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ENDOCRINE AND DIABETES
Departments of Neurosciences (M.R., M.T.D., F.G., G.U.C., R.M.) and Psychiatry, Neurobiology, Pharmacology, and Biotechnology (M.L.T.) and Department of Endocrinology, Centre of Excellence for the Study of Damage to the Nervous and Endocrine Systems Produced by Environmental, Alimentary, and Pharmacological Agents, AmbiSEN (A.D., P.A., A.P., P.V., M.T.), University of Pisa, Pisa, Italy
Received September 7, 2006; accepted October 20, 2006.
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2 adrenergic receptor in COS-7 cells. Conversely, Aroclor 1254 inhibited completely forskolin activity in CHO-K1 cells but not in COS-7 cells. Furthermore, it did not prevent accumulation of cAMP as induced by NECA in A2a transfected cells. The analog of DDT, diphenylethylene, was inactive against bTSH-induced increase in cAMP in CHO-K1 cells stably transfected with the TSHr. We interpreted these results as indicating that DDT and possibly Aroclor 1254 may have an uncompetitive inverse agonist activity for the TSHr.
). Experimental evidence demonstrating the existence of this constitutive activity comes from studies on heterologous expression systems, where receptors are expressed with high densities or mutated in such a way as to make their effector pathways more efficiently activated. Certain ligands may decrease this basal effector activity for having an intrinsically negative efficacy or for producing responses opposite to those of the agonist. These ligands are termed negative antagonists or inverse agonists that are, in turn, classifiable as either competitive or noncompetitive (Costa and Cotecchia, 2005). Competitive inverse agonists are known to interact with orthosteric sites of the receptor, whereas uncompetitive inverse agonists interact with allosteric sites of the receptor.
One of major effects caused by administration of competitive or uncompetitive inverse agonists is the suppression of a natural agonist input. This is likely to have a minor negative effect, unless the receptor constitutive activity is so relevant that only antagonists with negative effect can extinguish its activity. Such cases include natural activating mutations (the list of which is increasing; see Birnbaumer, 1995; Parnot et al., 2002) but also virally encoded G-protein-coupled receptors (Rosenkilde et al., 2001; Smit et al., 2003).
The precise molecular mechanisms by which glycoprotein hormones act on receptor binding and activation are essentially unknown. As other glycoprotein hormone receptors, the thyrotropin (TSH) receptor (TSHr) has a large extracellular domain (ECD), accounting for approximately half the size of the receptor, and a seven transmembrane domain (TMD) (Vassart et al., 2004). High-affinity hormone binding occurs at the ECD (Seetharamaiah et al., 1994; Smits et al., 2003), whereas G-protein coupling takes place at the TMD. Unlike other glycoprotein hormone receptors, TSHr has a constitutive receptor activity (Cetani et al., 1996; Vlaeminck et al., 2002). TSHr has been found to be activated by a wide spectrum of gain-of-function mutations. Germline TSHr mutations cause hereditary toxic thyroid hyperplasia, whereas a number of somatic mutations have been found to be responsible for the majority of autonomous thyroid adenomas (Tonacchera et al., 1998).
The hypothesis that the ectodomain might exert an inhibitory effect on a noisy rhodopsin-like serpentine domain is supported by early data showing that TSHr-expressing cells, mildly treated with trypsin, undergo partial activation of the receptor (Van Sande et al., 1996). That such an effect may really occur was actually demonstrated by Zhang et al. (2000), who found that activation of the TSHr is secondary to "beheading" in N-terminal truncated mutants. Recently, Chen et al. (2003) have narrowed down the inhibitory effect of the ectodomain to its C terminus, indicating a cluster of lysine (Lys287, Lys290, Lys291) and arginine (Arg293) residues as a possible target for the functional effect of trypsin.
The fact that little is known about the mechanisms governing glycoprotein hormone action is partly due to lack of pharmacological tools capable of interacting directly with these receptors. During a screening of several environmental factors that alter thyroid homeostasis, Santini et al. (2003) found that some of these compounds disrupt TSHr function and were consequently defined as thyroid disruptors. Aroclor 1254 (a complex mixture of polychlorinated biphenyls) and the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) are two of the several compounds they tested that are thus further analyzed in this article. Both Aroclor 1254 and DDT were previously shown to prevent TSH-induced accumulation of cAMP in Chinese hamster ovary (CHO)-K1 cells stably transfected with TSHr. However, in spite of this inhibitory effect, they did not interfere with 125I-bovine TSH (bTSH) binding to the receptor, nor did they affect the immunometric assay of the TSH hormone. This latter is an assay devised to test TSH antigenicity; as such, it is generally taken to indicate the occurrence of possible conformational changes in the TSH protein. These results suggest that Aroclor 1254 and DDT may inhibit TSHr function by interacting with receptor sites other than the TSH recognition site and that their inhibitory activity does not occur via alteration of the TSH structure. In this study, we aimed at verifying whether the inhibitory activity exerted by these thyroid disruptors on thyroid homeostasis may occur via interaction with the TSH receptor.
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Materials and Methods |
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Eukaryotic Expression Vectors. Wild-type and truncated human TSH receptor cDNA inserted into the pcDNA3.1Zeo(–) vector were a kind gift from Dr. M. Szkudlinski (Trophogen, Inc., Rockville, MD). The truncated TSH receptor (TSHr trunk) was modified by deletion of the entire extracellular domain until amino acid Glu409, except for the 22 amino acids of the signal peptide that were joined to Glu409. Both the wild type and the TSHr trunk contain an HA epitope inserted at the N terminus adjacent to the signal peptide. The construction of wild-type and truncated TSH receptors has been described by Zhang et al. (2000). The construction of the TSHr(I486M) receptor mutant inserted in a pSVL expression vector has been described by Parma et al., (1993). The human adenosine A2a receptor cloned in a pcDNA3.1(–) vector was purchased from the Unité Mixte de Recherche cDNA Resource Center. The cDNA for type V adenyl cyclase, subcloned into a pXMD1 vector, was kindly provided by Dr. Z. Vogel (Weizman Institute of Sciences, Rehovot, Israel).
Cell Culture and Transfection. In this study, seven cell types were employed: African green monkey kidney (COS)-7 cells, CHO-K1 cells, and CHO-K1 cells stably transfected with the TSH, FSH, or LH wild-type receptor (CHO-TSHr, CHO-FSHr, and CHO-LHr) (Perret et al., 1990) and with the dopamine D1 (CHO-D1) or the adenosine A2a (CHO-A2a) receptor. COS-7 cells were transiently transfected with one of the following receptors: TSHr, TSHr trunk, TSHr(I486M), or adenosine A2a. COS-7 and CHO-K1 cells were incubated at 37°C in a humidified atmosphere (containing 5% CO2) in Dulbecco's modified Eagle's medium, which was supplemented with 5% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2% L-glutamine, and 1% nonessential amino acids. COS-7 cells were seeded at a density of 7.5 x 105 cells in 100-mm Petri dishes and, 24 h later, were transfected using the DEAE-dextran and chloroquine method. The amount of cDNA transfected was 2 µg for each plasmid. With the exception of one set of experiments (see below), A2a, TSHr, and TSHr trunk receptor plasmids were always cotransfected with adenylyl cyclase (AC)-V plasmid to increase the sensitivity of the cAMP assay. Twenty-four hours after transfection, COS-7 cells were seeded into 24-well plates at a density of 6.5 x 104 cells in each well. CHO-TSHr, CHO-FSHr, CHO-LHr, CHO-A2a, CHO-D1, and CHO-K1 cells were grown directly on 24-well plates (1.0 x 105 cells for each well).
cAMP Accumulation in Intact Cells. To measure cAMP accumulation in intact cells, two procedures were used: as a routine, an inexpensive separation column and, in selected experiments, a much more sensitive, although expensive, RIA method. The first assay was performed in quadruplicate. The experimental protocol for this kind of functional assay was different for CHO and COS-7 cells transfected with the TSHr trunk (see also Results). CHO-K1, CHO-TSHr, CHO-FSHr, CHO-LHr, CHO-A2a, CHO-D1, and COS-7 cells transfected with A2a or TSHr plus adenyl cyclase V (ACV) were preincubated for 2 h with 250 µl of medium supplemented with 1.25 µCi of [3H]adenine at +37°C in a humidified atmosphere. By the end of the preincubation period, cells were stimulated with bTSH, NECA, or forskolin at the indicated concentrations in the presence of the phosphodiesterase inhibitors Ro 20-1724 (0.5 mM) and IBMX (1 mM). Aroclor 1254 and DDT were added during the 2-h preincubation period and together with the bTSH hormone for 10 min at the indicated concentrations. When measuring constitutive activity, Aroclor 1254 and DDT exposure was limited to 2 h. Reaction was blocked with perchloric acid. A two-step column separation procedure was used to resolve [3H]cAMP (Johnson and Salomon, 1991).
In COS-7 cells transiently transfected with the TSHr trunk, assays were performed for a 2-h period in the presence of two phosphodiesterase inhibitors, Ro 20-1724 and IBMX, to detect the weak constitutive activity of this receptor. To equilibrate in the medium, Aroclor 1254 and DDT were added 30 min prior to the phosphodiesterase inhibitors to the beginning of the experiment, for a total of 2.5 h of incubation period. Data were expressed as [3H]cAMP/50 µg of protein. The average protein content of each sample was 50.3 ± 3.4 µg.
In another set of experiments, COS-7 cells transiently transfected with the TSHr or the TSHr(I486M) receptor (and no AC5 plasmid) were seeded at the concentration of 25,000 cells/well into 96-well plates containing 50 µl of medium and used for the assay the following day. Cells were washed with Hanks' balanced salt solution and incubated for 1 h at 37°C in hypotonic buffer. The cell lines were then exposed for 2 h to different concentrations of Aroclor 1254 and DDT. Assays were performed in triplicate for each experiment. Extracellular cAMP accumulation in the medium was determined by a inhouse RIA (Vitti et al., 1993), using a commercially available anti-cAMP antibody.
Extracellular cAMP was measured because cell lysate was shown to interfere with RIA. Vitti et al. (1993) have shown that extracellular and intracellular cAMP equilibrates rather quickly when cells are allowed to swell in hypotonic buffer.
Adenylate Cyclase Assay on Membranes. Confluent CHO-TSHr cells were scraped off the plate in a hypotonic solution (1 mM HEPES/2 mM EDTA) containing a cocktail of protease inhibitors (1.5 µM pepstatin, 4 µM leupeptin, 0.01 M aprotinin, and 500 µM phenylmethylsulfonyl fluoride) and centrifuged at 4°C for 15 min at 15,000g. CHO-TSHr membranes were then resuspended in 50 mM HEPES/NaOH, pH 7.4, plus the protease inhibitor cocktail and used in AC assay. AC activity was measured by monitoring the conversion of [
-32P]ATP to [-32P]cAMP, using a previously reported method (Johnson and Salomon, 1991). In brief, enzyme activity was routinely assayed in 100 µl of reaction mixture containing 50 mM HEPES/NaOH, pH 7.4, 0.1 mM ATP, 0.1 mM cAMP, 1 mM dithiothreitol, 2 mM MgCl2, 0.1 mg/ml bacitracin, 0.5 mg/ml creatine phosphate, 0.1 mg/ml creatine phosphokinase, 1 mM EGTA, 0.5 mM Ro 20-1724 and 1 mM IBMX (as phosphodiesterase inhibitors), 10 µM GTP, 1 µCi [-32P]ATP, and tested compounds. Incubation started by addition of CHO-TSHr membranes (20 µg of proteins) and carried out for 10 min at 30°C. The reaction was terminated by placing assay tubes in an ice bath followed by addition of 0.5 ml of stop solution containing 120 mM Zn(C2H3O2)2/[3H]cAMP (10,000 cpm/sample) and 0.5 ml of 144 mM Na2CO3. The total radiolabeled cAMP was purified on columns of Dowex 50 ion-exchange resin and alumina, as described. In these experiments, we tested the ability of both compounds, DDT (1–100 µM) and Aroclor (1–100 µM), to modulate AC activity in the absence or in the presence of TSH (1 mU/ml). The compounds were dissolved in dimethyl sulfoxide and diluted in HEPES buffer so that the final dimethyl sulfoxide concentration never exceeded 1% of the total volume.
Statistical Analysis. Analysis of variance + Scheffe's post hoc tests were employed to ascertain the significance of data belonging the same experimental group. In every experiment, the significance level was set at p < 0.05.
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), these two compounds should prevent cAMP accumulation in CHO-TSHr cells when administered together with bTSH (1 mU/ml per 10 min). At 10 µM concentration, DDT already began to inhibit cAMP activity, whereas at 100 µM concentration, the inhibition was 45.8%. In contrast, the effect of Aroclor 1254 was significant only at 100 µM with a 62.7% inhibition (Fig. 1B). The IC50 of DDT against bTSH (1 mU/ml)-stimulated cAMP accumulation was 17.5 ± 6.41 µM. The IC50 for Aroclor 1254 could not be determined with accuracy because dimethylsulfoxide, a solvent employed to dissolve the drug, affected TSHr activity at concentrations higher than 100 µM (see Materials and Methods). The inhibitory effect did not result from alteration of cell viability, as demonstrated by the fact that full bTSH activity could be completely restored following Aroclor 1254 and DDT washout (data not shown). As we mentioned in the introduction, TSHr has a high level of constitutive activity when expressed in eukaryotic cells. In CHO-TSHr cells, phosphodiesterase inhibitors lead to a marked cAMP increase in the absence of bTSH (Fig. 1C), but not in control CHO-K1 cells (Fig. 1C, insert). Both Aroclor 1254 and DDT inhibited significantly the TSHr constitutive activity, starting from a minimum concentration of 30 µM (Fig. 1C).
Effect of Aroclor 1254 and DDT on Forskolin-Induced cAMP Accumulation in CHO-TSHr and CHO-K1 Cells. To test the possibility that this inhibitory effect may be directly targeted to the TSHr and not to downstream effectors, we analyzed the inhibitory effect exerted by both compounds against 10 µM forskolin in CHO-TSHr and CHO-K1 cells. Results showed that application of these compounds to CHO-TSHr (Fig. 2A) and to CHO-K1 cells (Fig. 2B) prevented the forskolin-induced cAMP accumulation. Under these conditions, DDT effect was much more pronounced in CHO-TSHr cells than in CHO-K1 cells, whereas Aroclor 1254 prevented forskolin-induced cAMP accumulation to the same extent in both cell lines. We also noticed that forskolin application caused cAMP to increase in a substantially different manner in the two cell lines. To further investigate this difference, the dose-response curves of forskolin-induced cAMP accumulation were compared in the two cell lines. As it can be clearly seen in Fig. 2C, forskolin was much more effective in inducing cAMP accumulation in CHO-TSHr cells than in CHO-K1 cells, but it showed similar EC50s (1.21 ± 0.17 and 0.79 ± 0.20 µM in CHO-TSHr and CHO-K1 cells, respectively). It is interesting to note that forskolin was equally effective in CHO-A2a cells (Fig. 2C), although in this latter cell type, 100 µM DDT did not prevent any forskolin-induced cAMP accumulation (Fig. 2C, insert). These data suggest that TSHr (and A2a) may activate Gs synergistically with forskolin through their constitutive activity. If this is the case, the observed reduction in forskolin-induced cAMP accumulation in CHO-TSHr by DDT could then depend, at least partially, upon inhibition of the TSHr constitutive activity.
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Effect of Aroclor 1254 and DDT in CHO-TSHr Cells after Light Digestion with Trypsin. To establish which domain of the TSHr (ECD or TMD) may interact with DDT and possibly Aroclor 1254 to produce their inhibitory effects, we treated CHO-TSHr cells for 1 min with 0.2% trypsin at +25°C in the presence of phosphodiesterase inhibitors. Trypsin is expected to increase the TSHr constitutive activity by removing the inhibitory effect of the ECD. In line with data by Van Sande et al. (1996) and Chen et al. (2003), trypsin increased significantly the TSHr constitutive activity, but it did not reduce DDT and Aroclor 1254 inhibitory effects (Fig. 3). These data suggest, but do not prove, that DDT and possibly Aroclor 1254 might exert their inhibitory effect on the TMD of the TSHr. Trypsin digestion did not entirely abolish the bTSH stimulatory effect, indicating that a fraction of the ECDs were still functional (Fig. 3).
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), the experiment was repeated in cells transfected with the ACV alone. After 2 h of incubation with phosphodiesterase inhibitors, any induced cAMP variation was found to be too low to be significant (Fig. 6A, insert). Based on these observations, we can conclude that the amount of cAMP accumulated in COS-7 cells, cotransfected with TSHr trunk and ACV in the presence of phosphodiesterase inhibitors, is directly correlated with the TSHr constitutive activity.
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Effect of Aroclor 1254 and DDT in COS-7 Transiently Transfected with the TSHr Trunk and ACV or the Wild-Type TSHr and ACV. In cells transfected with the TSH trunk and ACV, both DDT and Aroclor 1254 were shown to reduce the constitutive activity of the TSH trunk (Fig. 5A). At 30 µM, Aroclor 1254 exerted the strongest inhibition, whereas at 100 µM, its inhibitory effect was consistently less pronounced. By contrast, DDT reached the maximal inhibition at 100 µM. Notice that in COS-7 cells transfected with wild-type TSHr and ACV, Aroclor 1254 and DDT exhibited the same inhibitory activity against bTSH (10 mU/ml)-induced cAMP accumulation, even though the effect was much stronger (Fig. 5B).
Effect of Aroclor 1254 and DDT in COS-7 Cells Transiently Transfected with the Wild-Type TSHr or the Mutant TSHr(I486M). In these experiments, we tested the effect of Aroclor 1254 and DDT on the constitutive activity of the wild-type TSHr and the mutant TSHr(I486M) (Parma et al., 1993) transiently transfected in COS-7 cells. In the TSHr(I486M) mutant, the inhibitory effect of the two thyroid disruptors could be better characterized due to its higher level of constitutive activity. Because ACV was not cotransfected to amplify the signal, cAMP accumulation was determined by the much more sensitive RIA method (see Materials and Methods). As shown in Fig. 5C, the constitutive activity of the TSHr(I486M) mutant was inhibited by Aroclor 1254 and DDT from a minimum concentration of 10 up to 100 µM. In contrast, the basal activity of the wild-type TSHr was inhibited by Aroclor 1254 at only 30 µM and by DDT at 30 and 100 µM.
Effect of Aroclor 1254 and DDT in COS-7 Transiently Transfected with ACV Alone and in CHO-K1 Cells. The effect of Aroclor 1254 and DDT was further tested on COS-7 cells transfected with ACV alone to see whether these two compounds could have any effect other than inhibit the TSHr. To our surprise, both compounds clearly stimulated cAMP accumulation (Fig. 6A), although the effect of Aroclor 1254 and DDT in COS-7 cells could be due to inhibition of phosphodiesterases above the level observed in the presence of Ro 20-1724 and IBMX. To exclude this possibility, we tested the effect of Aroclor 1254 and DDT in the absence of Ro 20-1724 and IBMX. As shown in the insert of Fig. 6A, in the absence of these phosphodiesterase inhibitors, Aroclor 1254 and DDT did not induce any cAMP accumulation over the basal level.
In CHO-K1 cells, at variance with COS-7 cells, Aroclor 1254 and DDT did not stimulate cAMP accumulation (Fig. 6B). The mechanism of cAMP accumulation, as caused by Aroclor 1254 and DDT, was not further investigated in this study because it was considered beyond the scope of the present project.
Effect of Aroclor 1254 and DDT on Forskolin- or NECA-Induced cAMP Accumulation in COS-7 Transiently Transfected with ACV Alone or with the Adenosine A2a Receptor Plus ACV, Respectively. These experiments were intended to test the specificity of the inhibitory effect exerted by Aroclor 1254 and DDT on the TSHr. To this purpose, we analyzed the effect of the two thyroid disruptors on cAMP accumulation induced by forskolin or NECA in COS-7 transiently transfected with ACV alone or the adenosine A2a receptor plus ACV, respectively. In sharp contrast with what was previously shown in CHO-K1 cells, Aroclor 1254 did not prevent forskolin (1 µM)-induced accumulation of cAMP in COS-7 cells transfected with ACV alone for up to 100 µM concentration. On the contrary, DDT showed a slight but significant inhibition at 100 µM (Fig. 7A). Neither compound inhibited the stimulation of cAMP accumulation as induced by NECA (1 µM) in COS-7 cells cotransfected with the adenosine A2a receptor and ACV (Fig. 7A).
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s-coupled receptors, the 2-adrenergic receptor endogenously expressed in COS-7 cells and the dopamine D1 receptor stably transfected in CHO-K1 cells (CHO-D1). As for A2a, DDT did not inhibit the isoproterenol (1 µM)- and dopamine (10 µM)-induced increase in cAMP in COS-7 and CHO-D1 cells, respectively.
Effect of Aroclor 1254 and DDT on hrFSH- and hCG-Dependent cAMP Accumulation in CHO-FSHr and CHO-LH Cells. These experiments were performed to verify whether or not Aroclor 1254 and DDT could exert their inhibitory effect on other members of the glycoprotein hormone receptor family. To this purpose, we tested the inhibitory effect of Aroclor 1254 and DDT on CHO-K1 cells stably transfected with the other two glycoprotein hormone receptors (CHO-FSHr and CHO-LHr). Both Aroclor 1254 and DDT inhibited the hrFSH (100 mU/ml)- and hCG (10 mU/ml)-induced cAMP accumulation in CHO-FSHr and CHO-LHr cells, respectively. Interestingly, DDT inhibition began at 1 µM concentration in CHO-FSHr cells (Fig. 8A) and at 0.1 nM concentration in CHO-LHr cells (Fig. 8B). Of the two compounds, DDT at the 100 µM concentration was most efficient by causing a 41 and 39% reduction in cAMP accumulation in CHO-FSHr and CHO-LHr cells, respectively.
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Effect of Diphenylethylene and Bisphenol A on bTSH-Dependent cAMP Accumulation in CHO-TSHr Cells. This last set of experiments was carried out to verify whether such a membrane perturbing effect as lipophilicity could account for the inhibitory effect of DDT. To this purpose, we tested two analogs of DDT for their inhibitory effect on bTSH-dependent cAMP accumulation in CHO-TSHr cells: diphenylethylene, which lacks the Cl substituents, and bisphenol A, in which the para-Cl atoms are substituted by hydroxyl (OH)-groups, whereas up to 100 µM, diphenylethylene did not inhibit cAMP accumulation, because induced by bTSH (1 mU/ml), bisphenol A was found to retain partly its inhibitory effect (Fig. 9).
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). The inhibitory effect of both these compounds was shown to occur via a noncompetitive mechanism, in view of the fact that they did not alter TSH binding to the receptor. Furthermore, they were also shown to exert a negative effect on forskolin-induced cAMP accumulation, suggesting that they may act downstream from the TSHr. In this article, we analyzed in detail how Aroclor 1254 and DDT exert their inhibitory activity, with the aim of defining their specific target(s). Initially, we reproduced the inhibitory effect of Aroclor 1254 and DDT in CHO-TSHr cells. Under these conditions, we showed that both compounds reduced the bTSH-induced accumulation of cAMP, with DDT exhibiting the highest inhibitory activity from 10 µM onwards, and Aroclor 1254 most efficiently inhibiting more than 60% at 100 µM. Likewise, Aroclor 1254 and DDT inhibited the TSHr constitutive activity by roughly the same extent. This conclusion is in line with what previously shown by Santini et al. (2003), although in our hands, DDT and Aroclor 1254 differ in the extent that they counteract forskolin-induced accumulation of cAMP in CHO-TSHr and CHO-K1 cells. Although DDT was much more effective against forskolin in CHO-TSHr cells (55% inhibition) compared with CHO-K1 cells (15%), Aroclor 1254 inhibition was equivalent in the two cell lines. This great difference in the inhibitory effect of DDT against forskolin is likely to depend upon the role played by TSHr. In fact, the extent of cAMP increase as induced by this AC activator is much higher in CHO-TSHr cells than in CHO-K1 cells, indicating that forskolin may act synergistically with the Gs protein when activated constitutively by TSHr. Synergism between Gs and forskolin has been shown in the activation of several adenylyl cyclase isoforms (Feinstein et al., 1991; Gao and Gilman, 1991; Taussig et al., 1994). Taken together, these data suggest quite strongly that much of the effects exerted by DDT on CHO-TSHr cells are due to the inhibition of the TSHr constitutive activity. This view is further corroborated by the observation that the constitutive activity of a highly effective TSHr(I486M) receptor mutant (Parma et al., 1993) was reduced by exposure to DDT. The absence of any DDT effect against forskolin-induced cAMP elevation in CHO-K1 cells transfected with the adenosine A2a receptor give further support to the view that TSHr is the real target of this compound and not any other downstream effectors. Data obtained in CHO-A2a confirm that the adenosine A2a receptor has constitutive activity as high as that of TSHr (Klinger et al., 2002). As expected, forskolin exposure in these cells induces a much more pronounced cAMP increase than in CHO-K1 cells. However, unlike with CHO-TSHr cells, DDT was ineffective against forskolin-induced accumulation of cAMP in CHO-A2a cells.
Notwithstanding the above considerations, Aroclor 1254 and DDT may influence cAMP generation by indirect mechanisms not related to TSHr inhibition. For example, there is evidence that DDT might influence Na+ channels (Narahashi, 1992) and ATP synthase (Younis et al., 2002); furthermore, cell toxicity could also lead to cAMP leakage from cells. All of these effects might be most evident when intracellular cAMP levels are high rather than low. To exclude these confounding factors as potential causes of cAMP reduction, Aroclor 1254 and DDT were shown to maintain their inhibitory effect on cell membrane AC. This observation gives further support to the hypothesis that these compounds may act directly on the TSHr site.
If indeed TSHr is the target of the inhibitory activity of DDT and Aroclor 1254, this inhibition could in principle be exerted on either ECD or TMD of the receptor, although in the absence of any modification in [125I]bTSH binding, as seen by Santini et al. (2003), TMD is likely the real target. To verify whether or not DDT and Aroclor 1254 may act at the TMD level, we used two different approaches: TSHr was either slightly trypsinized or truncated of most ECD. TSHr trypsinization did not reduce the inhibitory activity of DDT and Aroclor 1254, suggesting that the integrity of ECD is not required for their effect. Additional evidence in favor of this view came from experiments with the TSHr trunk. The basal activity of this receptor was clearly inhibited by DDT and Aroclor 1254.
In COS-7 cells transfected with ACV alone, both compounds increased adenyl cyclase activity in the same range of concentrations that were shown to inhibit the TSHr trunk. At 100 µM concentration, Aroclor 1254 stimulated ACV more robustly. As pointed out in the Results, this effect could be due to inhibition of phosphodiesterases above the level observed in the presence of Ro 20-1724 and IBMX. This consideration is because of the fact that phosphodiesterase isoforms are not equally sensitive to most inhibitors, and some are totally insensitive (Fisher et al., 1998). For example, to attain stronger inhibition, two inhibitors were used in our experiments, but this does not guarantee a 100% inhibition of the enzyme(s) activity. Based on the result that no cAMP increase was observed in the absence of Ro 20-1724 and IBMX, we can conclude that the effect of DDT and Aroclor 1254 on COS-7 cells transfected with the sole ACV is not due to an additional inhibition of phosphodiesterases above the level observed with Ro 20-1724 and IBMX. Even though we did not further investigate this phenomenon, it is plausible that stimulation of ACV may counteract the inhibitory activity of Aroclor 1254 and DDT for the TSHr trunk (or the wild-type TSHr) in COS-7 cells cotransfected with TSHr trunk (or the wild-type TSHr) and ACV. This dual effect on cAMP accumulation, i.e., inhibitory in the presence of the TSHr trunk (or the wild-type TSHr) + ACV and stimulatory in the presence of the sole ACV, strengthens the idea that the inhibition exerted by Aroclor 1254 and DDT on COS-7 cells is targeted to the TSHr trunk rather than downstream from the receptor. This is also confirmed by the observation that Aroclor 1254 did not prevent accumulation of forskolin-induced cAMP in COS-7 cells transfected with ACV, whereas DDT produced only a slight inhibition. This stimulatory effect on AC exerted by Aroclor 1254 and DDT was not observed in CHO-K1 cells.
Another indication that both Aroclor 1254 and DDT may act through the TSHr comes from experiments with the adenosine A2a receptor. Both compounds proved unable to inhibit cAMP accumulation as induced by the adenosine agonist NECA in COS-7 cells transiently cotransfected with A2a and ACV. This finding rules out the possibility that Aroclor 1254 and DDT might interfere with the capacity of Gs to interact with adenyl cyclase. This view is further supported by DDT being unable to inhibit cAMP accumulation by the other two Gs-coupled receptors, the 2-adrenergic receptors endogenously expressed in COS-7 cells and the dopamine D1 receptor stably expressed in CHO-K1 cells.
However, the idea that TSHr may be a target for DDT is confirmed by data from CHO-TSHr and transiently transfected COS-7 cells, whereas for Aroclor 1254, only by data from COS-7 cells. The most parsimonious explanation of these observations is that both DDT and Aroclor 1254 act on TSHr, whereas Aroclor 1254 in CHO-K1 cells might have additional effects at the inhibitory doses that may mask TSHr inhibition. However, this interpretation remains a mere speculation.
DDT is known to have a membrane-perturbing effect due to its lipophilicity (Patton et al., 1984) that could well account for the observed inhibitory effect within the micromolar range of concentrations. To exclude this possibility, we tested two analogs of DDT, diphenylethylene and bisphenol A, that should have a similar perturbing effect as DDT. The diphenylethylene analog lacks the Cl substituents, and the bisphenol A analog has the para-Cl atoms substituted by hydroxyl (OH)-groups. Diphenylethylene did not inhibit cAMP accumulation in CHO-TSHr cells as stimulated by bTSH, supporting the concept that lipophilicity per se does not explain the effect of DDT but rather that the negatively charged Cl substituents exert an important role in docking DDT to its binding site. This interpretation is partly sustained by the observation that bisphenol A may equally inhibit cAMP accumulation, although to a much less extent. The hydroxyl (OH)-group substituents in bisphenol A have a net negative charge that could engage the same cationic residues in the TSHr as the Cl substituents in DDT. Nonetheless, lipophilicity could be important for DDT because it could facilitate its delivery to the site of action in the plasma membrane.
The TSHr together with the FSHr and LHr belong to the glycoprotein receptor family and share a high degree of amino acid sequence homology. Aroclor 1254 and DDT exert on the FSHr and LHr the same inhibitory activity as that observed with the TSHr, suggesting a common inhibitory mechanism for all these receptors.
As we mentioned in the introduction, Aroclor 1254 and DDT have been classified as environmental factors capable of causing thyroid dysfunction (Brucker-Davis, 1998). However, to interpret our findings as toxicologically significant, the range of concentration at which Aroclor 1254 and DDT exert their inhibitory effects must be compared with those actually detectable in the environment. Information on Aroclor 1254 levels in subjects exposed to polychlorinated biphenyls is scarce, even though available data indicate that it may reach at most 1 µM concentration (Karmaus et al., 2002). Comprehensive literature is available for DDT due to the worldwide use of this compound as an insecticide. Serum levels of 10 µM have been measured in subjects chronically exposed to DDT (Chen et al., 2005; De Jager et al., 2006). This being the case, it is predictable that any thyroid dysfunction following chronic exposure to DDT could be caused, at least in part, by a direct interaction of this insecticide with the TSHr (Brucker-Davis, 1998). Furthermore, the observed effects on the reproductive system following occupational exposure to Aroclor 1254 and DDT could indeed be explained by inhibition of LHr and FSHr (Queiroz and Waissmann, 2006).
Our data suggest that DDT, and possibly Aroclor 1254, might be uncompetitive inverse agonists rather than antagonists for the TSHr, as shown by the fact that they reduce the constitutive activity of the TSHr and the mutant TSHr(I486M). Furthermore, they indicate that both compounds may act through an allosteric mechanism of action at the TMD level. Nevertheless, a direct effect on adenylyl cyclase is also possible, as shown by the minor effect of DDT or by the major effect of Aroclor 1254 on forskolin-induced cAMP accumulation.
Compounds with uncompetitive inverse agonist activity, acting at sites different from the natural ligand recognition site, have been already reported for metabotropic glutamate receptors (Gasparini et al., 2002). As for TSHr, glutamate, the natural ligand of metabotropic glutamate receptors, does not bind to TMD, but rather it binds to a site located in the large N-terminal extracellular domain. Unlike competitive ligands that bind to the glutamate binding site located in the large N-terminal extracellular domain, several modulators have been shown to act as uncompetitive antagonists, uncompetitive inverse agonists, or positive modulators by binding to specific residues in the seven-transmembrane domain. These modulators have provided novel pharmacological tools that may prove useful to gain new insights into the mechanism activating metabotropic glutamate receptors (Pin et al., 2004).
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Footnotes |
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ABBREVIATIONS: TSH, thyrotropin; TSHr, TSH receptor; ECD, extracellular domain; TMD, transmembrane domain; DDT, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane; CHO, Chinese hamster ovary; bTSH, bovine TSH; Ro 20-1724, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone; IBMX, 3-isobutyl-1-methylxanthine; NECA, 5'-(N-ethyl-carboxamido)-adenosine; FSH, follicle-stimulating hormone; hrFSH, human recombinant FSH; hCG, human chorionic gonadotropin; A2a, adenosine type 2a receptor; COS, African green monkey kidney fibroblast; LH, luteinizing hormone; FSHr, FSH receptor; LHr, LH receptor; ACV, adenyl cyclase V; AC, adenylyl cyclase; RIA, radioimmunoassay; Aroclor 1254, a complex mixture of polychlorinated biphenyls.
Address correspondence to: Dr. Roberto Maggio, Department of Experimental Medicine, University of L'Aquila, Via Vetoio (Coppito 2), 67010 L'Aquila-Coppito, Italy. E-mail: roberto.maggio{at}univaq.it
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