Molecular Human Reproduction, Vol. 7, No. 9, 799-809,
September 2001
© 2001 European Society of Human Reproduction and Embryology
Reproductive endocrinology |
Relaxin signalling links tyrosine phosphorylation to phosphodiesterase and adenylyl cyclase activity
Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany
Abstract
The relaxin receptor has so far avoided molecular cloning and characterization. We have therefore characterized the signalling events activated by relaxin (RLX), using two different cell culture-based bioassay systems: primary human endometrial stromal cells from the cycle (ESC) and the human monocyte cell line THP-1. Upon RLX stimulation, both cell types showed a rapid increase in cAMP accumulation, which could be inhibited by an inhibitor of G-protein activation, GDP-ß-S. However, evolutionarily one would expect the RLX receptor, like those for the structurally related hormones insulin and insulin-like growth factor-I, to involve tyrosine kinase activity. The specific tyrphostins AG 1478, AG 527 and AG 879 inhibited the RLX-stimulated cAMP response in human ESC and THP-1 cells in a dose-dependent manner, though the potent broad range tyrphostin AG 213 had no effect. Also, treatment of THP-1 cells with the potent phosphotyrosine phosphatase inhibitors bpV(phen) and mpV(pic) increased RLX-stimulated cAMP accumulation in a dose-dependent manner. The effect of the general tyrosine kinase inhibitor genistein (which can also inhibit some phosphodiesterases) on RLX-mediated cAMP accumulation strongly depended on the activity status of phosphodiesterase. In the absence of a phosphodiesterase inhibitor, genistein enhanced RLX-stimulated cAMP accumulation in both bioassays. When phosphodiesterase was inhibited by isobutylmethylxanthine, this effect was not observed. The results imply that activation of the RLX receptor uses tyrosine kinase signalling to control phosphodiesterase activity, and hence to up-regulate intracellular cAMP.
decidualization/endometrium/relaxin receptor
Introduction
The mammalian peptide hormone, relaxin (RLX), belongs structurally to the family of insulin-like hormones which includes, besides insulin and relaxin, insulin-like growth factor-I (IGF-I), IGF-II, the relaxin-like factor (RLF/INSL3) and several novel insulin-like peptides (INSL4–INSL6). Other members of this family have been described throughout the animal kingdom, including Caenorhabditis elegans (Duret et al., 1998
), molluscs and insects (Sherwood, 1994; Schwabe and Büllesbach, 1998). To date, receptors for members of this family have been cloned only for insulin, IGF-I and IGF-II, though structurally related molecules have also been identified in the C.elegans genome (DAF2), as well as in insects, annelids and Hydra. Additionally, an orphan receptor (IRR, insulin-related receptor) has been cloned from mammals. Except for the so-called IGF-II receptor, which has been characterized as a mannose-6-phosphate receptor, all of these molecules belong to a subgroup of membrane-associated receptor tyrosine kinase. They share an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular C-terminus with tyrosine kinase activity. For the insulin and IGF-I receptors, the extracellular domain is post-translationally cleaved but remains covalently linked to the transmembrane and intracellular domains. Although the hormone RLX and its physiology have been described for many decades, there is still no detailed molecular description of a RLX receptor.
Using ligand-binding assays and receptor autoradiography, a single high-affinity binding site for RLX can be defined with a Kd in the low nanomolar range (Osheroff and Phillips, 1991; Osheroff et al., 1992; Büllesbach et al., 1995; Palejwala et al., 1998). Specific binding is observed in those tissues where RLX is known to have a physiological effect, namely in the uterus and cervix, particularly during pregnancy (Mercado-Simmen et al., 1980; Weiss and Bryant-Greenwood, 1982; Osheroff et al., 1990), and in the mammary gland (Kuenzi and Sherwood, 1995; Min and Sherwood, 1996; Kohsaka et al., 1998), brain (Osheroff and Phillips, 1991) and heart (Osheroff et al., 1992). In uterine cells, moreover, activation of the RLX receptor has been shown to be associated with specific tyrosine phosphorylation (Palejwala et al., 1998). Attempts to use ligand-binding to define the size of the RLX receptor have yielded varying and inconsistent results with sizes between 36 and 200 kDa (Büllesbach et al., 1995; Osheroff, 1995; Osheroff and King, 1995; Parsell et al., 1996; Palejwala et al., 1998).
Until recently, RLX activity was measured mostly using either whole animal or organ bioassays [e.g. mouse cervix, rat heart (Sherwood, 1994)]. Two cell systems have, however, now become established as suitable bioassays to determine relaxin bioactivity: the human monocyte cell-line THP-1 (Parsell et al., 1996) and primary stromal cells from fresh human endometrium of the cycle (ESC) (Fei et al., 1990; Gellersen et al., 1994). Although both cell types have relatively few RLX receptors as determined by ligand-binding [THP-1 cells: 260 receptors/cell (Parsell et al., 1996); endometrial stromal cells of the cycle (ESC) cells: ~1000 receptors/cell (Osheroff and King, 1995)], they are able to provide a relatively robust intracellular response to RLX stimulation. Surprisingly, the response is an elevation of cAMP (Fei et al., 1990; Parsell et al., 1996; Telgmann and Gellersen, 1998). This is not a peculiar feature of these cell types, since it has also been shown that RLX exerts its classic effect to suppress contractility of rat myometrial smooth muscle cells by means of a sustained cAMP response involving Gs (Sanborn et al., 1995; Dodge and Sanborn, 1998). Because there is an obvious conflict between such results and the notion of the RLX receptor being related to those for insulin and IGF-I, which are membrane-bound tyrosine kinases, we have begun a detailed characterization of RLX-induced signal transduction making use of the two discrete cell types, THP-1 and primary ESC cells. In the past, a number of groups have tried unsuccessfully to clone the RLX receptor, probably because it does not conform to conventional paradigms. It seems likely therefore that the new knowledge gained about the RLX receptor will be of great value for designing new strategies with which to search for and eventually clone the RLX receptor.
Materials and methods
Cell culture
Human ESC cells from premenopausal and cycling women (35–50 years old) undergoing hysterectomy for leiomyoma were prepared as previously described (Gellersen et al., 1994) to routinely obtain primary cultures of >95% purity. All patient samples were collected in accord with the Helsinki declaration and the authorization of the local ethical committee. Briefly, endometrial tissue was minced thoroughly and digested in Dulbecco's modified Eagle's medium (DMEM) with 0.5 mg/ml collagenase–dispase (Boehringer Mannheim, Mannheim, Germany) and 2.5 mg/ml DNase I (Sigma, Deisenhofen, Germany) for up to 2 h, with gentle pipetting every 20–30 min. Myometrial contamination and undispersed material were removed by sieving samples through a sterile nylon stocking and subsequently through a steel sieve (38 µm pore size). ESC cells were allowed to attach to T80 flasks (Nunc, Roskilde, Denmark) for 30–45 min before epithelial cells were washed away. Basal medium contained DMEM–Ham's F-12 at a 1:1 ratio, 10% fetal calf serum (FCS) that had been depleted of steroids by treatment with dextran–coated charcoal, 100 IU/ml penicillin, 100 µg/ml streptomycin, 1 µg/ml insulin and 10–9 mol/l 17ß-oestradiol (Sigma). Medium was changed every 48–72 h. Confluent monolayer cultures were trypsinized and passaged at a ratio of 1:3. Cells used in this study were taken between passage 2 and passage 6, and finally transferred to 12-well plates (Nunc) for the individual experiments. Where indicated, relaxin (>85% pure porcine relaxin; courtesy Dr O.D.Sherwood) was added to the medium at a concentration of 100 ng/ml dissolved in 0.3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Purity of the relaxin used was tested by polyacrylamide gel electrophoresis followed by Coomassie blue staining, where only a single stained band was ever detected. Previous studies had shown the EC50 for relaxin in these cells to be ~2 nmol/l (equivalent to 12 ng/ml porcine relaxin) (Zarreh-Hoshyari-Khah et al., 2001).
The human monocyte cell-line THP-1 was purchased from the American Type Culture Collection (ATTC) and cultured according to their recommendations. Briefly, non-adherent cells were grown in T175 flasks (Nunc) in Roswell Park Memorial Institute 1640 culture medium, containing 2 mmol/l glutamine, 20 µmol/l ß-mercaptoethanol and 10% fetal calf serum. Cell cultures were split at a density of ~5x105 cells/ml. After two to four such passages, cells were seeded at ~5x105 cells per 1 ml culture medium into 24-well plates (Nunc) for subsequent experimentation. Relaxin was applied as described above for ESC cells.
cAMP determination
ESC cells in 12-well plates were incubated with or without relaxin and a subset of different inhibitors of signal transduction as described below. For determination of extracellular cAMP, 500 µl of supernatant was collected and incubated at a final concentration of 70% ethanol at –20°C overnight. For measurement of intracellular cAMP, confluent monolayers were washed twice with ice-cold PBS and extracted in 1 ml 70% ethanol at –20°C overnight. The non-adherent THP-1 cells were transferred to 24-well plates at a concentration of 5x105 cells per well and incubated with or without relaxin and a similar set of signal transduction inhibitors as for the ESC cells. In general for THP-1 cells, 500 µl of the cell suspension was collected and frozen overnight at –20°C in a final concentration of 70% ethanol. In order to measure extracellular cAMP, cells were first centrifuged for 4 min at 200 g. Supernatants were collected and incubated in a final concentration of 70% ethanol at –20°C overnight. For determination of intracellular cAMP, cell pellets were washed twice with ice-cold PBS, resuspended in 1 ml 70% ethanol and frozen at –20°C overnight. cAMP content was measured using a specific enzyme-linked immunosorbent assay (ELISA) as described previously (Zarreh-Hoshyari-Khah et al., 2001).
Membrane preparations
THP-1 cells were harvested by centrifugation at 200 g for 4 min and finally resuspended in hypotonic homogenization buffer: 10 mmol/l Tris/HCl, pH 7.5, 1 mmol/l EDTA, 1 mmol/l dithiothreitol supplemented with protease inhibitor cocktail (completeTM; Boehringer Mannheim, Mannheim, Germany). ESC cells were harvested from cell culture dishes by scraping with a rubber policeman, and after centrifugation (200 g, 4 min) pellets were resuspended in homogenization buffer. Cell suspensions were allowed to swell for 8 min on ice and then homogenized with 10 strokes in a Dounce homogenizer with a tight-fitting pestle. The homogenate was then made isosmolar by addition of sucrose to a final concentration of 0.25 mol/l. After centrifugation at 200 g for 15 min, supernatants were then centrifuged for 30 min at 30 000 g. The washed pellets were finally resuspended in homogenization buffer. Protein concentration was determined using the Bio-Rad (Munich, Germany) kit, with bovine serum albumin (fraction V; Sigma) as standard and assays for adenylyl cyclase activity were performed immediately.
Adenylyl cyclase activity was analysed according to a published method (Dix et al., 1982) with some modifications. Incubations were performed at 30°C in a final volume of 50 µl for 30 min. The standard incubation mixture contained 40 mmol/l Tris–HCl (pH 7.5), 5 mmol/l MgCl2, 1 mmol/l EDTA, 0.5 mmol/l isobutylmethylxanthine (IBMX; Sigma), 1 mmol/l dithiothreitol, 0.1% bovine serum albumin, 1.0 mmol/l ATP, 10 mmol/l phosphocreatine, 13.2 units/ml creatine phosphokinase, and 10–15 µg membrane protein. When indicated in the text or figure legends, incubation mixtures were also supplemented with GTP, hormones or signal transduction inhibitors. Reactions were stopped by the addition of 1 ml ethanol and stored at –20°C overnight. After evaporation of ethanolic solutions to dryness, cAMP was analysed by ELISA as mentioned above. cAMP production was expressed as pmol/min/mg membrane protein.
Inhibitors of signal transduction
The effects of a subset of different inhibitors of signal transduction on relaxin-induced cAMP accumulation were studied in cell culture experiments and by adding reagents to membrane preparations of cultivated cells. For cultures of ESC and THP-1 cells, these were either pretreated or not with the non-selective phosphodiesterase inhibitor IBMX. Preincubation with IBMX was performed for 30 min using a range of concentrations from 50 µmol/l to 1 mmol/l as indicated. The isoflavone genistein (Calbiochem, La Jolla, CA, USA), known to act as a general inhibitor of protein tyrosine kinases (Akiyama et al., 1987), was dissolved in dimethylsulphoxide (DMSO) and added to ESC cell cultures at a concentration of 100 µmol/l 30 min prior to the addition of relaxin. For THP-1 cells, genistein was applied in concentrations ranging from 1 to 100 µmol/l for 30 min prior to incubation with relaxin. As a negative control, 100 µmol/l of daidzein (Calbiochem), an inactive analogue of genistein, was added to THP-1 cells. Tyrphostins AG1478, AG527, AG879, AG213, AG1295 and AG9 (Calbiochem) are synthetic analogues of the microbial inhibitor, erbstatin, with selective inhibitory activities on various tyrosine kinases (Yaish et al., 1988; Gazit et al., 1989, 1991). Tyrphostins were dissolved in DMSO just before use. Equivalent amounts of DMSO vehicle were included in all assays. Prior to administration of relaxin, cell cultures were preincubated with the tyrphostins for 30 min in a concentration range of 0.1–100 µmol/l for THP-1 cells and 100 µmol/l for hESC cells. Membrane preparations of THP-1 cells were co-incubated with relaxin and 100 µmol/l of either AG 527, AG 213 or AG 9 for 20 min. The peroxovanadium compounds bpV(phen) and mpV(pic) are potent activators of insulin receptor kinase by inhibiting phosphotyrosine phosphatase (Posner et al., 1994). THP-1 cell cultures were preincubated for 30 min with 0.1–100 µmol/l bpV(phen) or mpV(pic) prepared from aqueous stocks just before use. Pertussis toxin (List Biological Laboratories, Campbell, CA, USA) inactivates inhibitory G-proteins by ADP ribosylation. Where indicated, ESC cells were pretreated with pertussis toxin (stock solution: 100 µg/ml in 0.1 mol/l sodium phosphate, 0.5 mol/l NaCl, pH 7.0) diluted to a final concentration of 100 ng/ml for 3 h. The non-hydrolysable GDP analogue GDP-ß-S (Calbiochem) was redissolved in 19 mmol/l Tris–HCl containing 1 mmol/l EDTA (pH 7.9) and co-incubated with membrane preparations of ESC and THP-1 cells for 20 min in the presence or absence of relaxin and/or different tyrphostins at concentrations indicated in the text.
All experiments were carried out at least in triplicate and data were treated by analysis of variance. Where significance was detected between groups, these were submitted to a post-hoc unpaired Student's t-test. Differences were considered significant when P < 0.05.
Results
Time course of the relaxin-induced increase in cAMP production by cultured THP-1 and ESC
The time course of adenylyl cyclase induction by relaxin was first investigated in both the human monocyte cell-line, THP-1, growing as suspension culture, and in highly pure (>95% stromal cells) monolayers of primary ESC cells derived from uteri of pre-menopausal women undergoing elective hysterectomy for non-endocrine pathologies. A single concentration of 100 ng/ml pure (>85%) porcine relaxin was applied (~15 nmol/l), and cAMP was measured in both medium (extracellular) and cell pellets (intracellular) (Figure 1A and B). The addition of relaxin resulted in a rapid (<5 min) increase in intracellular cAMP in both cell types, reaching a maximum at 20–30 min, followed by a decline. cAMP was subsequently secreted into the medium with a delayed kinetic, and maxima at 1–3 h. For convenience, in all later experiments total cAMP (extracellular and intracellular) was measured for the THP-1 cells in suspension culture (Figure 1B), and intracellular cAMP was measured for the monolayer cultures of the primary ESC cells.
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Bioassays measuring net cAMP production could suffer from low sensitivity because of intracellular phosphodiesterase activity effectively degrading the cAMP. To check this effect, experiments were carried out with preincubation in the absence and presence of the general phosphodiesterase inhibitor, IBMX (Figure 1C and D
). Since preliminary experiments had shown no significant difference in the effect of this inhibitor on the ESC cell cultures at concentrations ranging from 50 to 1000 µmol/l (data not shown), the lowest concentration (50 µmol/l) was applied to these cells, consistent with its inhibitory effect in membrane preparations (see later). For the THP-1 cells, IBMX showed a dose-dependent effect on cAMP catabolism with optimal cAMP yields at a concentration of IBMX in the medium of 1 mmol/l, without this being toxic to the cells. Whereas IBMX had no significant effect on the cAMP yields in the ESC cells (Figure 1C), phosphodiesterase inhibition increased the total cAMP production by THP-1 cells (Figure 1D).
Augmentation of RLX-stimulated cAMP generation in THP-1 cells by inhibitors of tyrosine phophatase
Peroxovanadium compounds are the most potent phosphotyrosine phosphatase inhibitors described to date (Posner et al., 1994). bpV(phen) and mpV(pic) are both potent insulin receptor kinase activators showing excellent insulin mimetic effects in vivo and in vitro. In contrast to bpV(phen), mpV(pic) shows greater potency as an inhibitor of dephosphorylation for the insulin receptor than for the epidermal growth factor (EGF) receptor. Preincubation of THP-1 cells with either bpV(phen) or mpV(pic) (0–100 µmol/l) augmented the RLX-stimulated cAMP accumulation at moderate to high concentrations (Figure 2). The more general phosphotyrosine inhibitor bpV(phen) (Figure 2A) enhanced the RLX response in THP-1 cells at significantly lower doses (50 and 100 µmol/l) than the more insulin specific mpV(pic) which was only effective at the highest concentration used (0.5 mmol/l). Neither compound had a significant effect on basal cAMP accumulation. Preincubation of THP-1 cells with 1 mmol/l IBMX (Figure 2B) led to an overall increase in cAMP accumulation (with or without RLX) but had no effect on the peroxovanadate-dependent enhancement of the RLX response, indicating that the tyrosine phosphorylation event involved is independent of IBMX-sensitive phosphodiesterase activity. As a further control, THP-1 cells were also incubated with okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A; this had no effect on RLX-induced cAMP accumulation (data not shown).
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Suppression of RLX-stimulated cAMP generation in ESC and THP-1 cells by inhibitors of tyrosine kinase
Tyrphostins are synthetic low molecular weight inhibitors of tyrosine kinases modelled on the microbial inhibitor erbstatin (Yaish et al., 1988
). In a first set of experiments, THP-1 cells were treated with a subset of different tyrphostins prior to incubation with RLX (Figure 3). Basal cAMP production was unaffected at all concentrations (0.1–100 µmol/l) of tyrphostins applied. Tyrphostin AG 1478, which is considered highly potent and specific for the EGF receptor tyrosine kinase, showed a marked dose-dependent inhibition of RLX-stimulated cAMP production (Figure 3A). Tyrphostin AG 527, known to be most specific for EGF receptor-type kinases, and tyrphostin AG 879, a nerve growth factor (NGF)-specific tyrphostin, inhibited the RLX-stimulated cAMP accumulation in a similar dose-dependent manner (Figure 3B and C). These effects were not at the level of the phosphodiesterase, since preincubation of the THP-1 cells with 1 mmol/l IBMX (Figure 3F–H) had no effect on the tyrphostin-dependent inhibition. The tyrphostins AG 213, known as a general broad-range tyrosine kinase inhibitor, and AG 1295, a PDGF receptor-specific tyrphostin, showed no significant effects on RLX-stimulated cAMP response even at the highest concentration (Figure 3D and E), nor was this influenced by IBMX (Figure 3I and J).
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Applying a single maximal concentration of 100 µmol/l tyrphostins to the ESC cell cultures gave rise to essentially similar results (Figure 4
). Of those tested, only tyrphostin AG 527 was effective, causing an almost complete inhibition of the RLX-stimulated increase in intracellular cAMP, irrespective of whether or not the cells were preincubated with 50 µmol/l IBMX (Figure 4B). The inactive tyrphostin AG 9 was additionally used as a further negative control.
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RLX-stimulated cAMP production is G-protein dependent
When ESC cells were pretreated with pertussis toxin (100 ng/ml for 3 h) to inhibit any effects of Gi proteins in these cells (Figure 5
), there was an increase in the basal cAMP production, implying a basal inhibition of adenylyl cyclase involving a Gi protein. Subsequent incubation of the cells with two different concentrations of relaxin led to a further increase in cAMP production, which was additive to that caused by the pertussis toxin alone, and showed that a Gi protein was not mediating any of the RLX-dependent effects.
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cAMP is generated in most cells by activation of membrane-bound adenylyl cyclase through the mediation of G-proteins and usually ligand-activated membrane receptors of the heptahelical kind. The insulin family of peptide hormone receptors appears not to belong to this group, suggesting the possible involvement of other intracellular signal transducing systems. In order to analyse this aspect, membrane preparations were made from both THP-1 and ESC cells. In ESC cells, the membrane-bound adenylyl cyclase can be activated by vasoactive intestinal peptide (VIP), acting through a typical G-protein-coupled receptor (Bajo et al., 1993
). We could verify this as a positive control using membrane preparations of THP-1 cells stimulated in vitro by VIP in the presence of 1 mmol/l GTP as G-protein substrate and 100 mmol/l IBMX (Figure 6). VIP stimulated cAMP generation in a dose-dependent manner, consistent with the involvement of a membrane receptor linked via G-proteins to the adenylyl cyclase. Treatment of these membrane preparations with either RLX or, as a further control, IGF-I, indicated for both hormones small, but significant increases in GTP-dependent cAMP generation (Figure 6). However, unlike that caused by VIP, these increases are not dose dependent. In repeated experiments using membranes from both THP-1 and ESC cells, RLX and IGF-I consistently caused small, but statistically significant increases in cAMP only at concentrations of 1 or 10 nmol/l; at higher and lower concentrations no consistent and significant differences from the controls were detected (data not shown).
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When THP-1 cell membranes were additionally co-incubated with the non-hydrolysable analogue of GDP, GDP-ß-S, an inhibitor of G-protein activation (Figure 7A
), the stimulatory effect of GTP alone as substrate, as well as the additional effects due to RLX, IGF-I and VIP, were all suppressed to below control levels. This experiment was also repeated with membrane preparations of human ESC cells (Figure 7B), where GDP-ß-S suppressed the RLX-, IGF-I- and VIP-induced cAMP accumulation to a similar extent.
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Finally, these experiments were repeated in the presence of the tyrphostin AG 527, which had been shown to inhibit the RLX-induced cAMP increase in whole cells. Without exception, the stimulating effects of GTP, RLX, IGF-I and VIP were all further augmented by tyrphostin AG 527 (Figure 8
). Basal unstimulated cAMP generation was not affected by tyrphostin AG 527. These results imply that the strongly inhibitory effect of tyrphostin AG 527 on the RLX-induced stimulation of cAMP generation in intact cells must involve a cytoplasmic component not present in the membrane preparations.
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Effects of phosphodiesterase activity on RLX-stimulated cAMP generation in ESC and THP-1 cells
The isoflavone genistein is shown to inhibit tyrosine kinase activities in cells with an EC50 in the range 20–30 µmol/l without affecting the activity of serine/threonine kinases (Akiyama et al., 1987
). In the absence of IBMX, 100 µmol/l genistein for 30 min failed to have any inhibitory effect on cAMP production (either basal or RLX-induced) in ESC cells (Figure 9). In contrast, genistein caused an increase in the RLX-induced cAMP production. However, when these cells were pretreated with 1 mmol/l IBMX to block phosphodiesterase activity (Figure 9), this enhancing effect was completely lost. There was no effect on basal cAMP production either by genistein or by IBMX, suggesting that RLX may be acting in this system at the phosphodiesterase level.
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Similar results were obtained using the THP-1 cells (Figure 10
). In the absence of IBMX (Figure 10A), genistein, but not the inactive daidzein, augmented the stimulatory effect of RLX in a dose-dependent manner, with 100 µmol/l genistein causing a doubling of the RLX-induced cAMP production. When the cells were additionally preincubated with 1 mmol/l IBMX (Figure 10B), both basal and RLX-stimulated cAMP yields increased as expected. In these conditions genistein did not augment the effect of RLX, instead it caused a dose-dependent partial inhibition of the cAMP yields compared to that attained by RLX in the presence of the inactive control compound daidzein. Again basal cAMP levels are unaffected by genistein.
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Discussion
Taking all results together from two quite different cell types presents a relatively homogeneous picture of RLX-induced signal transduction. To date, there is to our knowledge no evidence that a conventional membrane-type tyrosine kinase receptor can cause an activation of adenylyl cyclase with such a rapid time course as that shown here for RLX. The fact that the general tyrosine kinase inhibitor genistein, the broad range tyrphostin AG 213 and the PDGF receptor kinase-specific tyrphostin AG1295 were unable to inhibit the effects of RLX, even at moderately high concentrations (100 µmol/l), implies that the RLX receptor does not appear to belong to any of the more common tyrosine kinase types of receptor. Genistein, however, is also known to inhibit phosphodiesterase activity (Ho et al., 1995; Ogiwara et al., 1997; Stringfield and Morimoto, 1997). This would explain the genistein-associated augmentation of RLX-mediated cAMP accumulation seen for both THP-1 and ESC cells, and the absence of such an effect in the additional presence of the phosphodiesterase inhibitor IBMX. Several reports support this principle of phosphodiesterase–inhibition mediating an enhancement of hormone responsiveness (Ho et al., 1995: Stringfield and Morimoto, 1997). Ogiwara et al. (1997), for example, showed that tyrosine kinase inhibitors, by their action on phosphodiesterase, were able to enhance gonadotrophin-releasing hormone-stimulated cAMP accumulation in rat anterior pituitary cells.
While genistein may influence phosphodiesterase activity, like other general tyrosine kinase inhibitors it had no effect on the RLX receptor. Instead, the tyrphostin AG 1478, which is considered to be a highly potent and specific inhibitor of the EGF receptor kinase (Fry et al., 1994; Ward et al., 1994), did inhibit RLX-stimulated cAMP generation with an IC50 of ~10 µmol/l. Inhibition was also observed with another inhibitor of EGF receptor autophosphorylation, tyrphostin AG 527 (IC50 10–20 µmol/l), as well as with tyrphostin AG 879 (IC502–3 µmol/l), which is supposedly specific for the NGF receptor kinase. These effects were not influenced by IBMX, suggesting that unlike the effect of genistein, they represent inhibition close to the receptor itself. Supporting these findings, the potent phosphotyrosine phosphatase inhibitors, bpV(phen) and mpV(pic), known to suppress insulin receptor dephosphorylation, enhanced the RLX-stimulated cAMP accumulation in THP-1 cells independently of phosphodiesterase inhibition. The more general inhibitor bpV(phen), which also inhibits EGF-receptor dephosphorylation (Ho et al., 1999), augmented the response to RLX with greater potency (IC50 30– 60 µmol/l) than mpV(pic) (IC50 >300 µmol/l) which displays a higher relative specificity for the inhibition of insulin receptor dephosphorylation (Posner et al., 1994).
Taken together, these results imply that the RLX receptor may be a membrane tyrosine kinase more closely related to the EGF receptor than to the currently known group of insulin-related receptors. This could explain why, in spite of intensive searching of various DNA databases using homology algorithms based on insulin-related receptors, no new receptors have appeared, although the number of insulin-related hormones has increased steadily.
As with most other adenylyl cyclase-activating systems, RLX required the involvement of a stimulatory G-protein (Gs), whose activity was unaffected by pertussis toxin but was inhibited by GDP-ß-S. In another study, it has been shown that the EGF receptor protein tyrosine kinase is able to phosphorylate Gs
on tyrosine residues, thereby activating this protein. This involves physical interaction of the EGF receptor with Gs (Poppleton et al., 1996; Sun et al., 1997). Such a direct interaction between the RLX receptor and Gs appears unlikely, since in this case one would have expected a clear dose–response relationship in membrane preparations, similar to that seen for VIP, assuming that the functionality of the RLX receptor is not in any way perturbed by the physical process of membrane preparation. It also remains possible that an adenylyl cyclase is influenced independently of Gs, for example, through Ca2+ acting on a Ca2+/calmodulin-dependent enzyme; RLX has been shown to induce Ca2+ transients in human granulosa-lutein cells (Mayerhofer et al., 1995). Further research is in progress to examine such pathways.
Given our current knowledge of signal transducing systems, the following working hypothesis of the action of RLX in different cell types can be postulated: adenylyl cyclase is probably basally activated either by Gs alone, or by Gs acting together with a constitutively active or autocrine functioning G protein-coupled heptahelical receptor, though a direct effect of the RLX receptor still cannot be excluded. Alternatively, intracellular Ca2+ may be involved in this step. The resulting cAMP is catabolized rapidly in THP-1 cells by an IBMX-sensitive phosphodiesterase, and less rapidly in ESC cells, which appear to be IBMX-insensitive. The RLX receptor, in analogy to the receptors for insulin and IGF-I, is probably a membrane-associated tyrosine kinase which can be inhibited by tyrphostins AG 1478, AG 527 or AG 879 either directly or indirectly, but not by genistein, nor by tyrphostins AG213 or AG 1295. This potential RLX receptor with tyrosine kinase characteristics would explain very well the observed augmentation of a RLX-mediated cAMP response upon co-incubation with the peroxovanadium compounds bpV(phen) and mpV(pic), which are specific inhibitors of insulin receptor dephosphorylation. One of the targets of the RLX-stimulated phosphorylation cascade is likely to be an IBMX-insensitive phosphodiesterase which is thereby inhibited, thus causing an elevation in cellular cAMP. This inhibition is most effective in intact cells, and less effective in membranes because some components of the RLX-stimulated kinase cascade are probably cytoplasmic.
Several pieces of evidence support this hypothesis. Recently, Fisher et al. (1998) and Hayashi et al. (1998) have described a new family of phosphodiesterases (PDE). The family members PDE8A and PDE8B can be distinguished from all so far known high affinity cAMP-specific phosphodiesterases, by being insensitive to IBMX (Soderling and Beavo, 2000). PDE8A was originally cloned from THP-1 cells (Fisher et al., 1998), while the second member of the family PDE8B was identified by searching the EST database (Hayashi et al., 1998). PDE8A is expressed in a wide variety of tissues, whereas PDE8B gene transcripts are present in uterus, placenta, prostate, aorta, brain and thyroid gland, thereby showing a striking similarity in their expression pattern to data available from studies on RLX receptor expression (Osheroff and Phillips, 1991; Osheroff et al., 1992; Yang et al., 1992; Osheroff and King, 1995; Palejwala et al., 1998; Tan et al., 1999).
There are also several reports suggesting candidate cytoplasmic components of a RLX-stimulated kinase cascade, including MAP kinase and raf. Increasing evidence suggests a link between the MAP kinase and cAMP pathways at the level of a phosphodiesterase. For example, the MAP kinase ERK2 inhibits the cAMP-specific human PDE4D3 by phosphorylating the protein at Ser579 (Hoffmann et al., 1999). Moreover, incubation of both HEK293 and F422A cells with the ERK kinase inhibitor PD98059 blocks the EGF-induced phosphorylation of PDE4D3 (Hoffmann et al., 1999). In preliminary studies, we have also found that RLX-stimulated cAMP accumulation in THP-1 cells appears to be inhibited by the ERK kinase inhibitors PD 98059 and UO126 in a dose-dependent manner (O.Bartsch and R.Ivell, unpublished data). Moreover, the involvement of a tyrosine kinase cascade in the action of RLX, with raf as one of the possible phosphorylation targets, has recently been postulated (Goldsmith, 2000).
The data presented here indicate a novel route through which RLX may serve to effect an increase in intracellular cAMP levels, namely by a phosphorylation cascade downstream of a RLX receptor with tyrosine kinase characteristics finally targeting a phosphodiesterase. In this model, inhibition of phosphodiesterase activity by RLX is sufficient, firstly, to increase cAMP levels possibly in concert with either RLX-dependent or RLX-independent processes able to activate adenylyl cyclase through Gs, and secondly, to sustain elevated cAMP levels during the decidualization of human endometrial cells [which are known to be IBMX insensitive (Telgmann et al., 1997)] or during RLX-dependent inhibition of myometrial contraction. The results suggest that the RLX receptor possibly belongs to a new subgroup of tyrosine kinase receptors with more EGF-like than insulin-like properties as well as several quite novel characteristics. This could explain why it is still proving so difficult to clone or datamine the RLX receptor.
Acknowledgements
We are very grateful to Dr Ralph Telgmann for helpful discussions throughout this study. Our thanks also go to Dr O.David Sherwood for the kind gift of highly pure porcine relaxin, and to Professor Freimut Leidenberger for his continued support of this difficult project. We should especially like to thank Professors Pauli and Lindner, their staff and patients at the Elim Krankenhaus, Hamburg for the excellent cooperation in helping us to obtain tissue samples. This study was in part financed by the Deutsche Forschungsgemeinschaft (grant Iv7/9-1).
Notes
1 To whom correspondence should be addressed. E-mail: bartsch{at}ihf.de 
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Submitted on April 10, 2001; accepted on June 26, 2001.
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