Molecular Endocrinology, doi:10.1210/me.2006-0255
Molecular Endocrinology 20 (12): 3179-3195
Copyright © 2006 by The Endocrine Society
Identification of Signaling Molecules Mediating Corticotropin-Releasing Hormone-R1
-Mitogen-Activated Protein Kinase (MAPK) Interactions: The Critical Role of Phosphatidylinositol 3-Kinase in Regulating ERK1/2 But Not p38 MAPK Activation
Anu Punn,
Michael A. Levine and
Dimitris K. Grammatopoulos
Endocrinology and Metabolism (A.P., D.K.G.), Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom; and Division of Pediatrics (M.A.L.), The Children’s Hospital of The Cleveland Clinic Foundation, Cleveland, Ohio 44195
Address all correspondence and 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, United Kingdom. E-mail: d.grammatopoulos{at}warwick.ac.uk.
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ABSTRACT
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In most target cells, activation of the type 1 CRH receptor (CRH-R1) by CRH or urocortin (UCN I) leads to stimulation of the Gs-protein/adenylyl cyclase/protein kinase A cascade. Signal transduction of CRH-R1 also involves alternative pathways such as phosphorylation of ERK1/2 and p38 MAPK, two members of the MAPK family that mediate important pathophysiological responses. The intracellular pathways by which CRH-R1 activates these MAPK are only partially understood; here we characterized further signaling mechanisms and molecules involved in CRH-R1-mediated ERK1/2 and p38 MAPK activation. In human embryonic kidney 293 cells overexpressing recombinant CRH-R1
, UCN I induced ERK1/2 and p38 MAPK activation was dependent on signaling molecules involved in agonist-induced CRH-R1 trafficking and endocytosis. Furthermore, time course studies and use of selective inhibitors demonstrated that ERK1/2 activation occured within 5 min, was sustained for at least 60 min, and was dependent on both phosphatidylinositol 3-kinase (PI3-K)/Akt activation and epidermoid growth factor receptor transactivation involving matrix metelloproteinases. UCN I effect on p38 MAPK phosphorylation was more transient, returned to basal within 40 min and was dependent on epidermoid growth factor receptor transactivation, but not PI3-K/Akt activation. Overexpression of G-transducin, showed that Gß
-subunit activation is only partially required for ERK1/2 phosphorylation and does not play a role in p38 MAPK phosphorylation, whereas overexpression of a dominant-negative Ras (Ras N17) attenuated both ERK and p38 MAPK activation. In conclusion, a complex signaling network appears to mediate CRH-R1-MAPK interactions; PI3-K might play a critical role in the regulation of CRH-R1 signaling selectivity and cellular responses.
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INTRODUCTION
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CRH RECEPTORS ARE critical for the development and integration of the mammalian response to stressful stimuli as well as many other pathophysiological processes (1, 2, 3). Two classes of mammalian CRH-Rs, termed R1 and R2, have been identified, encoded by unique genes; studies utilizing receptor knockout models (4) have clearly demonstrated that the CRH-R1 is principally responsible for mediating the CRH-driven stress response by activating the pituitary corticotrophs and ACTH secretion. The CRH-R1 has also been implicated in several pathophysiological mechanisms including depression and obesity (3).
The CRH-R1 (its human homolo termed CRH-R1), which recognizes and binds CRH as well as CRH-related peptides such as urocortin I (UCN I), belongs to the class B receptor superfamily, which includes receptors for CRH/PTH/calcitonin/pituitary adenylate cyclase activating polypeptide/GH-releasing factor/glucagon/glucagon-like peptide/secretin (5). CRH-R1 is widely expressed throughout the body and in most tissues signal transduction of CRH-R1 primarily involves coupling to the Gs-adenylyl cyclase system with subsequent cAMP generation and protein kinase A activation (2). In addition, the CRH-R1, like most heptahelical G protein-coupled receptors (GPCRs), can interact with multiple G proteins to relay signals to diverse intracellular effectors, in an agonist- and tissue-specific manner (2). Given that the CRH-R1 represents a potentially important therapeutic target, understanding the modes of signaling is of crucial importance. One of the most important signaling molecules activated by the CRH-R1 is the family of MAPK. Phosphorylation and activation of MAPK, and in particular the ERK1/2 and p38MAPK, have been implicated in mediating important pathophysiological processes regulated by CRH and CRH-related peptides, such as induction of POMC in corticotrophs (6), IL-6 production in aortic smooth muscle cells (7), Fas ligand production and apoptosis (8), RhoA/ROK and myosin light chain phosphorylation in pregnant myometrium (9), protection against glutamate-induced hippocampal neurotoxicity (10) and cardioprotective effects against ischemia reperfusion injury (11).
The intracellular pathways mediating CRH-R1-MAPK interactions are not fully understood; similar to many other GPCRs it is likely to involve multiple and diverse signling cascades. Previous studies in cells derived from human pregnant myometrium and human embryonic kidney (HEK) 293 cells overexpressing CRH-R1 have shown dependency of UCN I-induced ERK1/2 activation on the phospholipase C/protein kinase C (PKC) pathway (12). Further studies have also identified MEK1 and phosphatidylinositol 3-kinase (PI3-K) as important mediators of UCN I or CRH-ERK1/2 activation (13). The present study in HEK293 cells stably overexpressing CRH-R1, was designed to investigate the spatiotemporal characteristics of MAPK activation and characterize signaling pathways involved in UCN I-induced ERK1/2 and p38 MAPK activation.
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RESULTS
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Spatiotemporal Characteristics of UCN I-Induced ERK1/2 and p38 MAPK Activation in 293-R1 Cells
Treatment of HEK293 cells stably expressing CRH-R1 receptor (293-R1 cells) with UCN I (1–100 nM) for 5 min, increased immunoreactivity of both phospho-ERK1/2 and p38 MAPK in a dose-dependent manner (Fig. 1
, A and B). Maximal activation of phospho-ERK1 and ERK2 (by 42 ± 5 and 50 ± 7-fold, respectively) and phospho p38 MAPK (by 8 ± 2-fold), was induced with 100 nM UCN I (Fig. 1, A and B). The significant difference in the maximal level of ERK1/2 activation observed in this series of experiments compared with our previously reported findings (12) probably reflects the modified protocol employed to generate the 293-R1 cells, using the mammalian expression vector pcDNA 3.1(–), that leads to a 5- to 8-fold increase in CRH-R1 receptor expression levels [dissociation constant (Kd) 1.3 ± 0.3 nM; and maximum binding capacity (Bmax) 35–50 nmol/mg protein] (data not shown) and the higher sensitivity of the method used for detection of protein phosphorylation that employs antibodies conjugated to IRDye 800 and Alexa Fluor [near-infrared (IR) fluorophore dyes]. Time course studies demonstrated that UCN I (100 nM) effect on both ERK1/2 and p38 MAPK activation was evident within 2 min and reached maximum after 5–10 min of treatment. However, ERK1/2 activation (especially ERK2) was sustained for at least 60 min (Fig. 1C, upper panel), whereas p38 MAPK activation declined within 40 min of treatment (Fig. 1C, bottom panel). We also examined the spatiotemporal characteristics of MAPK activation using indirect immunofluorescence confocal microscopy with phospho-specific MAPK antibodies, to monitor the relative subcellular distribution of activated MAPK and CRH-Rs after agonist stimulation. In unstimulated cells, low levels of activated (phosphorylated) ERK1/2 were found, primarily localized in the cytoplasm with a small fraction also present in the nucleus (Fig. 2). As expected, CRH-R1 was primarily localized in the plasma membrane. Agonist-induced activation of CRH-R1 led to a significant increase in the amount of fluorescent signal for phospho-ERK1/2 indicating increased ERK1/2 activity. The majority of UCN I-activated phospho-ERK1/2 was persistently retained in the cytoplasm, both at an early (5 min) and late (30 min) time period of agonist stimulation, although a small fraction of phospho-ERK1/2 immunoreactivity was also found in the nucleus. Furthermore, during the early phase of ERK1/2 activation, a small amount of phospho-ERK1/2 immunoreactivity was translocated to the plasma membrane, whereas in the later stages of ERK1/2 activation, a significant pool of phosho-ERK1/2 was found to be colocalized with internalized CRH-R1. These observations were confirmed by relative quantification of cellular fluorescence spectra of multiple individual cells that were randomly selected (Fig. 2, inset).
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Fig. 1. Time and Dose-Dependent ERK1/2 and p38 MAPK Activation by UCN I in 293-R1 Cells
Left panels are representative Western blots of cells stimulated with UCN I (1, 100 nM) for 5 min (A and B) or 100 nM UCN I for various time points (2–60 min) (C). After cell lysis and centrifugation, supernatants were subjected to SDS-PAGE and immunoblotted with antibodies for phospho- and total-ERK1/2 to determine the phosphorylated/activated ERK1/2 and secondary antibodies conjugated to IRDye 800 and Alexa Fluor 680 IR fluorophore dyes as described in Materials and Methods. Alternatively, samples were immunoblotted with antibody for phospho- and total p38 MAPK. The data in right panels represent the mean ± SEM of three estimations from three independent experiments. *, P < 0.05 compared with basal.
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Interestingly, investigations of the pattern of phospho-p38 MAPK subcellular distribution revealed some notable differences. Under basal (unstimulated) conditions, phospho-p38 MAPK was barely detectable (Fig. 3). Acute UCN I treatment for 5 min significantly increased phospho-p38 MAPK immunoreactivity primarily in the cytoplasmic compartment, although a small fraction of phospho-p38 MAPK immunoreactivity was also found in the nucleus, and this pattern was not altered after 30min of agonist stimulation. Similar to ERK1/2 activation, during the early phase of p38 MAPK activation, a small amount of phospho-p38 MAPK appeared to target the plasma membrane. In contrast, during its activation period, phospho-p38 MAPK did not associate with internalized CRH-R1. These observations were confirmed by relative quantification of cellular fluorescence spectra of multiple individual cells that were randomly selected (Fig. 3, inset).
Role of CRH-R1 Internalization on UCN I-Induced ERK1/2 and p38 MAPK Activation
The confocal microscopy data raised the possibility that common mechanisms are involved in both agonist-induced CRH-R1 internalization and ERK1/2 and p38 MAPK activation. This was further investigated by inhibitors of GPCR endocytosis. The internalization of GPCRs can be inhibited by diverse agents, such as concanavalin A (con A) (14), which blocks receptor clustering, monodansylcadaverine (MDC) (15), which interferes with clathrin-mediated internalization by stabilizing clathrin-coated vesicles, and phenylarsine oxide (PAO), a general inhibitor of endocytosis forming stable ring structures with vicinal sulfhydryl groups on membrane protein (16). In preliminary experiments (Fig. 4), we confirmed that pretreatment of 293-R1 cells for 30 min with either con A (0.25 mg/ml), PAO (2.5 µM), or MDC (300 µM) significantly impaired CRH-R1 endocytosis in response to 100 nM UCN I treatment for 30 min. Furthermore, maximal activation of ERK1/2 and p38 MAPK induced by UCN I (100 nM for 5min) was attenuated by an average of 77 ± 5% and 69 ± 5% respectively, in cells pretreated with endocytosis inhibitors (Fig. 5A), suggesting that CRH-R1-MAPK interaction is dependent on mechanisms involved in receptor internalization. The possibility of nonspecific effects on the receptor function by altering the receptor-membrane interactions was examined by determination of UCN I-induced cAMP production in 293-R1 cells pretreated with endocytosis inhibitors. Our results (Fig. 5B) showed that these inhibitors did not alter the Gs-protein mediated CRH-R1 signaling and adenylyl cyclase activation, confirming that their action primarily targeted the receptor endocytosis intracellular machinery.
We also investigated the potential of ERK1/2 and p38 MAPK pathways to modulate CRH-R1 internalization through a feedback mechanism. Pretreatment of cells with 10 µM UO126 (a MEK inhibitor) for 2 h or 10 µM SB203580 (a p38 MAPK inhibitor) for 30 min did not affect CRH-R1 internalization, induced by treatment of 293-R1 cells with 100 nM UCN I for 30 min (Fig. 5C
), suggesting that activation of MAPK pathways is not required for CRH-R1 endocytosis.
G Protein-Mediated Pathways Involved in ERK1/2 and p38 MAPK Activation: Epidermoid Growth Factor (EGF)-Receptor (EGF-R) Transactivation
Our studies on the various CRH-R1 variants signaling properties have shown that although the signaling impaired receptor variant, CRH-R1ß (2) exhibits intact internalization properties (17), it is unable to activate the MAPK pathway. Thus, it is unlikely that receptor internalization is the only pathway involved in MAPK activation. In support of this, we have previously reported that in 293-R1 cells, G protein (Gq/11) and protein kinase (PKC)-dependent pathways are crucial for UCN I-induced ERK1/2 activation (12). Because the CRH-R1 can potentially relay multiple intracellular signals through interaction with different G proteins (2), we investigated contribution of alternative pathways involving receptor tyrosine kinase (RTK) such as the EGF-R.
Studies using Western blot analysis demonstrated that 293-R1 cells endogenously express EGF-Rs, and a specific anti-EGF-R antibody recognized an immunoreactive protein with apparent molecular mass of 170 kDa (data not shown). Also indirect immunofluorescence confocal microscopy studies using an EGF-R antibody showed that EGF-Rs were primarily localized on the cell surface (data not shown). The functional activity of native EGF-Rs in our cellular model, was assessed by measurement of tyrosine phosphorylation by immunoprecipitation of EGF-Rs from cell lysates and immunoblotting with an antiphosphotyrosine antibody (PY20). Indeed, EGF treatment (20 ng/ml for 5 min) induced a rapid increase in tyrosine phosphorylation levels of EGF-R (data not shown); an effect that was blocked when cells were pretreated with AG1478 (1 µM for 30 min) (a specific EGF-R kinase inhibitor). Interestingly, the immunoblotting experiments revealed phospho-EGF-R heterogeneity because the PY20 Ab recognized two protein bands with slightly different molecular weight, possibly representing distinct phosphorylation states of the immunoprecipitated EGF-R (data not shown). Furthermore, the native EGF-R appeared to be functionally linked to ERK1/2 and p38 MAPK activation, because stimulation of 293-R1 cells with EGF (20 ng/ml for 5 min) led to a rapid increase in both ERK1/2 and p38 MAPK phosphorylation (data not shown).
To test the role of EGF-R in UCN I-induced MAPK activation, 293-R1 cells were treated with AG1478 (1 µM for 30 min) before MAPK stimulation assay. In preliminary experiments it was established that AG1478 did not affect CRH-R1 Gs-signaling because pretreatment of 293-R1 cells with AG1478 did not alter UCN I-induced adenylyl cyclase activation (Fig. 6A). In contrast, pretreatment of cells with AG1478, significantly attenuated UCN I-induced maximal phosphorylation of both ERK1/2 (by 76 ± 5%) and p38 MAPK (by 66 ± 9%) (Fig. 6B), suggesting that transactivation of endogenous EGF-Rs is important for UCN I-MAPK interactions. This was further reinforced by experiments using a specific antibody, raised against the EGF-R N terminus, as a neutralizing agent to inhibit EGF-R activation (Fig. 6C); preincubation of 293-R1 cells with this antibody (10 µg/ml for 2 h), significantly impaired UCN I-induced phosphorylation of both ERK1/2 and p38 MAPK (by 70 ± 9% and 73 ± 6%, respectively). The EGF-R neutralizing Ab had similar effects on EGF-induced phosphorylation of both ERK1/2 and p38 MAPK (data not shown). Preincubation with a IgG1 Ab (isotype control) (10 µg/ml for 2 h) had no effect on UCN I activated ERK1/2 and p38 MAPK, confirming the specificity of the anti-EGF-R neutralizing Ab (data not shown).
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Fig. 6. Dependence of UCN I-Induced cAMP, ERK1/2, and p38 MAPK Activation on EGF-R Transactivation in 293-R1 Cells
A, cAMP production from HEK293 cells stably expressing CRH-R1 receptor subtype after stimulation with 1 or 100 nM UCN I for 10 min in the absence or presence of pretreatment with AG418 (1 µM) for 30 min. Results are expressed as the mean ± SEM of three estimations from three independent experiments. *, P < 0.05 compared with basal. B and C, Inhibition of UCN I-induced activation of ERK1/2 (top panel) and p38 MAPK (bottom panel) by the EGF-R kinase inhibitor, AG1478 or EGF-R neutralizing antibody. 293-R1 cells were pretreated with or without AG1478 (1 µM) for 30min or neutralizing Ab (10 µg/ml for 2 h) and were subsequently stimulated with or without UCN I (100 nM) for 5 min. After cell lysis and centrifugation, supernatants were subjected to SDS-PAGE and immunoblotted with antibodies for phospho- and total-ERK1/2 and secondary antibodies conjugated to IR fluorophore dyes as described in Materials and Methods. Alternatively, samples were immunoblotted with antibody for phospho- and total p38 MAPK. D, Effects of UCN I and EGF treatment on tyrosine phosphorylation of EGF-R. 293-R1 cells were stimulated with or without UCN I (100 nM) or EGF (20 ng/ml) for 5 min. EGF-R was immunoprecipitated (IP) from cell lysates and the phosphotyrosine levels were analyzed by immunoblotting (IB) (B, top panel). Lysates were also immunoblotted for EGF-R to ensure equal protein loading (B, bottom panel). Alternatively, cell lysates were immunoblotted by using antibodies for specific phospho-tyrosine EGF-R residues (E). The data shown are representative Western blots of three independent experiments.
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Addition of UCN I to serum-starved cells induced a striking increase in tyrosine phosphorylation of the EGF-R, indicative of EGF-R transactivation, shown by EGF-R immunoprecipitation using specific anti-EGF-R antibodies and Western blot analysis using the PY20 antibody (Fig. 6D). To define which EGF-R tyrosine residues are phosphorylated in response to UCN I/CRH-R1-induced transactivation, we used antibodies to specific phospho-tyrosine residues. As shown in Fig. 6E, EGF-R activation (by either EGF or UCN I) was associated with a rapid increase (within 5 min) in receptor phosphorylation at position Y845 (a direct Src phosphorylation site) (18), as well as Y1068 and Y1148 of the EGF-R C terminus, which are typical autophosphorylation sites (19) (Fig. 6E).
Tyrosine phosphorylation of EGF-R C terminus leads to EGF-R internalization (20). Thus, we investigated whether CRH-R1 interactions with EGF-Rs can also induce heterologous internalization of the latter. We used indirect immunofluorescence confocal microscopy with specific CRH-R and EGF-R antibodies to monitor subcellular distribution of CRH-R1 and EGF-R in response to either EGF or UCN I treatment of 293-R1 cells for various time intervals. As shown in Fig. 7A in the absence of agonist stimulation, both receptors were primarily localized on the cell surface of 293-R1 cells. Treatment of cells with EGF (20 ng/ml) elicited a redistribution of EGF-R cellular immunostaining, indicative of EGF-R internalization, without affecting CRH-R1 localization (Fig. 7, top panel). Within 5 min of EGF treatment, a significant increase in signal inside the cell was evident and after 15 min of EGF treatment the majority of EGF-Rs were found in the cytoplasm. Recycling of EGF-Rs to the plasma membrane was achieved within 45–60 min of EGF treatment indicated by a significant increase in green fluorescence at the plasma membrane (seen as yellow color in the overlap images). Activation of CRH-R1 by UCN I (100 nM) induced a similar effect on EGF-R subcellular localization; however, studies on the kinetics of EGF-Rs heterologous internalization revealed some marked differences. UCN I-induced EGF-R internalization was slower and was apparent only after 15 min of UCN I treatment (Fig. 7, bottom panel). Furthermore, after 1 h of UCN I treatment, the majority of EGF-Rs were still present in the cytoplasm and only a small fraction of EGF-Rs recycled back to the plasma membrane. UCN I treatment also induced CRH-R1 endocytosis, in a time-dependent manner, within 15–30 min of agonist treatment, in agreement with previous reports (21).
We also investigated some of the intracellular mediators of CRH-R1-EGF-R-MAPK interactions. In many RTK-MAPK interactions, the p21(Ras) family of small GTPases (H-Ras, K-Ras, N-Ras, and Rap1), is a crucial downstream intermediate by linking RTKs and all isoforms of Raf that activate MEK and MAPK (22). The involvement of Ras in CRH-R1-MAPK interactions was tested by using the inactive S17N mutant of p21(Ras) (23). Accordingly, overexpression of the dominant-negative mutant p21(Ras) led to a strong inhibition of EGF and UCN I-induced ERK1/2 phosphorylation (Fig. 8A), by 59 ± 5 and 64 ± 7-fold respectively. The inactive S17N mutant of p21(Ras) had similar effects on EGF and UCN I-induced p38 MAPK phosphorylation (data not shown), indicating that Ras is a potential mediator of both ERK1/2 and p38 MAPK activation.
One of the major mechanisms involved in the GPCR-mediated transactivation of the EGF-R is matrix metalloproteinases (MMP)-dependent release of heparin-binding EGF, which binds to and phosphorylates the EGF-R causing activation of Shc/Grb/Sos (24). This mechanism appears to be involved in CRH-R1 EGF-R interactions because pretreatment of 293-R1 cells with the broad-spectrum MMP inhibitor, GM6001 (20 µM) for 30 min attenuated, but not completely blocked UCN I-induced EGF-R phosphorylation (Fig. 8B, top panel) as well as ERK1/2 (Fig. 8B, bottom panel) and p38 MAPK (data not shown) activation by 46 ± 5% and 36 ± 3%, respectively.
Intracellular Mediators Involved in ERK1/2 and p38 MAPK Activation: the Role of PI3-K/Akt
In addition to EGF-R transactivation, we evaluated to role of PI3-K in UCN I-MAPK interactions. Pretreatment of cells with specific PI3-K inhibitors, LY294002 (50 µM for 20 min) or wortmannin (50 nM for 30 min), significantly attenuated (73 ± 6%) UCN I induced maximal (5 min) ERK1/2 phosphorylation (Fig. 9A). The role of PI3-K on UCN I-induced p38 MAPK activation was also investigated; interestingly, the ability of UCN I to activate the p38 MAPK pathway was not affected by either LY294002 or wortmannin pretreatment (Fig. 9B), suggesting that in HEK293 cells PI3-K might mediate ERK1/2 but not p38 MAPK activation. Furthermore, EGF-induced ERK1/2, but not p38 MAPK, activation appears to be PI3-K dependent because pretreatment of 293-R1 cells with LY294002 impaired by 80 ± 4% the ability of EGF (20 ng/ml for 5 min) to activate ERK1/2, without affecting EGF-induced p38 MAPK (Fig. 9C).
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Fig. 9. Effect of PI3-K Inhibition on ERK1/2 (A and C) or p38 MAPK (B) Activation by UCN I and EGF in 293-R1 Cells
HEK293 cells stably expressing CRH-R1 were pretreated with or without PI3-K inhibitors, LY294002 (50 µM) or wortmannin (50 nM) for 30 min and were subsequently stimulated with or without UCN I (100 nM) for 5 min (A and B) or EGF (20 ng/ml) for 5 min (C). After cell lysis and centrifugation, supernatants were subjected to SDS-PAGE and immunoblotted with antibodies for phospho- and total-ERK1/2 (A and C) and secondary antibodies conjugated to IRDye 800 and Alexa Fluor 680 IR fluorophore dyes as described in Materials and Methods. Alternatively, samples were immunoblotted with antibody for phospho- and total p38 MAPK (B and C). The data shown are representative Western blots of three independent experiments. Data shown in right panel of panel A represent the mean ± SEM of three estimations from three independent experiments. *, P < 0.05 compared with basal.
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Several classes of PI3-K have been identified in mammalian cells (25). Heterodimeric PI3-K and PI3-Kß, consist of p110 catalytic subunits and p85-related adapter-regulatory molecules and are regulated by receptors with tyrosine kinase activity. In contrast, PI3-K, has distinct regulatory partners (p101 or p84) and can be activated by GPCRs. HEK293 cells endogenously express multiple PI3-K catalytic subunits (namely p110, p110ß, and p110) as well as the p85-related adapter molecules, shown by immunoblotting of cell lysates using specific antibodies for the different PI3-K subunits (data not shown). To obtain some information about the PI3-K subunits involved in CRH-R1-ERK1/2 interaction, we employed indirect immunofluorescence confocal microscopy using the same Abs and monitored each PI3-K subunit subcellular localization. Our results showed that under basal conditions all catalytic subunits tested as well as p85 adapter molecule were primarily found in the cytoplasm (Fig. 10, left column). UCN I treatment (100 nM for 5 min) increased translocation to the plasma membrane of native p110 and its partner p85 as well as p110, but not p110ß (Fig. 10, right column), indicating that multiple PI3-K subunits are activated by UCN I and might mediate the intracellular actions of UCN I. These observations were confirmed by relative quantification of cellular fluorescence spectra of 20 individual cells which were randomly selected (Fig. 10, inset). These results were additionally confirmed by using a manual scoring of protein movement (0, no staining; 5, substantial membrane staining) by an independent observer. Similar experiments were performed with EGF as the stimulation agent and results showed that only p110 and p85 subunits were activated in response to EGF treatment (data not shown).
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Fig. 10. PI3-K Subunit Subcellular Distribution after UCN I Treatment in 293-R1 Cells: Visualization by Confocal Microscopy
293-R1 cells were stimulated with or without UCN I (100 nM) for 5 min. PI3-K subunits distribution was monitored over the ensuing time period by indirect immunofluorescence using specific primary antibodies for each PI3-K subunit and Alexa-Fluor 633 secondary antibody (red) as described in Materials and Methods. Cell nuclei were stained with the DNA-specific dye DAPI (blue). Inset, Representative profiles of fluorescence intensity (measured as arbitrary units) generated along the lines depicted in overlap image, by using the Image J software. Identical results were obtained from four independent experiments and at least 20 cells were examined in each experiment. Scale bar, 20 µm.
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GPCRs can activate PI3-K and ERK1/2 through a mechanism involving Gß-subunits (26). We investigated their potential role on the signaling pathways involved in CRH-R1-PI3-K-ERK1/2 interactions by employing the Gß-subunit scavenger -transducin. Increasing concentrations of an expression plasmid DNA for -transducin (0.1–16 µg) were transiently transfected and the UCN I-induced ERK1/2 phosphorylation was determined. This resulted in increasing protein levels of -transducin as determined by immunoblotting using a specific -transducin antibody (Fig. 11A). The ERK1/2 phosphorylation assay showed that expression plasmid for -transducin at concentrations greater than 1 µg significantly inhibited UCN I-induced ERK1/2 activation in a dose-dependent manner (Fig. 11B). The maximum effect (40 ± 4%) was observed at 4 µg concentration of transfected plasmid (Fig. 12B, inset) and greater concentrations did not have any additional effect despite higher levels of -transducin protein expression. Adenylyl cyclase stimulation assays showed that transfection of expression plasmid for -transducin at concentrations greater than 1 µg significantly enhanced UCN I activated cAMP production by 35 ± 6%, probably as a result of Gß-subunit sequestration and removal of their inhibitory effect on adenylyl cyclase (Fig. 11B, bottom panel). Furthermore, -transducin expression attenuated UCN I-induced translocation to the plasma membrane of native p110, shown by indirect immunofluorescence confocal microscopy (Fig. 12), without affecting UCN I-effect on p110 cellular distribution (data not shown). In agreement with our previous findings, overexpression of -transducin had no effect on UCN I-induced p38 MAPK activation (data not shown).
We also investigated the potential role of protein kinase B (PKB)/Akt as a signaling intermediate downstream of PI3-K. Using immunoblotting with a phospho-specific Akt antibody (phospho-Ser473), it was demonstrated that UCN I (100 nM) increased Akt phosphorylation in a time-dependent manner (Fig. 13A). UCN I effect was evident within 1 min, reached maximum after 2 min of treatment and was sustained for at least 10 min. Activation of Akt was required for UCN I effect on ERK1/2 phosphorylation because pretreatment of cells with a specific Akt inhibitor [IL-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate] (27) reduced by 77 ± 9%, UCN I potency to phosphorylate ERK1/2 (Fig. 13B) without affecting p38 MAPK activation (data not shown). Furthermore, pretreatment of cells with the Akt inhibitor did not affect PMA-induced ERK1/2 activation confirming the specificity of its effects (Fig. 13C).
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DISCUSSION
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The CRH-R1, like many other GPCRs, can activate ERK1/2 through a plethora of signaling pathways, involving various G proteins and intracellular mediators (12, 13). Another member of the MAPK family, p38MAPK, can also be regulated by CRH-R1 agonists (8, 28); however, the signaling pathways are not well known. Studies in mast cells suggested that cAMP and protein kinase A are important for p38 MAPK activation by CRH (29). Our study in a receptor overexpression system provides novel evidence that the human CRH-R1 has the ability to activate ERK1/2 and p38 MAPK through multiple, G protein-dependent and -independent pathways. Furthermore, signaling cascades leading to ERK1/2 or p38 MAPK activation appear to require distinct intracellular molecules, and we identified for the first time that PI3-K/Akt is important for ERK1/2 but not p38 MAPK activation by CRH-R1 agonist, suggesting a crucial role for PI3-K/Akt in regulating CRH-R1 signaling selectivity.
CRH-R1 ability to activate both types of MAPK is directly linked to receptor trafficking and endocytosis, as shown by the inhibitory effects of different pharmacological inhibitors of CRH-R1 endocytosis. This appears to be important especially for activated ERK1/2 because our experiments on the spatial distribution of phospho-ERK1/2 demonstrated significant colocalization with internalized CRH-R1 receptors. This characteristic might be an essential determinant of the duration of UCN I-induced ERK1/2 activation as well as subcellular compartmentalization and potential selection of downstream (mainly cytoplasmic) targets and ultimately ERK1/2 biological effects. In contrast, although UCN I-activated p38 MAPK also requires intact intracellular machinery involved in receptor internalization, it seems to follow a distinct pathway that does not require anchoring to the internalized CRH-R1, which might thus allow targeting of both nuclear and cytoplasmic substrates. Many GPCRs regulate MAPK activity through ß-arrestin-dependent pathways (30); thus, it is possible that ß-arrestin, which is involved in CRH-R1 desensitization and internalization (21) and also colocalizes with internalized CRH-R1 (17), plays a role in CRH-R1-ERK1/2 and p38 MAPK interactions. This will be addressed in future studies.
Receptor endocytosis is not the sole mechanism involved in CRH-R1-ERK1/2 and p38 MAPK interactions because the CRH-R1 variant, R1ß which exhibits intact internalization properties (17) is unable to stimulate MAPK pathways (12). Searching for other potential intracellular molecules mediating CRH-R1-MAPK interactions, we found that an intact EGF-R tyrosine kinase activity and downstream activation of p21(Ras) were critical for UCN I-induced ERK1/2 and p38 MAPK activation. Convergence of GPCRs and RTK signaling pathways at various points to achieve MAPK activation is now well established (31). Furthermore, our studies suggest that the robust cross-talk between CRH-R1 and EGF-R involves rapid phosphorylation of specific tyrosine residues (Y845, Y1068, and Y1148) in the EGF-R C terminus, which represent major phosphorylation sites (18, 19). EGF exerted a similar phosphorylation effect raising the possibility that CRH-R1-mediated EGF release might precede EGF-R transactivation. Tyrosine phosphorylation at specific Tyr residues leads to EGF-R endocytosis (20), and indeed UCN I action initiated heterologous EGF-R internalization, mimicking EGF-dependent internalization of the EGF-R. It should be noted that some GPCRs such as the dopamine D3R (32) are unable to induce heterologous EGF-R internalization although they use EGF-R transactivation to regulate EK1/2 activation.
Comparative studies between UCN I and EGF-induced EGF-R internalization characteristics revealed some distinct differences; EGF-R trafficking was slower in response to heterologous (UCN I induced) internalization compared with EGF. Also, the majority of heterologously internalized EGF-R failed to recycle to the plasma membrane and were retained in the cytoplasm. Evidence suggests that the fate of internalized EGF-Rs is dependant upon the specific Ser/Thr residues phosphorylated and PKC-dependent phosphyration at Thr654 diverts internalized EGF-R from a degradative lysosomal pathway to recycling endosomes (33). Therefore, it is possible that UCN I- and EGF effects involved distinct kinases pathways and EGF-R phosphorylation phenomena. Interestingly, unlike the AT(1)R (34), internalized CRH-R1 and EGF-Rs did not colocalize suggesting that the EGF-R does not participate in the protein scaffolds between internalized CRH-R1 and phospho-ERK1/2 described above.
EGF-R transactivation seems to occur through multiple pathways and for many GPCRs involves membrane-bound MMPs and subsequent processing of EGF-like precursor molecules present on the plasma membrane release of heparin-binding EGF (35). Our study provided preliminary evidence that MMPs are indeed important for CRH-R1-mediated EGF-R phosphorylation, transactivation, and ERK1/2 and p38 MAPK activation. The precise mechanisms regulating CRH-R1-MMPs interactions are unknown; other GPCRs such as the lysophosphatidic receptors activate MMPs through PKC-dependent mechanisms (36), thus, it is possible that the CRH-R1, which also activates PKC (12), uses similar mechanisms. However, the relative weak effect of the MMP inhibitor used, on UCN I-induced EGF-R transactivation and MAPK phosphorylation suggests the involvement of MMP-independent pathways. Additional mechanisms of RTK transactivation by GPCRs have been described which are MMP-independent and use different cascades such as Fyn kinase, Gi-protein/Src and protein scaffolds comprising of G protein subunits, ß-arrestin and adaptor proteins (37, 38, 39).
GPCR-dependent activation of RTKs such as EGF-R often leads to recruitment of the family of lipid kinases, PI3-Ks and activation of PKB/Akt (40), which has been shown to mediate important antiapoptotic and cell survival effects of UCN I in cardiac cells (41) In agreement with previous studies (13) our results confirm that activation of PI3-K and its downstream target Akt (through phosphorylation at Ser473 in the carboxy-terminal regulatory region) plays a major role in the signaling pathway mediating CRH-R1-dependent ERK1/2 phosphorylation. Most importantly, we provide novel evidence that these signaling molecules are not involved in CRH-R1-dependent p38 MAPK activation, thus pointing toward a crucial role for PI3-K/Akt in directing CRH-R1 signaling selectivity. This selective involvement of PI3-K/Akt in UCN I-induced ERK1/2 but not p38 MAPK activation is also present in native cells such as the uterine smooth muscle cells endogenously expressing CRH-R1 receptors (Punn, A., and D. K. Grammatopoulos, unpublished data). Interestingly, the PI3-K/Akt plays a central role in both ERK1/2 and p38 MAPK activation by Gi-coupled receptors such as the human adenosine A3 receptor (42), demonstrating the specificity of signaling pathways employed by different GPCRs to activate the MAPK and induce different cellular responses.
Most cellular systems including the HEK293 cells express multiple catalytic subunits (p110) of PI3-K, namely p110 p110ß and their regulatory subunit p85 as well as p110, offering considerable signaling versatility. Our preliminary results suggest that UCN I and CRH-R1 effects involve mobilization and translocation to the plasma membrane of at least two p110 subunits, p110 and p110. The latter pathway is dependent on Gß-subunits release, possibly through a similar mechanism originally described for muscarinic m2 receptors (26). However, the modest effect of the Gß-subunits scavenger -transducin, on UCN I-induced ERK1/2 activation compared with PI3-K inhibitors, LY294002 and wortmannin, suggests that the CRH-R1-Gß-p110-ERK1/2 pathway is not the major pathway involved in UCN I-induced ERK1/2 activation. It is possible that PI3-K activation and downstream ERK1/2 phosphorylation primarily might involve the EGF-R and p110 in a linear signaling cascade and potentially other, as yet unidentified, signaling cascades. The CRH-R1 can potentially activate multiple G proteins, including Gq/11 proteins; other Gq/11-coupled GPCRs such as the AT(1)R when overexpressed in HEK293 cells, are able to stimulate PI3-K and ERK1/2 through EGF-R-independent mechanism (24), therefore it is possible that similar mechanisms regulate CRH-R1-PI3-K subunit interactions.
In summary, we demostrated that CRH-R1-MAPK interactions involve multiple G protein-dependent and -independent pathways (Fig. 14). Receptor endocytosis, EGF-R transactivation, and downstream Ras are important for both ERK1/2 and p38 MAPK activation. Activation of ERK1/2, but not p38 MAPK, also involves activation of multiple PI3-K subunits potentially activated via distinct intracellular mechanisms, a finding that places PI3-K in a key position in modifying CRH-R1 signaling toward distinct intracellular pathways and ultimately biological responses.
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MATERIALS AND METHODS
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Plasmids
cDNAs for -transducin and dominant-negative p21(Ras)-S17N subcloned into pcDNA 3.1(+) (Invitrogen Ltd., Paisley, Scotland, UK) were purchased from Guthrie cDNA Resource Center (University of Missouri-Rolla, Rolla, MO).
Chemicals
Urocortin I (human) was obtained from Bachem UK Ltd. (St. Helens, Merseyside, UK). Antibodies for EGF-R, phospho-ERK 1/2 (Thr202/Tyr204), ERK1/2, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-Akt (Ser 473) and Akt were purchased from Cell Signaling Technology, Inc. (Danvers, MA). The mammalian expression vector pcDNA3.1(+ and –) and Lipofectamine were obtained from Invitrogen. Antibodies for the EGF-R (Western analysis), p110 catalytic subunits of PI3-K (p110, p110ß, and p110) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phosphatase and protease inhibitor cocktail sets, anti- transducin subunit antibodies. Akt inhibitor, recombinant human EGF, AG1478, LY 294002 and wortmannin, were from Calbiochem (EMD Biosciences, Inc., San Diego, CA), antiphosphotyrosine PY20 was from Zymed (Invitrogen) and antiphospho-EGF receptor (Y845, Y1148, and Y1068) were purchased from Biosource International (Biosource International Inc, Camarillo, CA). The antibody for the p85 regulatory subunit of PI3-K was purchased from Upstate/Chemicon (Chandlers Ford, Hampshire, UK), Monodansylcadaverine, phenylarsine oxide and concanavalin A were all purchased from Sigma (Sigma-Aldrich Co. Ltd., Gillingham, Dorset, UK).
Stable Cell Lines Expressing CRH-R1
Human CRH-R1 cDNAs were transfected in HEK293 cells using Lipofectamine reagent as previously described (12). Cells were grown in a culture medium consisting of high-glucose DMEM (Invitrogen) containing Glutamax, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin. For generation of cell lines stably expressing CRH-R1 (293-R1), transfected cells were grown in DMEM in the presence of G418 (500 µg/ml) to select for transfected cells and those survived were subcultured and maintained in DMEM containing 200 µg/ml of G418 (Invitrogen). In some experiments, 293-R1 cells were transiently transfected with plasmids expressing dominant-negative H-Ras (S17N) (3 µg) or different amounts of -transducin using Lipofectamine 2000 Reagent and Opti-MEM-1 (Invitrogen) following the manufacturer’s instructions. Mock transfections were performed in parallel with empty vector.
cAMP Assays
cAMP stimulation assays of HEK293 cells stably expressing CRH-R1 receptors were carried out as previously described (12). cAMP production was measured using a cAMP RIA kit (Dupont-NEN, Stevenage, Hertfordshire, UK).
Treatments with Signaling Pathways Activators/Inhibitors-MAPK Phosphorylation Assay
Cells cultured in six-well or 100-mm dishes were grown to 70–80% confluency and serum starved for 18 h. On the day of treatment, the cells were washed in fresh DMEM and pretreated with the various inhibitors 20–30 min before ligand stimulation. PI3-K was inhibited with LY294002 (50 µM) or Wortmannin (50 nM); EGF-R tyrosine kinase was inhibited with AG 1478 (1 µM); Akt was inhibited with 1L-6 hydroxymethyl-chiro-inositol 2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (5 nM–5 µM). Receptor internalization was inhibited by pretreatment with phenylarsine oxide (2.5 µM), monodansylcadaverine (300 µM) or concanavalin A (0.25 mg/ml). Cells were stimulated with UCN I (1–100 nM) or EGF (20 ng/ml) for the indicated time points (see figure legends for description). All pretreatments and stimulations were carried out at 37 C in a humidified incubator at 5% CO2. After treatments, cells were washed with ice-cold PBS and lysed in RIPA lysis buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 0.25% Na-deoxycholate, 1% Nonidet P-40, 5 mM EDTA and 1:100 dilution of protease and phosphatase inhibitor cocktail set (Calbiochem, EMD Biosciences, Inc., San Diego, CA). After protein concentration determination for each sample (BCA protein assay kit, Pierce, Rockford, IL), cell lysates were mixed with Laemmli sample buffer. For MAPK activation assays, cells were lysed directly in Laemmli sample buffer. Cell lysates were then sonicated for 20 sec, heated to 95–100 C for 5 min, and then cooled on ice. Samples were resolved by SDS-PAGE (8–10%) and transferred to nitrocellulose membranes at 100 mA for 60 min as previously described (12).
For visualization of proteins with the Odyssey Infrared Imaging System (LI-COR Biosciences, Cambridge, UK), membranes were incubated with Odyssey blocking buffer (LI-COR Biosciences, for 1 h at room temperature and then incubated overnight at 4 C with a 1/1000 dilution of primary antibodies (for p110ß, p110, p85, phospho-p38 MAPK, and total p38 MAPK). For ERK1/2 activation, membranes were incubated simultaneously with phospho- and total-ERK1/2 antibodies, allowing the concomitant detection of phospho- and total-ERK1/2. Membranes were washed four times for 10 min with TBS containing 0.1% Tween 20 (TBS-T), then further incubated in the dark for 1 h at room temperature with IRDye 800-conjugated goat antirabbit IgG (Rockland Immunochemicals, Gilbertsville, PA) and/or Alexa Fluor 680 conjugated goat antimouse IgG (Molecular Probes, Invitrogen), diluted 1/6000 in Odyssey blocking buffer (LI-COR Biosciences). After four subsequent washes with TBS-T, proteins were detected and quantified with the Odyssey Infrared Imaging System (LI-COR Biosciences).
For detection of protein bands using enhanced chemiluminescence, membranes were blocked for 1 h at room temperature with 5% BSA in TBS-T, incubated overnight at 4 C with primary antisera diluted 1:500 (EGF-R) or 1:1000 (p110), washed four times for 10 min with TBS-T, then further incubated for 1 h at room temperature with horseradish peroxidase-conjugated Igs (1:2000) (DAKO, Glostrup, Denmark). Membranes were washed as before with TBS-T, with a final 5-min wash in TBS. Blots were then treated with enhanced chemiluminescence according to the manufacturer’s instructions (Amersham Biosciences, Buckinghamshire, UK) and exposed to x-ray film (Fuji, Tokyo, Japan).
Immunocytochemistry and Confocal Microscopy
HEK293-R1 cells, seeded on glass coverslips pretreated with 3-(aminopropyl)triethoxy silane, were grown in six-well plates until 70–80% confluency. After treatments, cells were fixed with 4% paraformaldehyde in PBS and nonspecific binding was reduced by incubating cells with 3% BSA in PBS-Triton X-100 (0.01%) for 1 h at room temperature. Cells were washed three times for 5 min with PBS-Triton X-100 (0.01%) then incubated with antiserum overnight. With the exception of phospho-MAPK antibodies, which were diluted 1/200 and incubated with fixed cells at room temperature, all other primary antibodies were diluted 1:100 and incubated at 4 C. Cells were then washed with PBS-Triton X-100 as before and incubated with a 1/400 dilution of Alexa Fluor 633 or 488 conjugated secondary antibodies (Molecular Probes, Invitrogen) for 1 h at room temperature. Coverslips were washed with PBS and mounted in SlowFade Gold antifade reagent (Molecular Probes, Invitrogen) or VECTASHIELD Mounting Medium with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc., Orton Southgate, Peterborough, UK) on microscope slides. Cells were examined under an oil immersion objective (x63) using a Leica model DMRE laser scanning confocal microscope (Leica, Milton Keynes, UK) with TCS SP2 scan head using the appropriate filters. Optical sections (0.5 µm) were taken and representative sections corresponding to the middle of the cells were presented. Fluorescence intensity was analyzed and quantified using Image J software developed at the National Institutes of Health (NIH) (http://rsb.info.nih.gov/ij/). No specific fluorescence was observed in untransfected HEK293 cells or in 293-R1 cells treated with secondary Alexa-Fluor antibodies only (data not shown).
For each treatment, between 20 and 30 individual cells in five random fields of view, randomly selected and examined. Fluorescence intensity profiles indicative of protein movement were generated along multiple line axes, analyzed and quantified using Image J software. In addition, qualitative (visual) examination of images and manual scoring of protein movement, (0-no staining, 5-substantial staining) was also carried out in a blind fashion by an independent Biomedical Laboratory Officer of the Molecular Pathology Laboratory, Division of Pathology, University Hospitals Coventry and Warwickshire, National Health Service Trust.
EGF-R Immunoprecipitation
After appropriate treatment, cell cultures grown on 100-mm plates were rinsed twice with ice-cold PBS and then lysed in RIPA lysis buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 0.25% Na-deoxycholate, 1% Nonidet P-40, 5 mM EDTA, 1 mM AEBSF, 0.8 µM aprotinin, 50 µM bestatin, 15 µM E-64, 20 µM leupeptin, 10 µM pepstatin, 1 mM NaF and 1 mM Na-orthovanadate. Cells were disrupted on ice by repeated passage through a 21-gauge needle, clarified by centrifugation at 8000 x g for 10 min, then precleared with agarose. After protein determination with a BCA protein assay kit, cell lysates were then incubated with specific antibodies and protein G agarose. The immunoprecipitates were collected, washed four times PBS before being resuspended in Laemmli sample buffer. After heating at 95 C for 5 min, the samples were centrifuged briefly and the supernatants analyzed by SDS-PAGE on 8% gels.
Statistics
The results obtained are presented as the mean ± SEM of each measurement. Data were tested for homogeneity and comparison between group means was performed by one or two-way ANOVA and probability values of P < 0.05 were considered to be significant.
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FOOTNOTES
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This work was supported by a Wellcome Trust University Award (to D.G.). A.P. is a Wellcome Trust VIP Fellow.
The authors have nothing to disclose.
First Published Online September 7, 2006
Abbreviations: CRH-R1, Type 1 CRH receptor; con A, concanavalin A; DAPI, 4',6-diamidino-2-phenylindole; EGF, epidermoid growth factor; EGF-R, EGF receptor; GPCRs, G protein-coupled receptors; HEK, human embryonic kidney; IR, near-infrared; MDC, monodansylcadaverine; MMPs, matrix metelloproteinases; PAO, phenylarsine oxide; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PKC, protein kinase C; PY20, antiphosphotyrosine antibody; RTK, receptor tyrosine kinase; UCN I, urocortin.
Received for publication June 20, 2006.
Accepted for publication August 30, 2006.
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ABSTRACT
INTRODUCTION
