To Örebro University

Rapid, androgen actions initiated at the cell surface have been reported in a variety of vertebrate cells, including several macrophage and prostate cancer cell lines that lack the nuclear androgen receptor. However, until recently the identity of the novel membrane androgen receptor (mAR) mediating these nonclassical androgen actions remained unknown. In 2014, a novel mAR unrelated to nuclear androgen receptors was identified in Atlantic croaker ovaries as the zinc transporter protein, ZIP9. ZIP9 is one of the 14 members of the ZIP (ZRT-and Irt-like Protein, SLC39A) family that regulates zinc homeostasis by transporting zinc across cell and organelle membranes into the cytoplasm. Zinc is a micronutrient critical for the maintenance of physiological and cellular processes, such as development, growth, protein assembly and activity, signaling, and apoptosis. Both croaker ZIP9 and human ZIP9 proteins have the binding characteristics of high affinity, specific mARs, and are coupled to G proteins. Testosterone induces apoptosis through ZIP9 in croaker granulosa cells and in human breast and prostate cancer cells by a unique mechanism involving increases in both second messengers and intracellular free zinc concentrations. ZIP9 also mediates testosterone regulation of tight junction formation in Sertoli cells and nonclassical testosterone signaling in spermatogenic cells. ZIP9 acts through several signal transduction pathways, a stimulatory G protein (Gs) in granulosa cells, an inhibitory one (Gi) in cancer cells, and a Gq11 one (Gnα11) in spermatogenic cells. ZIP9 has a very broad tissue distribution and is predicted to mediate numerous and diverse nonclassical androgen actions in vertebrates.

Metals released into the environment continue to be of concern for human health. However, risk assessment of metal exposure is often based on total metal levels and usually does not take bioavailability data, metal speciation or matrix effects into consideration. The continued development of biological endpoint analyses are therefore of high importance for improved eco-toxicological risk analyses. While there is an on-going debate concerning synergistic or additive effects of low-level mixed exposures there is little environmental data confirming the observations obtained from laboratory experiments. In the present study we utilized qRT-PCR analysis to identify key metal response genes to develop a method for biomonitoring and risk-assessment of metal pollution. The gene expression patterns were determined for juvenile zebrafish exposed to waters from sites down-stream of a closed mining operation. Genes representing different physiological processes including stress response, inflammation, apoptosis, drug metabolism, ion channels and receptors, and genotoxicity were analyzed. The gene expression patterns of zebrafish exposed to laboratory prepared metal mixes were compared to the patterns obtained with fish exposed to the environmental samples with the same metal composition and concentrations. Exposure to environmental samples resulted in fewer alterations in gene expression compared to laboratory mixes. A biotic ligand model (BLM) was used to approximate the bioavailability of the metals in the environmental setting. However, the BLM results were not in agreement with the experimental data, suggesting that the BLM may be overestimating the risk in the environment. The present study therefore supports the inclusion of site-specific biological analyses to complement the present chemical based assays used for environmental risk-assessment.

Rapid, cell surface-initiated, pregenomic androgen actions have been described in various vertebrate cells, but the receptors mediating these actions remain unidentified.We report here cloning and expression of a cDNA from Atlantic croaker (Micropogonias undulatus) ovaries encoding a 33kDa, 7-transmembrane protein with binding and signaling characteristics of a membrane androgen receptor (mAR) that is unrelated to any previously described steroid receptor. Instead croaker mAR has 81–93 % amino acid sequence identity with zinc transporter ZIP9 (SLC39A9) subfamily members, indicating it is a ZIP9 protein. Croaker ZIP9 is expressed in gonadal tissues and in brain, and is upregulated in the ovary by reproductive hormones. ZIP9 protein is localized to plasma membranes of croaker granulosa cells and human breast cancer (SKBR-3) cells stably transfected with ZIP9. Recombinant croaker ZIP9 has a high affinity (Kd 12.7 nM), limited capacity (Bmax 2.8nM/mgprotein), displaceable, single binding site specific for androgens, characteristic of steroid receptors. Testosterone activates a stimulatory G protein coupled to ZIP9, resulting in increased cAMP production. Testosterone promotes serum starvation-induced cell death and apoptosis in transfected cells and in croaker ovarian follicle cells that is associated with rapid increases in intracellular free zinc concentrations, suggesting an involvement of zinc in this nonclassical androgen action to promote apoptosis. These responses to testosterone are abrogated by treatment with ZIP9 siRNA. The results provide the first evidence that zinc transporter proteins can function as specific steroid membrane receptors and indicate a previously unrecognized signaling pathway mediated by steroid receptors involving alterations in intracellular zinc.

Recently we discovered a cDNA in teleost ovarian follicle cells belonging to the zinc transporter ZIP9 subfamily encoding a protein with characteristics of a membrane androgen receptor (mAR). Here we demonstrate that human ZIP9 expressed in MDA-MB-468 breast cancer cells and stably overexpressed in human prostate cancer PC-3 cells (PC-3-ZIP9) also displays the ligand binding and signaling characteristics of a specific, high-affinity mAR. Testosterone treatment of MDA-MB-468 and PC-3-ZIP9 cells caused activation of G proteins and second messenger pathways as well as increases in intracellular free zinc concentrations that were accompanied by induction of apoptosis. [3H]-Testosterone binding and these responses were abrogated in MDA-MB-468 cells after ZIP9 siRNA treatment and absent in PC-3 cells transfected with empty vector, confirming that ZIP9 functions as an mAR. Testosterone treatment caused upregulation of proapoptotic genes Bax, p53 and JNK in both cell lines and increased expression of Bax, Caspase 3 and cytochrome C proteins. Treatment with a zinc chelator (TPEN) or a MAP kinase inhibitor (PD98059) blocked testosterone induced increases in Bax, p53 and JNK mRNA expression. The results suggest that both androgen signaling and zinc transporter functions of ZIP9 mediate testosterone promotion of apoptosis. ZIP9 is widely expressed in human tissues and upregulated in malignant breast and prostate tissues, suggesting it is a potential therapeutic target for treating breast and prostate cancers. These results provide the first evidence for a mechanism mediated by a single protein through which intracellular steroidandzinc signaling pathways interact to regulate physiological functions in mammalian cells.

Recent studies by several research groups have shown that G protein estrogen receptor-1 (GPER) formerly known as GPR30, mediates 17beta-estradiol (E2) activation of signal transduction pathways in a variety of human cancer cells and displays E2 binding typical of a membrane estrogen receptor. However, the importance of GPER as an estrogen receptor has been questioned by Otto and co-workers. Some of the pitfalls in investigating the functions of recombinant steroid membrane receptors that may explain the negative results of these investigators are discussed. The characteristics of GPER have also been investigated in a teleost fish, Atlantic croaker, where it has been shown to mediate E2 inhibition of oocyte maturation. Investigations on newly discovered homologous proteins from distantly related vertebrate groups are valuable for determining their fundamental, evolutionarily conserved functions. Therefore, the functions of croaker and human GPERs were compared. The comparisons show that croaker and human GPER have very similar estrogen binding characteristics, typical of estrogen membrane receptors, and activate the same estrogen signaling pathways via stimulatory G proteins (Gs) resulting in increased cAMP production. These results suggest that the estrogen binding and estrogen signaling functions of GPER arose early in vertebrate evolution, prior to the divergence of the teleosts from the tetrapods, more than 200 million years ago. The finding that estrogen membrane signaling through GPER has been conserved for such a long period in two distantly related vertebrate groups, mammals and fish, suggests that this is a fundamental function of GPER in vertebrates, and likely its major physiological role.

Spiggin is an adhesive glycoprotein produced in the kidney of sticklebacks during the breeding season and is subsequently secreted into the urinary bladder from where it is employed for nest building. Since the production of the protein has been shown to be under androgenic control, spiggin has been suggested to be a useful biomarker for androgenic substances in the environment. In this study, two polyclonal spiggin antibodies based on synthetic peptides and one polyclonal antibody directed against native spiggin have been characterized. The antibodies ability to identify spiggin was investigated by quantitative immunoassay. For both peptide antibodies the quantification range was determined to be between 1 and 80 ng spiggin and determination of renal spiggin levels from immature and mature males displayed a 15-fold increase in total spiggin content of the kidney resulting in a 6-fold increase in male kidney weight due to hypertrophy. The kidney somatic index (KSI) was found to correlate well with the total renal spiggin content and therefore it appears that KSI in sticklebacks could be used as an initial method to identify substances displaying androgenic effects. Furthermore, western blot analysis revealed that the polyclonal antibodies recognize different spiggin isoforms and that spiggin can be detected in the urinary bladder and kidney of both males and female sticklebacks. In order to develop a quantitative detection method for native spiggin it is necessary to produce a standard that can be used in a bioassay. Due to the adhesive and polymerization characteristics of spiggin the protein is difficult to use as a standard in bioassays. So far spiggin has been shown to exist in at least 14 isoforms, all of which contain polymerization domains. To overcome the solubility problem we have produced recombinant spiggin gamma, with only one polymerization domain, that can be expressed in E. coli. Western blot analysis demonstrated that the polyclonal antibodies were able to detect recombinant spiggin gamma protein in bacterial cell lysate, suggesting that it may be developed into a useful source of standard spiggin to be used for quantitative determination of androgen induced spiggin production in sticklebacks.

Background: Reported incidences of prostate cancer and masculinization of animals indicate a release of compounds with androgenic properties into the environment. Large numbers of environmental pollutants have been screened to identify such compounds; however, not until recently was 1,2-dibromo-4-(1,2-dibromoethyl)cyclohex​ane (TBECH) identified as the first potent activator of the human androgen receptor (hAR). TBECH has been found in beluga whales and bird eggs and has also been found to be maternally transferred in zebrafish.

Objectives: In the present study we investigated interaction energies between TBECH diastereomers (α, β, γ, and δ) and the hAR, and their ability to activate the receptor and induce prostate-specific antigen (PSA) expression in vitro.

Methods: We performed computational modeling to determine interaction energies between the ligand and the AR ligand-binding site, and measured in vitro competitive binding assays for AR by polarization fluorometry analysis. We used enzyme-linked immunosorbent assays to determine PSA activity in LNCaP and HepG2 cells.

Results: We found the γ and δ diastereomers to be more potent activators of hAR than the α and β diastereomers, which was confirmed in receptor binding studies. All TBECH diastereomers induced PSA expression in LNCaP cells even though the AR present in these cells is mutated (T877A). Modeling studies of LNCaP AR revealed that TBECH diastereomers bound to the receptor with a closer distance to the key amino acids in the ligand-binding domain, indicating stronger binding to the mutated receptor.

Conclusions: The present study demonstrates the ability of TBECH to activate the hAR, indicating that it is a potential endocrine disruptor.

The identities of the membrane receptors mediating the majority of rapid, cell surface-initiated, nongenomic (i.e. nonclassical) steroid actions described to date are unclear. Two novel 7-transmembrane spanning proteins, representing two distinct classes of steroid membrane receptors, membrane progestin receptor alpha (mPRalpha) and a membrane estrogen receptor (mER), GPR30, have recently been identified in several vertebrate species. Evidence that both receptors activate G-proteins and function as G-protein coupled receptors (GPCRs) is briefly reviewed. New data on progestin actions on fish gametes suggest a widespread involvement of mPRalpha in oocyte maturation and sperm hyperactivity in this vertebrate group. Information on the second messenger pathways activated upon estrogen binding to a membrane estrogen receptor in croaker gonads and preliminary evidence for the presence of a GPR30-like protein in fish gonads are discussed. Finally, initial characterization of the ligand binding, G-protein activation and molecular size of a membrane androgen receptor (mAR) in croaker ovaries suggests the presence of a third unique steroid receptor in fish gonads that also may function as a GPCR.

The fate of chiral organochlorine compounds (OCs) and selected metabolites in exposed Arctic char (Salvelinus alpinus) was investigated. The contaminants alpha-hexachlorocyclohexane (alpha-HCH), cis-chlordane, 13C4-heptachlor, o,p'-DDT, and the atropisomeric chlorinated biphenyls (CBs) 95, 132, 136, 149, and 174 were solved in peanut oil and injected into the peritoneal cavity. The exposed fish were sampled three times during a five-week period, and the OC residues and detected metabolites (heptachlorexo-epoxide) were quantified in muscle and liver tissues by chiral and achiral gas chromatography-mass spectrometry and gas chromatography-electron-capture detection. Peak concentrations were reached after one to two weeks, and thereafter, the levels declined. At the end of the experiment, liver concentrations had decreased 76 to 92% relative to peak concentrations, whereas muscle concentrations showed a moderate decline (5-38%), with the exception of alpha-HCH (91%). Hydrophobicity and steric hindrance were shown to influence the assimilation process, and a significant linear relationship between the product of the steric hindrance coefficients and the inverse of the octanol-water partition coefficients (Kow) versus peak concentration was found for the CBs (r2 = 0.86, p = 0.02). The assimilation of the contaminants into muscle and liver tissues generally resulted in racemic mixtures, whereas elimination was enantioselective for alpha-HCH, cis-chlordane, o,p'-DDT, CB-132, and CB-136. The chiral heptachlor metabolite 13C4-heptachlor-exo-epoxide was formed in the fish. The enantiomeric composition of the formed metabolite indicated racemic formation, whereas the elimination process appeared to be enantioselective.

Membrane progestin receptors are involved in oocyte maturation in teleosts. However, the maturation-inducing steroid (MIS) does not appear to be conserved among species and several progestins may fulfill this function. So far, complete biochemical characterization has only been performed on a few species. In the present study we have characterized the membrane progestin receptor in Arctic char (Salvelinus alpinus) and show that the 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) receptor also binds several xenobiotics, thus rendering oocyte maturation sensitive to environmental pollutants. We identified a single class of high affinity (Kd, 13.8 +/- 1.1 nM), low capacity (Bmax, 1.6 +/- 0.6 pmol/g ovary) binding sites by saturation and Scatchard analyses. Receptor binding displayed rapid association and dissociation kinetics typical of steroid membrane receptors, with t1/2 s of less than 1 minute. The 17,20beta-P binding also displayed tissue specificity with high, saturable, and specific 17,20beta-P binding detected in ovaries, heart and gills while no specific binding was observed in muscle, brain or liver. Changes in 17,20beta-P binding during oocyte maturation were consistent with its identity as the oocyte MIS membrane receptor. Incubation of fully-grown ovarian follicles with gonadotropin induced oocyte maturation, which was accompanied by a five-fold increase in 17,20beta-P receptor binding. In addition, competition studies with a variety of steroids revealed that receptor binding is highly specific for 17,20beta-P, the likely maturation-inducing steroid (MIS) in Arctic char. The relative-binding affinities of all the other progestogens and steroids tested were less than 5% of that of 17,20beta-P for the receptor. Several ortho, para derivatives of DDT also showed weak binding affinity for the 17,20beta-P receptor supporting the hypothesis that xenobiotics may bind steroid receptors on the oocyte's surface and might thereby interfere with oocyte growth and maturation.