Urocortin, but Not Corticotropin-Releasing Hormone (CRH), Activates the Mitogen-Activated Protein Kinase Signal Transduction Pathway in Human Pregnant Myometrium: An Effect Mediated via R1{alpha} and R2ß CRH Receptor Subtypes and Stimulation of Gq-Proteins

Dimitris K. Grammatopoulos, Harpal S. Randeva, Michael A. Levine, Efrosini S. Katsanou and Edward W. Hillhouse

Sir Quinton Hazell Molecular Medicine Research Centre (D.K.G., H.S.R., E.S.K., E.W.H.) Department of Biological Sciences University of Warwick Coventry, CV4 7AL, United Kingdom
The Johns Hopkins University School of Medicine Division of Pediatric Endocrinology and the Ilyssa Centre for Cellular and Molecular Endocrinology Department of Pediatrics Baltimore, Maryland 21287


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CRH and CRH-related peptides such as urocortin mediate their actions in the human myometrium via activation of two distinct classes of CRH receptors, R1 and R2. These heptahelical receptors are able to stimulate a number of different intracellular signals; one key mediator of G protein-activated intracellular signaling is the cascade of p42/p44, mitogen-activated protein kinase (MAPK). We therefore hypothesized that activation of MAPK might mediate CRH and or/urocortin actions in the myometrium.

In cultured human pregnant myometrial cells, urocortin but not CRH was able to induce MAPK phosphorylation and activation, suggesting that in the human myometrium these two peptides have distinct actions and biological roles. To identify the particular receptor subtypes mediating this phenomenon, all known CRH receptors present in the human myometrial cells were stably expressed individually in HEK293 and CHO cells, and their ability to activate MAPK was tested. The R1{alpha} and R2ß, but not the R1ß, R1c, or R1d, receptor subtypes were able to mediate urocortin-induced MAPK activation. The signaling components were further investigated; activation of Gs, Go, or Gi proteins did not appear to be involved, but activation of Gq with subsequent production of inositol triphosphates (IP3) and protein kinase C (PKC) activation correlated with MAPK phosphorylation. Studies on Gq protein activation using [{alpha}-32P]-GTP-{gamma}-azidoanilide and IP3 production in cells expressing the R1{alpha} or R2ß CRH receptors demonstrated that urocortin was 10 times more potent than CRH. Moreover, urocortin (UCN) generated peak responses that were 50–70% greater than CRH in activating the Gq protein and stimulating IP3 production.

In conclusion, UCN acting thought multiple receptor subtypes can stimulate myometrial MAPK via induction of the Gq/phospholipase C/IP3/PKC pathway, whereas CRH-induced activation of this pathway appears to be insufficient to achieve MAPK activation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In mammals, CRH, the hypothalamic peptide that regulates the stress response via activation of the pituitary adrenal axis (1), is also involved in the neuroendocrine control of immune response and can regulate cardiovascular function, cognitive function, ingestive behavior, reproductive function, and pregnancy and labor (2, 3, 4, 5, 6). The multiple actions of CRH in various tissues are mediated by specific CRH receptors that are distributed throughout the brain and peripheral tissues. Distinct CRH actions require interactions of ligands to two classes of CRH receptors, termed R1 and R2 (7, 8) that are encoded by unique genes. Alternative processing of nascent transcripts from each of these genes generates several protein variants (9, 10, 11). CRH belongs to a family of peptides that include the recently discovered mammalian peptide urocortin (UCN) (12), the frog peptide sauvagine, and the fish peptide urotensin I. The existence of these structurally related peptides enables a diversity of CRH receptor actions in specific target tissues. Interestingly, UCN binds and activates the CRH-R2 family of receptors with higher binding affinity and potency than CRH, suggesting that UCN may be the preferred or native ligand for these receptors.

During human pregnancy, CRH derived from the placenta and intrauterine tissues (13, 14, 15) is secreted into the maternal circulation. Our studies on the human myometrium have led us to hypothesize that during pregnancy circulating CRH has a "protective" role, by preventing uterine contractions (16). Human myometrium expresses an extensive variety of CRH receptor subtypes, both types R1 and R2 (17), and CRH effects are mediated primarily via activation of Gs proteins and stimulation of the adenylate cyclase-cAMP second messenger system (18). Additional evidence suggests that UCN is also expressed in placental and other intrauterine tissues (19, 20) but is not secreted into the maternal circulation, raising the possibility that UCN acts in a local and paracrine manner via CRH receptors and may also modulate myometrial contractility (21).

We recently showed that native CRH receptors in human myometrium (22) and stably expressed CRH-R1{alpha} in HEK293 cells (23) can activate multiple G proteins, namely Gs{alpha}, Gi{alpha}, Go{alpha}, Gq{alpha}, and Gz{alpha}, suggesting that CRH may regulate diverse signaling pathways. The CRH-R1{alpha} and the CRH-R2{alpha} receptor subtypes have been shown to activate the p42/p44 mitogen-activated protein kinase (MAPK) signaling cascade after stimulation with sauvagine (24). MAPK belongs to a family of serine-threonine kinases comprised of p44 and p42 MAPKs, which are activated via phosphorylation of both tyrosine and threonine residues by diverse stimuli and are expressed ubiquitously in eukaryotic cells where they play an important role in the regulation of processes such as cell proliferation, differentiation, and apoptosis (25, 26). In addition, MAPK has been proposed to be involved in the regulation of myometrial contractility by uterotonins (27, 28), as oxytocin-stimulated uterine contractions can be partially inhibited by MAPK inhibitors and MAPK appears to be involved in the activation of phospholipase A2, which stimulates the release of arachidonic acid from cellular lipids with subsequent stimulation of prostaglandin production.

In view of these findings we sought to investigate the role of CRH and UCN in the activation of MAPK in human myometrial cells. In addition, to characterize in detail this interaction and identify the particular receptor subtypes mediating this phenomenon, all known CRH receptor subtypes present in the human myometrial cells (29) were stably expressed individually in HEK293 or CHO cells and investigated for their ability to activate the MAPK signaling pathway.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MAPK Activation in Cultured Human Pregnant Myometrial Cells
Cultured human myometrial cells that had been obtained from lower segment myometrial biopsies at term before the onset of labor were incubated with various peptides (oxytocin,10 nM; CRH,100 nM; UCN, 100 nM; or sauvagine, 100 nM) for 10 min, and cell lysates were assayed for MAPK activation. Treatment with UCN and sauvagine led to an increase in phospho-MAPK, with p42 appearing to be the predominant form (Fig. 1aGo). Similar results were obtained with oxytocin, which was used as a tissue-specific positive control based on previous studies that demonstrated oxytocin-stimulated increase in MAPK in human pregnant myometrium (30). Sauvagine was used as a receptor-specific positive control since it has been previously shown to activate the p42/p44 MAPK signaling cascade in HEK293 cells stably expressing CRH-R1 and -R2 receptor subtypes (24). Despite increased levels of phospho-MAPK, the total amount of immunoreactive p42/p44 MAPK was unchanged. CRH was unable to activate MAPK in these cells, however, even at concentrations up to 1 µM.



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Figure 1. Effect of (A) CRH, CRH-Like Peptides (upper panel), Oxytocin (lower panel), and (B) PKA or PKC Activators (Forskolin and PMA, Respectively) on MAPK Activation in Human Pregnant Myometrial Cells

Cells were stimulated with either CRH-agonists (100 nM) for 10 min, oxytocin (1–10 nM), forskolin (500 µM), and PMA (200 ng/ml) for 20 min. After cell lysis and centrifugation, supernatants were subjected to SDS-PAGE and immunoblotted with antibody for phospho-p44/42 to determine the phosphorylated/activated p44/42 MAPK as described in Materials and Methods. Alternatively, the same samples were immunoblotted with antibody for p44/42 MAPK to determine total MAPK as a control. Identical results were obtained from five independent myometrial cell preparations.

 
As MAPK can be activated by various signaling cascades (31), we used forskolin, which directly activates the adelylyl cyclase, and phorbol myristate acetate (PMA), an activator of PKC, to obtain information about the intracellular mechanisms involved in MAPK activation in these cultured cells. Although forskolin (500 µM) failed to stimulate MAPK, PMA (200 nM) caused a marked increase in MAPK p42/p44 activity (Fig. 1bGo).

The characteristics of UCN-stimulated MAPK activation in the cultured myometrial cells were further investigated. The effect of UCN was found to be time- and concentration-dependent. The UCN effect was maximal after 5–10 min of treatment and returned to basal levels after 60 min of treatment (Fig. 2aGo). The UCN-dependent increase in MAP activation was significant only at concentrations greater than 10 nM and was maximal at concentrations of 100 nM (Fig. 2bGo). Higher concentrations did not produce any further stimulation (data not shown), and there was no increase in the total amount of p42/p44 MAPK immunoreactivity. In addition, the CRH receptor antagonist astressin (1 µM) was able to significantly inhibit the stimulatory action of UCN (Fig. 2cGo), indicating that the UCN effect was mediated via activation of CRH receptors.



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Figure 2. Characteristics of UCN-Induced MAPK Activation in Myometrial Cells

Time course (A) and dose response (B) of UCN-induced MAPK activation in human pregnant myometrial cells. C, Effect of the CRH receptor antagonist astressin on UCN-induced MAPK activation in human pregnant myometrial cells. Cells were stimulated with either UCN (100 nM) for 5, 10, or 60 min or varying concentrations of UCN (100 pM to 100 nM) for 10 min or UCN (100 nM) in the presence or absence of astressin (1 µM) for 10 min. After cell lysis and centrifugation, supernatants were subjected to SDS-PAGE and immunoblotted with antibody for phospho-p44/42 to determine the phosphorylated/activated p44/42 MAPK as described in Materials and Methods. Alternatively, the same samples were immunoblotted with antibody for p44/42 MAPK to determine total MAPK as a control (A, lower panel). The intensity of the bands corresponding to p42 and p44 MAPK were quantitated with an imaging densitometer (B, lower panel). Identical results were obtained from five independent myometrial cell preparations. *, P < 0.05 compared with basal.

 
Since cultured human myometrial cells express at least five CRH-R subtypes (i.e. R1{alpha}, R1ß, R1c, R1d, and R2ß) (29), each CRH-R was stably expressed individually in HEK293 or CHO cells, and the activation of MAPK by CRH or UCN was further investigated.

MAPK Activation in Mammalian Cells Expressing Individual CRH Receptors
In HEK293 or CHO cells, untransfected or stably expressing different CRH-R subtypes, MAPK p42/p44 activity was stimulated by PMA (200 nM) but not by forskolin (500 µM) or choleragen (12 h, 500 ng/ml) (data not shown), confirming the existence of functional MAPK signaling cascades with similar characteristics to those in the cultured myometrial cells.

Studies in HEK293 or CHO cells expressing the CRH-R1{alpha} receptor subtype (293-R1{alpha} or CHO-R1{alpha}) showed that UCN but not CRH was able to increase phospho-MAPK p42/p44 immunoreactivity, with p42 appearing to be the predominant form (Fig. 3aGo). The UCN effect was found to be time- and concentration-dependent with characteristics similar to those found in the human myometrial cells (data not shown). Although CRH at concentrations up to 1 µM failed to stimulate MAPK, 125I-tyro-CRH bound to the R1{alpha} receptor subtype with binding affinity comparable to 125I-tyro-UCN, as assessed by Scatchard analysis (1.25 ± 0.4 vs. 1.05 ± 0.3 nM, respectively) (Fig. 3bGo). Subsequent studies in HEK293 cells expressing various CRH-R receptor subtypes revealed that UCN was able to activate MAPK in 293-R2ß cells, but not in cells expressing the CRH-R1ß, -R1c, or -R1d receptor subtypes, even at pharmacological concentrations of urocortin (10 µM). By contrast, CRH failed to activate MAPK in all receptor subtypes tested (Table 1Go). Furthermore, although UCN has been shown to have greater binding affinity [dissociation constant (Kd) for the R2ß than the R1{alpha} receptor subtype (0.6 ± 0.2 vs. 1.9 ± 0.6 for the R2ß and R1{alpha}, respectively], the sensitivity and potency of MAPK activation was found to be similar via both receptors (sensitivity: 10 nM of UCN; potency: 3.1 ± 0.4 times above basal at a UCN concentration of 100 nM) (data not shown). In addition, in 293-R1c cells UCN or CRH was found to have very low binding affinity for UCN or CRH, and these ligands stimulated only a small cAMP response, consistent with the finding that the 40-amino acid deletion in the N-terminal domain of CRH-R1c impairs the binding ability of this receptor. Both peptides could exert a significant cAMP production only at concentrations greater than 1 µM, and maximum cAMP responses ranged between 4 ± 1.1- and 5 ± 1.5-fold above basal (at a peptide concentration of 1 µM) (Fig. 4Go). Neither CRH nor UCN elicited an increase in IP3 production or MAPK activity.



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Figure 3. Comparison of CRH and UCN Effects on MAPK Activation and Binding to CRH-R1{alpha} Receptor

A, Effect of CRH and CRH-like peptides on MAPK activation in 293-R1{alpha} (upper panel) and CHO-R1{alpha} (lower panel) cells. Cells were stimulated with agonists (100 nM to1 µM) for 10 min. After cell lysis and centrifugation, supernatants were subjected to SDS-PAGE and immunoblotted with antibody for phospho-p44/42 to determine the phosphorylated/activated p44/42 MAPK as described in Materials and Methods. Alternatively, the same samples were immunoblotted with antibody for p44/42 MAPK to determine total MAPK as a control. Identical results were obtained from three independent experiments. B, Scatchard analysis of 125I-labeled [Tyr0]-oCRH or 125I-labeled-UCN specific binding to membranes prepared from HEK-293 cells stably transfected with CRH-R1{alpha}. Results indicate the presence of a single population of high affinity receptors with comparable dissociation constants (Kd) for both peptides. Data are representative from three independent experiments for each peptide.

 

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Table 1. Effect of CRH and UCN on MAPK and Adenylyl Cyclase Activation from Various CRH Receptor Subtypes (Expressed in Human Pregnant Myometrial Cells) Individually Expressed in HEK 293 Cells

 


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Figure 4. cAMP Release from HEK-293 Cells Stably Transfected with CRH-R1c Receptor Subtype (50 µg Protein) in the Presence of Different Concentrations of h/rCRH or Urocortin

Results are expressed as the mean ± SEM of four estimations from three independent experiments. *, P < 0.05 compared with basal.

 
No MAPK activation was observed upon addition of UCN (up to 1 µM) (data not shown) to untransfected HEK293 cells.

Characterization of the Intracellular Components Involved in MAPK Activation
Because activation of the CRH-R1{alpha} receptor by CRH or UCN leads to interaction with at least four G proteins, Gs, Gi, Gq, and Go (23), we sought to identify which G proteins were involved in UCN/CRH-R1{alpha}/MAPK activation. The involvement of Gs{alpha} proteins was excluded by the finding that activation of the Gs-adenylyl cyclase-cAMP pathway (cholera toxin or forskolin) in 293-R1{alpha} cells did not induce MAPK activation. To evaluate possible involvement of Gi or Go proteins, we used pertussis toxin (PTX) (2–12 h, final concentration, 100 ng/ml) to inactivate Gi/Go proteins via ADP ribosylation. Pertussis treatment of 293-R1{alpha} cells had no effect on UCN-induced MAPK activation (Fig. 5aGo); however, in all PTX incubation periods, a reduction (P < 0.05) in basal MAPK activity was found to be present. PTX inactivation of Gi and Go was confirmed by examining the ability of UCN to induce 32P-GTP-AA incorporation to these G protein {alpha}-chains. As expected, UCN-induced incorporation of 32P-GTP-AA into the PTX-sensitive G proteins, Gi and Go, was dramatically inhibited (Fig. 5bGo) by PTX at all incubation periods used (2–12 h).



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Figure 5. Investigation of Gi/Go Protein Involvement on UCN-MAPK Interaction

A, Effect of pertussis-toxin pretreatment on UCN-induced activation of MAPK in 293-R1{alpha} cells. Cells were pretreated with or without PTX (100 ng/ml for 2–12 h) at 37 C at 5% CO2 and were then washed and stimulated with UCN (100 nM) for 10 min. After cell lysis and centrifugation, supernatants were subjected to SDS-PAGE and immunoblotted with antibody for phospho-p44/42 to determine the phosphorylated/activated p44/42 MAPK (upper panel) as described in Materials and Methods. Alternatively, the same samples were immunoblotted with antibody for p44/42 MAPK to determine total MAPK as a control. The intensity of the bands corresponding to p42 MAPK was quantitated with an imaging densitometer (lower panel). Identical results were obtained from three independent experiments. *, P < 0.05 compared with untreated basal; +, P < 0.05 compared with untreated UCN-stimulated. B, Effect of PTX pretreatment (final concentration, 100 ng/ml) on UCN-induced photolabeling (with GTP-AA) of G{alpha} i and G{alpha}o proteins from membranes prepared from 293-R1{alpha} cells. Cells were cultured in six-well plates in the presence or absence of PTX for 6 h at 37 C at 5% CO2, followed by cell membrane preparation and addition of UCN (100 nM) for 5 min at 30 C. Then GTP-AA was added, followed by UV cross-linking and immunoprecipitation of the G{alpha}-subunits using specific antibodies. Proteins were resolved on SDS-PAGE gels, followed by autoradiography and densitometry scanning for quantitation of agonist-induced photolabeling of specific G{alpha}-subunits. Identical results were obtained from six independent experiments. *, P < 0.05 compared with untreated basal.

 
These data suggested that the UCN effect might be mediated via Gq proteins and implicated involvement of the phospholipase C (PLC)-IP3-PKC signaling pathway. To confirm this, 293-R1{alpha} cells were pretreated with the PLC inhibitor U73122 (20 min, 10 µM) before stimulation with UCN (100 nM). U73122 treatment led to a 60–80% reduction in UCN-induced MAPK activation (Fig. 6aGo), confirming that activation of PLC, likely through Gq-proteins, is required for UCN action. The role of PKC in UCN activation of MAPK was investigated after prolonged stimulation with PMA (12 h, 200 ng/ml) to deplete PKC (32). In this experimental paradigm the UCN-induced increase in phospho-MAPK p42/p44 immunoreactivity was reduced by 70–90% (Fig. 6bGo), confirming that UCN activates MAPK predominantly via a PKC-mediated phenomenon. Similar results were obtained with myometrial cells and 293-R2ß cells, suggesting a common signaling pathway of myometrial R1{alpha} and R2ß receptor subtypes after stimulation by UCN (data not shown).



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Figure 6. Effect of (A) PLC Inhibition Using U73122 or (B) PKC Depletion on UCN-Induced Activation of MAPK in 293-R1{alpha} Cells

Cells were pretreated with or without PMA (200 ng/ml for 12 h) or with or without U73122 (10 µM for 20 min). Cells were then washed and stimulated with UCN (100 nM) for 10 min. After cell lysis and centrifugation, supernatants were subjected to SDS-PAGE and immunoblotted with antibody for phospho-p44/42 to determine the phosphorylated/activated p44/42 MAPK (a, b, upper panel) as described in Materials and Methods. Alternatively, the same samples were immunoblotted with antibody for p44/42 MAPK to determine total MAPK as a control (b, lower panel). The intensity of the bands corresponding to p42 MAPK was quantitated with an imaging densitometer (a, lower panel). Identical results were obtained from three independent experiments. *, P < 0.05 compared with basal; +, P < 0.05 compared with untreated UCN-stimulated.

 
In previous studies we had shown that CRH treatment of 293-R1{alpha} stimulates Gq proteins and IP3 production (23), and yet in the present study CRH failed to stimulate MAPK activity. This apparent discrepancy was investigated in more detail in experiments studying the concentration of CRH or UCN needed to activate G protein signaling. Gq protein activation was investigated by using the nonhydrolyzable GTP analog 32P-GTP-AA to label receptor-activated G proteins, followed by immunoprecipitation with specific G protein {alpha}-chain antibodies (33). As shown in Fig. 7Go, CRH or UCN exhibited substantial differences in their ability to radiolabel the 40-kDa G{alpha}q protein in 293-R1{alpha} cells. Both CRH and UCN activated Gq at a threshold concentration of about 1 nM, with maximal 32P-GTP-AA incorporation obtained at a concentration of 100 nM. However, UCN was 80–100% more potent than CRH, both at submaximal (10 nM) and maximal (100 nM) concentrations.



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Figure 7. Comparison of CRH and UCN-Induced Photolabeling with GTP-AA of G{alpha}q Proteins from HEK-293 Cells Stably Transfected with CRH-R1{alpha} Receptor Subtype

A, Autoradiograph of CRH or UCN-induced photolabeling (with GTP-AA) of G{alpha}q- proteins from membranes prepared from 293-R1{alpha} cells. Membranes were incubated with GTP-AA and different concentrations of CRH or UCN followed by UV cross-linking and immunoprecipitation of the G{alpha}q-subunits using specific antibodies. Proteins were resolved on SDS-PAGE gels, followed by autoradiography. B, Quantitation of agonist-induced photolabeling of G{alpha}q -subunits using densitometry scanning. Identical results were obtained from four independent experiments. *, P < 0.05 compared with basal; +, P < 0.05 compared with each other.

 
In addition, activation of Gq protein by CRH or UCN correlates with activation of phospholipase C and increased generation of IP3 . We found that CRH or UCN treatment stimulated a significant increase in inositol phosphate production, with a threshold of 1 nM and a maximum response at 100 nM (Fig. 8aGo). Consistent with our previous findings, the response to UCN was 60–80% more potent than that to CRH (maximum 150 ± 21% of basal for CRH and 235 ± 34% of basal for UCN), which resulted in a 95 ± 17% stimulation of basal PKC activity, whereas CRH had no effect on PKC activity (Fig. 8aGo, inset). By contrast, both peptides have comparable potency, increasing cAMP with a threshold of 100 pM and maximum response at 100 (maximum 90 ± 14-fold above basal for CRH and 104 ± 25-fold above basal for UCN) (Fig. 8bGo).



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Figure 8. IP3 Accumulation (A) and cAMP Release (B) from HEK-293 Cells Stably Transfected with CRH-R1{alpha} Receptor Subtype (50 µg Protein) in the Presence of Different Concentrations of CRH or UCN

Inset, PKC activation by CRH or UCN in 293-R1{alpha} cells. Cells were stimulated with CRH or UCN (100 nM for 10 min) and after cell lysis and centrifugation the PKC activity was determined using a nonradioactive, ELISA-based protein kinase C assay kit. Results are representative of one receptor clone and are expressed as the mean ± SEM of four estimations from three independent experiments. *, P < 0.05 compared with basal; +, P < 0.05 compared with each other.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The purpose of this study was to determine whether CRH and CRH-related peptides are able to activate MAPK in human pregnant myometrium. In cultured human pregnant myocytes, UCN, but not CRH, was a potent stimulator of MAPK activity. This effect appears to be mediated via the R1{alpha} and R2ß CRH receptor subtypes as shown by studies in which all CRH-R subtypes known to be present in the cultured myocytes were individually transfected into HEK293 cells. This is the first demonstration that the CRH-R1{alpha} receptor subtype, which binds both CRH and UCN with equipotent affinity, can differentially activate intracellular signaling cascades based on the peptide ligand, leading to either selective activation (UCN-MAPK) or nonselective activation (UCN/CRH-adenylate cyclase). This might have wider implications for understanding the biological actions of the two peptides and their receptors. Identical results were obtained in experiments using CRH-R subtypes stably expressed in CHO cells, confirming that this finding is not due to artifacts of the heterologous expression system used. In addition, the CRH-R2ß receptor subtype appeared to possess similar properties; this, however, is less surprising considering that this receptor subtype preferentially binds UCN over CRH.

Although at present the mechanism whereby the R1 receptor can exhibit such selective activation in signaling cascades is unknown, it is likely that CRH and UCN bind to distinct domains within the receptor leading to conformational changes, which facilitate coupling to particular G proteins and activation of intracellular signaling molecules. Several studies have identified important sequences for high-affinity agonist binding to CRH-R1 in the N terminus (amino acids 42–50 and 76 to 84) and in the extracellular (EC) domains of the R1 receptor (34, 35). Also, regions important for high-affinity CRH binding have been localized to the second and fourth EC domain, the junctions of the third EC domain/fifth transmembrane domain (TMD), and second EC domain/third TMD (36, 37). The same three regions have been shown to influence the binding of UCN and sauvagine, but to different degrees (38). Regions in the third EC domain, such as Asp254, appear to be important for sauvagine but not CRH or urocortin binding (38), demonstrating that different agonists differentially interact with some of the same regions of the CRH-R1.

Previous studies have demonstrated that CRH and CRH-like ligands are capable of modulating the MAPK system; in ovine anterior pituitary cells, CRH can stimulate and in mouse pituitary tumor AtT20 cells inhibit MAPK activity (39). In addition, in CHO cells stably expressing the subtypes R1{alpha} or R2{alpha}, sauvagine has been shown to activate MAPK (24). Most interestingly, in primary cardiac myocyte cultures, UCN acting via activation of MAPK is able to protect against ischemic and reperfusion injury (40). However, very little is known about the intracellular pathways involved in these events initiated by CRH receptor activation. Studies in other 7TMD receptors/MAPK interactions have shown that in different cellular systems and under certain conditions several G proteins, such as G{alpha}s, G{alpha}q, G{alpha}i, G{alpha}12, and Gß{gamma}-subunits, can regulate MAPK activity (41). In earlier studies we showed that CRH-R1{alpha} receptors that are stimulated by CRH or UCN can couple to and activate at least four different G proteins, namely G{alpha}s, G{alpha}i, G{alpha}q, and G{alpha}o (23). The data presented in this study indicate that the UCN stimulation of MAPK via R1{alpha} and R2ß CRH receptor subtypes requires G{alpha}q-, but not G{alpha}s-, G{alpha}i-, or G{alpha}o-, proteins. Further support of this signaling pathway derives from our experiments showing that the CRH receptor subtypes, R1ß and R1d, which are unable to couple to Gq protein, are unable to activate the MAPK signaling pathway. The mechanism by which PKC directly activates the MAPK cascade remains unclear; it has been shown that activated PKC can directly phosphorylate and activate Raf1 (42), by a mechanism partially dependent on Ras, leading to stimulation of the MAPK cascade (43). Interestingly, neither PKC depletion nor PLC inhibition totally blocked MAPK activation, thus raising the possibility that additional mechanisms, such as activation of ß{gamma}-subunits of G proteins in a Ras-dependent, PKC-independent process (44), which has been found to be active in HEK293 cells (45), might be involved in the UCN/MAPK interaction.

Having established that the activated CRH-R1{alpha} receptor can interact with MAPK via a Gq/PLC/PKC signal transduction pathway, we investigated ligand-R1{alpha} receptor interactions to identify differences between CRH and UCN binding that might explain the inability of CRH to stimulate MAPK phosphorylation. Although CRH and UN were equipotent in binding to the R1{alpha} and activating the Gs/AC/cAMP pathway, UCN was more potent in activating the Gq proteins and IP3 production and subsequently PKC, reinforcing the view that binding of UCN to the R1{alpha} receptor subtype causes a distinctive conformational change that preferentially couples to Gq proteins and leads to activation of the PLC/IP3/PKC/MAPK cascade (Fig. 9Go). Although the stoichiometric details of this signal generating pathway are not known, CRH-induced activation of PLC appears to be insufficient to achieve activation of PKC or MAPK. Similar findings have been previously reported in the human pregnant myometrium at term (22). This raises the possibility that CRH and urocortin, acting via the same receptor subtypes, may have distinct functional roles during human pregnancy and labor. Thus, the ligand-receptor complex, rather than the ligand alone, may play a fundamental physiological role in determining signal specificity.



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Figure 9. Schematic Representation of the Proposed Mechanism For MAPK Activation by CRH-Related Agonists in Human Pregnant Myometrium

According to this model UCN can bind with equal or higher affinity with CRH to the CRH-R1{alpha} and CRH-R2ß receptor subtypes, respectively, initiating a series of diverse intracellular events. UCN acting via both receptor subtypes can stimulate the Gq-protein/ PLC/IP3 pathway leading to MAPK activation by a PKC-dependent mechanism. CRH acting via the same receptor subtypes is able to stimulate the same intracellular pathway but with reduced potency, which appears to be insufficient for the downstream stimulation of MAPK. Both peptides can stimulate the adenylate cyclase (AC) pathway via activation of Gs proteins (with equal potency when bound to CRH-R1{alpha} or greater for UCN when bound to CRH-R2ß) leading to elevation of cAMP, but this pathway appears not to be involved in MAP kinase activation. The modulators of the intracellular components used in this study are represented in italics.

 
The UCN effect on myometrial MAPK activation was rapid and transient. This is usually associated with cellular growth and proliferation, whereas prolonged activation is involved in cell differentiation (46, 47). Activation of myometrial MAPK has also been implicated in the regulation of myometrial contractility, particularly as a target of several uterotonins such as oxytocin, prostaglandins, and endothelin-1 (27, 28, 48). Myometrial MAPK has also been shown to be activated by oxytocin via a PTX-dependent but PKC-independent mechanism, which leads to cyclooxygenase-2 expression and increased production of prostaglandins (49). These data indicate that UCN may play an important and distinct role in the regulation of myometrial contractions during labor (21). The physiological circulating concentrations of UCN do not change during pregnancy and are in the range of 2.6–3.6 pM (50). However, local production of urocortin from intrauterine tissues (19) during pregnancy raises the possibility that myometrial cells might be exposed to much higher concentrations of urocortin.

Our data demonstrate that UCN and sauvagine can interact with specific CRH receptor subtypes to acutely stimulate myometrial MAPK in part through the Gq-coupled PKC pathway. In contrast, CRH can interact with the same receptor subtypes but is unable to generate a similar effect, raising the possibility that these two ligands can induce different conformations in the same receptor with different signaling consequences. In the human pregnant myometrium, activation of MAPK appears to be involved in the development of myometrial contractility, suggesting that UCN and CRH have distinct roles in the mechanism of human labor. Further molecular characterization of the receptor-ligand complex for CRH and related peptides will help to elucidate the functional role of these peptides and provide the basis for their proposed "dual" role (maintenance of myometrial relaxation and stimulation of contractility) (16) during pregnancy and labor.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals
Radioiodinated ovine-tyr-CRH, radioiodinated UCN, human UCN, human/rat CRH, sauvagine, and oxytocin were obtained from Peninsula Laboratories, Inc. (Merseyside, UK). The mammalian expression vector pCI-neo was obtained from Promega Corp. UK Ltd (Chilworth Research Centre, Southampton, UK). Dithiothreitol, GDP, forskolin, cholera and pertussis toxins, 2-[N-morpholino]ethane sulfonic acid, 1,4-dioxane, triethylamine, and all other chemicals were purchased from Sigma Chemical Company Ltd (Poole, Dorset, UK). Waters Sep-Pak C18 columns were obtained from Millipore Corp. Ltd (Watford, Herts, UK). The PhosphoPlus p44/42 MAPK antibody kit was purchased from New England Biolabs, Inc. Ltd (Hitchin, Hertfordshire, UK). Polyclonal G-protein rabbit antibodies, [3H]myo-inositol and the cAMP assay kits were obtained from NEN Life Science Products (Hertfordshire, UK). PMA and U73122 were obtained from Calbiochem (La Jolla, CA). Protein-A Sepharose beads (CL-4B) were purchased from Pharmacia Biotech (Uppsala, Sweden). [{alpha}-32P]-GTP, enhanced chemiluminescence (ECL), the DNA sequencing kit, and 35S-{alpha}-ATP were obtained from Amersham International (Amersham Place, Little Chalfont, Buckinghamshire, UK). 4-Azidoanilide-HCl, 1-(3-dimethylamino propyl)-3-ethylenecarbodiimide hydrochloride (NDEC) was purchased from Aldrich Chemical Co. (Dorset, UK). PCR and cloning reagents were purchased from Life Technologies Ltd (Renfrewshire, UK). The DNA 3'-end labeling kit was purchased from Roche Molecular Biochemicals (Bell Lane, UK). Synthetic oligonucleotide probes and enzymes were purchased from Life Technologies (Paisley, UK). All other chemicals were purchased from BDH (Poole, UK).

Subjects and Culture of Myocytes
Pregnant myometrial biopsies (n = 7) were obtained from women undergoing elective cesarean section at term before the onset of labor for nonmaternal problems. The biopsy site was standardized to the upper margin of the lower segment of the uterus in the midline. This provides the closest approximation to the upper segment of the uterus. These studies were approved by the local ethical committee, and informed consent was obtained from all patients.

The tissue was immediately placed in 20 ml of ice-cold DMEM culture medium containing 200 IU penicillin/ml, 200 mg streptomycin/ml. Myocytes were prepared by enzymatic dispersion as previously described (29). The cells were cultured at 37 C in a humidified atmosphere of 95% air and 5% CO2 until confluent.

Stable Transfection of HEK293 and CHO Cells
Human CRH-R1 or R2 receptor subtypes (23) were subcloned into the expression vector pCI-neo (Promega Corp.), which contains the human cytomegalovirus immediate early promoter. HEK293 or CHO cells were transfected using Lipofectamine reagent (Life Technologies). The cells were grown in DMEM in the presence of G418 (500 µg/ml) to select for transfected cells and those surviving were subcultured.

Membrane Preparation and CRH RRA
Confluent HEK293 or CHO cells were washed with PBS and lysed with 0.2% NaCl. The cells were homogenized in extraction buffer A (10 mM Tris-HCl, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 10 mM MgCl2, 0.1% BSA, and 0.1% bacitracin, pH 7.2). The homogenate was centrifuged at 600 x g for 30 min at 4 C to remove nuclei and unbroken cells. The supernatant was collected and centrifuged at 40,000 x g for 60 min at 4 C. The pellet was rinsed twice, resuspended in binding buffer B (10 mM Tris-HCl, 1 mM EDTA, 10 mM MgCl2, 0.1% BSA, and 0.1% bacitracin, pH 7.2), and aliquoted (50 µg in 50 µl aliquots) in microfuge tubes.

For receptor analysis, membranes (50–100 µg of protein) were incubated with 125I-oCRH or 125I-UCN (0.2–2 nM) and unlabeled r/h CRH or UCN (1,000 molar excess) in 50 µl of binding buffer B. The tubes were incubated at 22 C for 120 min. The reaction was terminated by adding 1 ml/tube of ice-cold 20% polyethylene glycol (PEG). After centrifugation at 10,000 x g for 15 min at 4 C, the pellets were washed once with 20% PEG and radioactivity was quantified in a {gamma}-counter (Packard Instruments, Meriden, CT) at 70% efficiency. Nonspecific binding was 18 ± 5% of the total added radioactivity.

The binding data were analyzed using the EBDA program (51) and LIGAND (52) (EBDA/LIGAND, Elsevier-Biosoft, Cambridge, UK).

MAPK Activation and Western Blot Analysis
Cells (myometrial, HEK293, or CHO) were cultured in six-well dishes for 2 days in DMEM containing 0.5% FCS. The confluent cells were washed with DMEM containing 0.5% FCS and incubated in fresh medium for a further 30 min before addition of agonists. At the end of the incubation the medium was aspirated and the cells were washed twice with PBS containing 1 mM NaF. Cells were lysed by the addition of 100 µl SDS-PAGE sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% (wt/vol) SDS, 10% glycerol, and 50 mM dithiothreitol. The solubilized material was then removed from dishes and sonicated for 15 sec, heated to 95–100 C for 5 min, and cooled on ice. Before electrophoresis the extracts were centrifuged at 4,000 rpm for 5 min to remove insoluble material. After electrophoresis through 10% PAGE gels, the resolved proteins were transferred to polyvinylidene difluoride membrane at 100 mA for 90 min. The membrane was then blocked with Tris-buffered saline containing 5% nonfat dry milk and 0.1% Tween 20 at room temperature for 1 h and subsequently incubated overnight at 4 C in primary antibody solution (phospho-MAPK, 1:1000, total MAPK, 1:2000 in TBS containing 5% nonfat dry milk, 0.1% Tween-20). Polyvinylidene difluoride membranes were washed three times with TBS-Tween 20 (0.1%) and incubated with goat antirabbit antibody conjugated with horseradish peroxidase (1:1000 in blocking solution) for 1 h at room temperature. After three washes with TBS-Tween 20 (0.1%), antibody binding was detected by enhanced chemiluminescence (ECL).

Second Messenger Studies
293-R1 or 293-R2 cells were seeded in 96-well dishes and cultured until 95% confluency. Before treatments, cells were washed once with 200 µl DMEM containing 0.1% BSA, followed by preincubation with DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine for 30 min. Cells were then stimulated with hCRH or hUCN (0.1–1000 nM) for 15 min at 37 C; reactions were terminated by addition of 0.1 M HCl. After an overnight freeze/thaw cycle, the cAMP levels were measured in the supernatants using RIA. The sensitivity of the assay was 0.025 pmol/liter and the precision was as follows: intraassay CV, 2.9%; and interassay CV, 9.7%.

For the inositol phosphate stimulation assay, cells were seeded in six-well dishes and subcultured in DMEM until 95% confluency. After incubation with inositol-free DMEM containing [3H]myo-inositol (10 µCi/well) for 24 h, cells were washed with inositol-free DMEM once and preincubated with inositol-free DMEM containing 0.1% BSA and 30 mM LiCl for 30 min at 37 C. Phosphoinositide turnover was stimulated with hCRH or UCN (1–1000 nM) in the presence of 30 mM LiCl, and the reactions were terminated by addition of chloroform/methanol/hydrochloric acid (50:100:1) at specified time intervals. After transferring to borosilicate glass tubes and centrifugation, the upper phase was applied to Prefilled Poly-Prep columns (AG 1-X8 resin 100–200 mesh chloride from Bio-Rad Laboratories, Inc. York, UK), and [3H]IPs were resolved and quantified as previously described (53, 54). The radioactivity was measured by a ß-counter.

Measurement of PKC Activity
293-R1{alpha} or CHO-R1{alpha} cells were seeded in six-well dishes and cultured until 90–95% confluency (density 107cells per well). Cells were incubated with CRH or UCN (100 nM) in DMEM, for 10 min at 37 C. Cells were then collected in ice-cold PBS and after centrifugation the pellet was suspended in sample preparation buffer containing 50 mM Tris-HCl, 10 mM benzamidine, 5 mM EDTA, 10 mM EGTA, 50 mM ß-mercaptoethanol, 1 mM phenylmethylsulfonylfluoride, pH 7.5, followed by sonication on ice four to five times for 10 sec. Samples were centrifuged at 100,000 x g for 60 min at +4 C, and the PKC activity in each sample was measured using a nonradioactive, protein kinase assay kit (Calbiochem, San Diego, CA) according to the manufacturer’s instruction. This enzyme-linked immunosorbent assay (ELISA)-based assay kit utilizes a immobilized synthetic PKC pseudosubstrate and a biotinylated monoclonal antibody that recognizes the phosphorylated form of the peptide.

Synthesis of GTP-AA and Photolabeling of G{alpha} Subunits
GTP-AA was synthesized following a method described previously (23). Fractions containing GTP-AA were combined, evaporated to dryness, and stored at -70 C for up to 1 month. The overall yield of GTP-AA varied from 30–50%. All procedures were performed in a darkened room.

293-R1 or 293-R2 cell membranes (100 µg) were incubated with or without varying concentrations of h/rCRH or UCN for 5 min at 30 C before the addition of 5 µCi of [{alpha}-P32]-GTP-AA in 120 µl of 50 mM HEPES buffer, pH 7.4, containing 30 mM KCl, 10 mM MgCl2, 1 mM benzamidine, 5 µM GDP, and 0.1 mM EDTA, in a darkened room. After incubation at 30 C, membranes were collected by centrifugation and resuspended in 100 µl of the above buffer containing 2 mM glutathione, placed on ice, and exposed to UV light (254 nm) at a distance of 5 cm for 5 min.

G Protein Immunoprecipitation
GTP-AA-labeled 293-R1 or 293-R2 cell membranes were precipitated by centrifugation, solubilized, immunoprecipitated, and electrophoresed following a method described previously (23). The gels were then stained with Coomassie Blue, dried using a slab gel dryer, and exposed to Fuji Photo Film Co., Ltd (Tokyo, Japan) x-ray film at -70 C for 2–5 days.

Statistical Analysis
Data are shown as the mean ± SEM of each measurement. Comparison between group means was performed by ANOVA, and P < 0.05 was considered significant. The relative density of the bands was measured by optical density scanning using the software Scion Image-Beta 3b for Windows (Scion Corp., Frederick, MD).


    FOOTNOTES
 
Address requests for reprints to: Dr. D. Grammatopoulos, Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, The University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK. E-mail: chdg{at}dna.bio.warwick.ac.uk

This work was supported by a Wellcome Trust Career Development Award to D.G. E.H. is the Warwickshire Private Hospitals Charitable Trust Chair of Medicine.

Received for publication June 19, 2000. Revision received August 25, 2000. Accepted for publication September 14, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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