in spleen, lung and testis and prostanoid EP1 receptor mRNA transcripts were predominantly expressed in the kidney. The rat prostanoid EP1 probes also detected additional and abundant transcripts present in all the tissues examined. These were found to be related to the expression of a novel protein kinase gene and not the prostanoid EP1 gene [Batshake, B., Sundelin, J., 1996. The mouse genes for the EP1 prostanoid receptor and the novel protein kinase overlap. Biochem. Biophys. Res. Commun. 227. 1329-1333].

Aggravation by prostaglandin E2 of interleukin-6-dependent insulin resistance in hepatocytes.

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 E2 (PGE2) 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 PGE2 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 PGE2 while insulin receptor autophosphorylation was barely affected. IL‐6 but not PGE2 induced suppressors of cytokine signaling (SOCS3). PGE2 but not IL‐6 activated extracellular signal‐regulated kinase 1/2 (ERK1/2) persistently. Inhibition of ERK1/2 activation by PD98059 abolished the PGE2‐dependent but not the IL‐6‐dependent attenuation of insulin signaling. In HepG2 cells expressing a recombinant EP3‐receptor, PGE2 pre‐incubation activated ERK1/2, caused a serine phosphorylation of insulin receptor substrate 1 (IRS1), and reduced the insulin‐dependent Akt‐phosphorylation. Conclusion: PGE2 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, PGE2 may synergize with IL‐6, which interrupted the insulin receptor signal chain, principally by an induction of SOCS, namely SOCS3. (HEPATOLOGY 2009.)

Molecular cloning and expression of a prostaglandin E2 receptor of the EP3β subtype from rat hepatocytes

Rat hepatocytes have previously been reported to possess prostaglandin E2 receptors of the EP3‐type (EP3‐receptors) that inhibit glucagon‐stimulated glycogenolysis by decreasing cAMP. Here, the isolation of a functional EP3β receptor cDNA clone from a rat hepatocyte cDNA library is reported. This clone can be translated into a 362‐amino‐acid protein, that displays over 95% homology to the EP3β receptor from mouse mastocytoma. The amino‐ and carboxy‐terminal region of the protein are least conserved. Transiently transfected HEK 293 cells expressed a single binding site for PGE2 with an apparent K d of 15 nM. PGE2 > PGF2α > PGD2 competed for [3H]PGE2 binding sites as did the EP3 receptor agonists M&B 28767 = sulprostone > misoprostol but not the EP1 receptor antagonist SC 19220. In stably transfected CHO cells M&B 28767 > sulprostone = PGE2 > misoprostol > PGF2α inhibited the forskolin‐elicited cAMP formation. Thus, the characteristics of the EP3β receptor of rat hepatocytes closely resemble those of the EP3β receptor of mouse mastocytoma.

Possible role for ligand binding of histidine 81 in the second transmembrane domain of the rat prostaglandin F2α receptor

For the five principal prostanoids PGD2, PGE2, PGF2α, 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 PGF2α receptor, which is conserved among all PGF2α 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 PGF2α 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 PGF2α. Wild type receptor and H81Q showed a shallow pH optimum for PGF2α binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF2α 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 PGF2α 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 prostaglandins carboxyl group but rather as a hydrogen bond donor.

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

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.

THE C-TERMINAL DOMAIN OF THE GS-COUPLED EP4 RECEPTOR CONFERS AGONIST-DEPENDENT COUPLING CONTROL TO GI BUT NO COUPLING TO GS IN A RECEPTOR HYBRID WITH THE GI-COUPLED EP3 RECEPTOR

Prostaglandin E2 receptors (EPR) belong to the family of G‐protein‐coupled receptors with 7 transmembrane domains. They form a family of four subtypes, which are linked to different G‐proteins. EP1R are coupled to Gq, EP2 and EP4R to Gs and EP3R to Gi. Different C‐terminal splice variants of the bovine EP3R are coupled to different G‐proteins. A mouse EP3R whose C‐terminal domain had been partially truncated no longer showed agonist‐induced Gi‐protein activation and was constitutively active. In order to test the hypothesis that the C‐terminal domain confers coupling specificity of the receptors on the respective G‐proteins, a cDNA for a hybrid rEP3hEP4R, containing the N‐terminal main portion of the Gi‐coupled rat EP3βR including the 7th transmembrane domain and the intracellular C‐terminal domain of the Gs‐coupled human EP4R, was generated by PCR. HEK293 cells transiently transfected with the chimeric rEP3hEP4R cDNA expressed a plasma membrane PGE2 binding site with a slightly lower K d value for PGE2 but an identical binding profile for receptor‐specific ligands as cells transfected with the native rat EP3βR. In HepG2 cells stably transfected with the chimeric rEP3hEP4R cDNA PGE2 did not increase cAMP formation characteristic of Gs coupling but attenuated the forskolin‐stimulated cAMP synthesis characteristic of Gi coupling. This effect was inhibited by pre‐treatment of the cells with pertussis toxin. Thus, the hybrid receptor behaved both in binding and in functional coupling characteristics as the native rat EP3βR. Apparently, the intracellular C‐terminal domain did not confer coupling specificity but coupling control, i.e. allowed a signalling state of the receptor only with agonist binding.

The human longevity gene homolog INDY and interleukin‐6 interact in hepatic lipid metabolism

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. (Hepatology 2017;66:616–630).

NF‐κB‐dependent IL‐8 induction by prostaglandin E2 receptors EP1 and EP4

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).

Identification by site-directed mutagenesis of amino acids contributing to ligand-binding specificity or signal transduction properties of the human FP prostanoid receptor

Prostanoid receptors belong to the class of heptahelical plasma membrane receptors. For the five prostanoids, eight receptor subtypes have been identified. They display an overall sequence similarity of roughly 30%. Based on sequence comparison, single amino acids in different subtypes of different species have previously been identified by site-directed mutagenesis or in hybrid receptors that appear to be essential for ligand binding or G-protein coupling. Based on this information, a series of mutants of the human FP receptor was generated and characterized in ligand-binding and second-messenger-formation studies. It was found that mutation of His-81 to Ala in transmembrane domain 2 and of Arg-291 to Leu in transmembrane domain 7, which are putative interaction partners for the prostanoids carboxyl group, abolished ligand binding. Mutants in which Ser-263 in transmembrane domain 6 or Asp-300 in transmembrane domain 7 had been replaced by Ala or Gln, respectively, no longer discriminated between prostaglandins PGF(2alpha) and PGD(2). Thus distortion of the topology of transmembrane domains 6 and 7 appears to interfere with the cyclopentane ring selectivity of the receptor. PGF(2alpha)-induced inositol formation was strongly reduced in the mutant Asp-300Gln, inferring a role for this residue in agonist-induced G-protein activation.

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

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.