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Endocrinology Vol. 140, No. 4 1525-1536
Copyright © 1999 by The Endocrine Society

ARTICLES

Corticotropin-Releasing Factor Type 1 and Type 2{alpha} Receptors Regulate Phosphorylation of Calcium/Cyclic Adenosine 3',5'-Monophosphate Response Element-Binding Protein and Activation of p42/p44 Mitogen-Activated Protein Kinase

C. J. Rossant, R. D. Pinnock, J. Hughes, M. D. Hall and S. McNulty

Parke-Davis Neuroscience Research Center, Cambridge, United Kingdom CB2 2QB

Address all correspondence and requests for reprints to: Dr. S. McNulty, Parke-Davis Neuroscience Research Center, Robinson Way, Cambridge, United Kingdom CB2 2QB. E-mail: shaun.mcnulty{at}wl.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CRF exerts a key neuroregulatory control on the function of the hypothalamic-pituitary-adrenal axis. These effects are thought to be mediated primarily through activation of Gs-coupled plasma membrane receptors. In the present study, we investigated the effects of activation of CRF receptors by sauvagine on signaling pathways that converge on phosphorylation of the transcription factor calcium/cAMP response element-binding protein (CREB). Studies were undertaken using CHO cell lines transfected with either rat CRF-1 or CRF-2{alpha} receptors. Signaling pathways were investigated using immunocytochemical, Western blot, and imaging techniques. Treatment with sauvagine increased phosphorylation of p42/p44, but not of p38 or stress-activated protein kinase (SAPK)/JUN N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases correlating with increased p42/p44 MAP kinase activity. Mobilization of intracellular Ca2+ stores was observed in cells treated with high concentrations (100 nM, 1 µM) of sauvagine. A time- and dose-dependent increase in phosphorylation of the transcription factor CREB was observed in cultures treated with sauvagine. Phosphorylation of CREB occurred at lower concentrations of sauvagine than those required to mobilize intracellular calcium stores, and phosphorylation was not blocked by the mitogen-activated protein kinase kinase inhibitor PD98059 at a concentration (1 µM) that fully inhibited phosphorylation of MAP kinase. Cotreatment of cultures with the protein kinase A inhibitor H89 (10 µM) blocked fully the stimulatory actions of sauvagine (0.1 nM, 1 nM) on phosphorylation of CREB, but not those on phosphorylation of MAP kinase. Phosphorylation of MAP kinase was partially blocked by the phosphoinositide 3-kinase inhibitor LY294002 (5 µM) and by the phosphoinositide-phospholipase C inhibitor U73122 (10 µM). These data demonstrate that cAMP-, Ca2+-, and MAP kinase-dependent signaling pathways are activated by stimulation of CRF-1 and CRF-2{alpha} receptors. However, in these cells, only protein kinase A-dependent pathways contribute significantly to enhanced phosphorylation of CREB. These represent the first reported observations of CRF receptor-mediated phosphorylation of the transcription factor CREB and activation of MAP kinase signal transduction pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRF, A 41-amino acid polypeptide originally isolated from ovine hypothalamus (1), plays a central role in controlling the function of the hypothalamic-pituitary-adrenal axis, mediating the endocrine response to stressful stimuli (2, 3, 4). Hypothalamic neurons release CRF into the hypophyseal portal system in response to stress, causing a release of ACTH from the pituitary, which, in turn, initiates the release of glucocorticoid from the adrenal gland (3, 5, 6). The cellular effects of CRF are mediated by high affinity receptors of two main types, CRF-1 and CRF-2, which exists in three alternative splice variant forms, CRF-2{alpha}, CRF-2ß, and CRF-2{gamma} (7, 8, 9). In addition to the anterior and intermediate lobes of the pituitary (3), CRF receptors are located in a wide variety of locations, including cerebellum, cerebral cortex, olfactory bulb, amygdala, and spleen (10, 11, 12, 13). The wide spread distributions of both CRF-1 and CRF-2{alpha} receptors in the central nervous system are consistent with a general neuromodulatory role within the brain in addition to control of the endocrine stress response.

The intracellular signaling pathways used by CRF in the anterior pituitary have been extensively studied (14, 15, 16). In this tissue, receptor activation by CRF causes Gs-mediated stimulation of adenylyl cyclase leading to increased levels of the intracellular second messenger cAMP. Further studies have shown that activation of CRF receptors within the central nervous system stimulates cAMP production (17, 18). In addition to increased production of cAMP, activation of CRF receptors has also been shown to increase hydrolysis of phosphatidylinositol 4,5-bisphosphate (19, 20), leading to elevated levels of intracellular free calcium ([Ca2+]i) (20). The hydrolysis of inositol lipids leads to the formation of several distinct second messengers, including diacylglycerol (21), raising the possibility of simultaneous CRF-mediated activation of multiple signaling pathways. CRF-stimulated production of cAMP has also been shown to increase [Ca2+]i indirectly through protein kinase A (PKA) modulation of voltage-sensitive calcium channels (22), demonstrating CRF-mediated cross-talk between distinct signaling pathways. However, the mechanisms by which CRF receptor agonists regulate cellular actions downstream from control of second messenger levels are poorly understood.

Increased cytosolic levels of cAMP and/or Ca2+ lead to the activation of several intracellular kinases, including PKA and Ca2+/calmodulin-dependent kinase II, which are able to phosphorylate the calcium/cAMP response element-binding protein (CREB) at Ser133 and thereby cause activation. This phosphorylated form of the transcription factor CREB (pCREB) is then able to regulate the transcription of genes containing the calcium/cAMP response element (CRE) (23, 24), including c-fos (25). In addition to cAMP- and calcium-dependent signaling pathways, activation of mitogen-activated protein (MAP) kinase pathways increases phosphorylation of CREB through activation of a distinct CREB kinase, RSK2 (26, 27). Activation of MAP kinase also leads to phosphorylation of the transcription factor Elk1. Modulation of gene expression by CREB may involve it binding to and interacting with additional nuclear proteins (28), including the CREB-binding protein. Therefore, both cAMP-dependent and cAMP-independent signaling pathways converge to modulate CREB phosphorylation (29). As CRF has been proven to activate signaling pathways that converge on cellular kinases known to phosphorylate CREB, it is possible that CREB provides a link between CRF receptor activation and control of changes in gene expression.

The aim of the present study was to investigate the effect of activation of both CRF-1 and CRF-2{alpha} receptors on phosphorylation of CREB and activation of MAP kinases and to characterize the possible signal transduction pathways responsible for mediating phosphorylation of CREB. Phosphorylation of CREB was examined using antisera raised against a synthetic peptide containing the phospho-Ser133 residue to perform Western blot and immunocytochemistry. Changes in [Ca2+]i were estimated by fluorescence imaging, and activation of MAP kinase was assessed using Western blot analysis. Experiments using binding studies and functional assays of cAMP production had confirmed the validity of the CHO-CRF-1 and CHO-CRF-2{alpha} cell lines. Competition binding studies using the radioligand [125I-Tyr0]sauvagine revealed rank orders of potency for the CRF-1 receptor of sauvagine = astressin = urocortin = rat/human CRF = ovine CRF > {alpha}-helical CRF, and for the CRF-2{alpha} receptor of sauvagine = astressin = urocortin > {alpha}-helical CRF > rat/human CRF > ovine CRF. Assays of receptor-mediated stimulation of cAMP demonstrated a rank order of potency for the CRF-1 receptor of sauvagine = rat/human CRF = ovine CRF, and for the CRF-2{alpha} receptor of sauvagine > rat/human CRF > ovine CRF, confirming the utility of the rat CRF-1 and rat CRF-2{alpha} cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell line preparation and growth conditions
Cells were transfected with either subcloned CRF-1 or CRF-2{alpha} receptors and were maintained in MEM{alpha} medium supplemented with 10% FCS at 37 C in an atmosphere containing 5% CO2. For immunocytochemical analysis and measurements of intracellular free calcium concentration, cells were seeded at a density of 1.5 x 105 onto sterile coverslips contained in six-well plates and incubated overnight at 37 C with 5% CO2 and 95% humidity to permit the cells to adhere. Cells were used after 48 h in culture. For Western blot analysis, cells were plated at a density of 2.5 x 105 into six-well plates and incubated overnight at 37 C with 5% CO2 and 95% humidity. Cells were used after 48 h in culture.

Western blot analysis of CREB and MAP kinase phosphorylation
Western blots for CREB immunoreactivity (CREB-ir) were performed using a validated, commercially available kit (New England Biolabs, Inc., Beverley, MA). Cells were maintained as described, then starved of serum by washing three times in serum-free medium and incubated in 2 ml medium for 30 min before experimentation. Stock solutions of agonists and antagonists were added to a given well, and cells were incubated for the required experimental period at 37 C and 5% CO2. At the end of this period medium was aspirated, and cells were washed once in PBS containing 1 mM NaF before extraction in 200 µl SDS-PAGE sample buffer. Extracts were then sonicated for 5 sec and centrifuged at 4000 rpm to remove debris. Samples (15 µl) were loaded onto the stacking gel lanes of 10% SDS-PAGE minigels (Novex, San Diego, CA), and proteins were separated using a Novex XCell II Mini-Cell electrophoresis system for 2 h at 100 V. Proteins were then transferred (25 V, for 2 h) to nitrocellulose membranes (Novex) before detection. Membranes were incubated for 1 h at 20 C in blocking solution (10 ml; Tris-buffered saline containing 5% dried milk and 0.1% Tween-20) and then incubated overnight at 4 C in primary antibody solution [10 ml; pCREB, 1:1000; total CREB, 1:2000; phospho-MAP kinase, 1:1000; total MAP kinase (phosphorylation state-independent antiserum, raised against residues 345–358 of rat p42 MAP kinase), 1:2000; phospho-Elk1, 1:1000 in TBS containing 5% BSA and 0.1% Tween-20]. The next day, antiserum was removed, and blots were washed three times in TBS (10 ml) containing 0.1% Tween before incubation with secondary antibody (10 ml; 1:1000 horseradish peroxidase-linked goat antirabbit, in blocking solution, New England Biolabs, Inc.) for 30 min at room temperature. Blots were then rinsed three times for 5 min each time in TBS (10 ml) and a further three times in washing buffer (10 ml; New England Biolabs, Inc.) before development using a modified enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL).

Western blot analysis of MAP kinase assay
Measurement of activity was carried out using the p44/42 MAP kinase immunoprecipitation assay kit (New England Biolabs, Inc.). After agonist stimulation, cells were rinsed with PBS, and 0.5 ml cell lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM sodium orthovanadate, and 1 µg/ml leupeptin] was added. The cells were incubated on ice for 5 min, then harvested. Cell lysates were transferred to tubes and sonicated four times for 5 sec each time. Cell debris was removed by centrifugation (12,000 x g, 4 C, 10 min). Phospho-MAP kinase p44/42 antiserum (Thr202, Thr204 phosphorylation site-specific antiserum, 1:100 dilution) was added to 200 µl supernatant and incubated overnight at 4 C with gentle shaking. Protein A-Sepharose beads were added (20 µl of a 50% bead solution in PBS), and the mixture was incubated for a further 3 h at 4 C. Samples were centrifuged (2400 x g, 1 min) and washed twice with 0.5 ml lysis buffer. The pellet was then washed twice with kinase buffer [25 mM Tris (pH 7.5), 5 mM ß-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate, and 10 mM MgCl2], and the pellet was suspended in 50 µl kinase buffer containing 100 µM ATP and 1 µg Elk1 fusion protein. Samples were incubated for 30 min at 30 C, and the reaction was terminated with 2 x SDS-PAGE sample buffer (Novex). Samples were Western blotted and probed using phospho-Elk1 antiserum specific for the Ser383-phosphorylated form of Elk1.

Analysis of Western blot immunoreactivity
Analysis was undertaken essentially as described previously (30). Briefly, individual bands were viewed on an image analysis system consisting of a monochrome video camera (Dage MTI CCD 72; MTI, Michigan City, IN) connected to an Inter Focus Ltd. (MCID Imaging Research Inc., St. Catharines, Ontario, Canada) image analysis system. To quantify the relative intensity of a given band, the entire band was selected, and the average intensity of signal above mean background was expressed as a relative optical density (ROD) value. Further measurements were made at other points on the lane free from identifiable immunoreactivity, and these values were subtracted from the measurements obtained for individual bands to assess the intensity of specific immunoreactivity.

Immunocytochemical analysis of CREB phosphorylation
Immunocytochemistry and analysis were undertaken as described previously (31, 32). Briefly, cultures were starved of serum for 30 min. To begin treatment, 2 ml of agonist were added to given wells. Incubations were terminated by rinsing coverslips twice with PBS and then fixing with 1 ml 4% paraformaldehyde for 30 min at room temperature. The cells were then washed twice for 5 min each time in 2 ml glycine solution (10 mM glycine in PBS) and then placed in 2 ml blocking solution (PBS containing 2% normal goat serum and 0.3% Triton X-100) for 30 min. Blocking solution was replaced with 1 ml primary antibody solution (pCREB, 1:1000; New England Biolabs, Inc.; in PBS containing 5% BSA and 0.3% Triton X-100). Coverslips were incubated overnight at 25 C, washed twice with PBS for 10 min, and 1 ml biotinylated secondary antibody (Vector Laboratories, Inc., Burlingame, CA) was added to each well for 60 min at room temperature. The coverslips were washed twice for 10 min each time in PBS, and immunoreactivity was detected by a standard Vectastain ABC reaction using diaminobenzidine as chromogen (Vector Laboratories, Inc.).

Densitometry of nuclear immunoreactivity in cell populations
Total nuclear pCREB-ir was quantified using image analysis and densitometry as described previously (30, 31, 32). Coverslips were viewed on an image analysis system consisting of a monochrome video camera (Dage MTI CCD 72) connected to an Inter Focus Ltd. MCID image analysis system. To quantify the relative degree of immunostaining, ROD measurements were made of the staining found in individual nuclei chosen at random from the total cell population on the coverslip. In all cases, the level of illumination was initially set so that the background measure through the coverslip in an area free of cells was consistent, in order that measurements between coverslips were comparable. The relative intensity of the reaction product in any given nucleus that passed through a random transect line was recorded from a total of 50 nuclei from any given coverslip. Measurements were also made along transect lines at further points free of cells to calculate an average background mean for each coverslip. These background values were subtracted from the individual measurements obtained from each nucleus sampled on a given coverslip. The final values obtained from experimental coverslips were expressed as mean (ROD) values ± SEM from a single representative experiment. All control and experimental cultures were processed identically and simultaneously for ICC so that direct experimental comparisons could be made. Experiments were undertaken at least four times with different cell preparations and produced ROD values that were consistent between as well as within studies and gave similar experimental outcomes. The effects of various treatments were determined by t test and ANOVA, and differences between experimental groups were assessed by post-hoc Dunnett’s t test. For a given result, * indicates significance at the P < 0.05 level, and ** corresponds to significance at the P < 0.01 level.

Analysis of changes in [Ca2+]i
Coverslips containing cells were prepared and maintained as described. Cultured cells (grown attached to 22-mm diameter coverslips) were washed twice in a Krebs-HEPES extracellular medium buffer (EM; NaCl, 118 mM; KCl, 4.7 mM; MgSO4, 1.2 mM; CaCl2, 1.2 mM; KH2PO4, 1.2 mM; HEPES, 10 mM; glucose, 11 mM; BSA, 0.1%; pH 7.2 at 20 C) (33) and then loaded with fura-2 (34) by incubation for 3 h at 20 C with EM containing fura-2/AM (2 µM; Molecular Probes, Inc., Eugene, OR). This procedure enables the cells to load with fura-2/AM, which becomes hydrolyzed to the free acid form once inside the intact cells. After loading, coverslips were mounted into imaging chambers and perfused with EM to remove extracellular fura-2/AM and to allow hydrolysis of intracellular fura-2/AM to occur. Measurements of changes in the free [Ca2+]i in individual cells were made from the fluorescence ratio (excitations, 340 nm/380 nm; emission, >510 nm) using a spectral Wizard monochromator, cooled integrating CCD camera, and a dedicated suite of software (Merlin, Life Sciences Resources, Cambridge, UK). Data are expressed as the ratio of 340/380 nm units.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of sauvagine on the phosphorylation of CREB
Experiments were undertaken to investigate whether receptor activation would cause increased phosphorylation of the transcription factor CREB. The effects of sauvagine on nuclear pCREB-ir assayed by immunocytochemistry are shown in Fig. 1Go (a–f). Basal unstimulated levels of pCREB-ir were low (Fig. 1aGo); however, stimulation of CHO-CRF-1 cultures with sauvagine (1 µM, 10 min) caused a large increase in immunoreactivity that was restricted to the cell nucleus (Fig. 1bGo). Identical results were observed after treatment of CHO-CRF-2{alpha} cultures with sauvagine (data not shown). Analysis of nuclear immunoreactivity demonstrated that sauvagine caused both a time (Fig. 1cGo)- and dose (Fig. 1eGo)-dependent increase in pCREB-ir in CHO-CRF-1 cultures and also in CHO-CRF-2{alpha} cultures (Fig. 1Go, d and f).



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  		Figure 1. Nuclear pCREB-ir of sauvagine-treated CHO-CRF-1 and CHO-CRF-2{alpha} cultures visualized by immunocytochemistry and quantified using image analysis techniques. a and b show representative photomicrographs of CHO-CRF-1 cells starved of serum for 30 min and then either incubated in serum-free medium (a) or treated with (1 µM) sauvagine for a 10-min period (b) before staining. Cells were stained using pCREB-specific antiserum. Scale bar = 10 µM. c and d show the RODs of nuclear pCREB-ir in CHO-CRF-1 and CHO-CRF-2{alpha} cells, respectively, treated with 10 nM sauvagine for varying time intervals. e and f show the RODs of nuclear pCREB-ir in CHO-CRF-1 and CHO-CRF-2{alpha} cells, respectively, treated with varying concentrations of sauvagine for a 15-min period. Values represent the mean ROD ± SEM (n = 50) from results obtained in a single experiment repeated at least three times with similar results.

 
Western blot analysis confirmed the identity of nuclear pCREB-ir (Fig. 1bGo) as the phosphorylated form of the protein CREB (Fig. 2Go, a–d). Blots probed using an antiserum that recognizes both phosphorylated and nonphosphorylated forms of CREB did not show a systematic increase in immunoreactivity on treatment of CHO-CRF-1 and CHO-CRF-2{alpha} cultures with 1 µM sauvagine for varying time intervals (Fig. 2Go, c and d). These results demonstrate that de novo CREB synthesis does not account for the observed increase in pCREB-ir. The consistency of the total CREB-ir band between cell extracts from a given experiment provided an index of equal extraction and preparation of samples and was used as an internal control in all experiments. Sauvagine caused a time-dependent increase in pCREB-ir in two major bands with apparent molecular masses of 43 and 38 kDa, consistent with published reports (31). The 43-kDa band was recognized by both CREB- and pCREB-specific antiserum, confirming its identity as native CREB; the lower molecular mass band probably represented an additional unidentified CREB family member, potentially phospho-ATF-1. The increase in phosphorylation of CREB was apparent by 1 min of stimulation, becoming maximal between 15–30 min, with immunoreactivity remaining elevated after 60 min of stimulation (Fig. 2Go, a and b). Treatment with sauvagine for different time periods was without effect on either CREB phosphorylation or total CREB protein in the parental CHO-Pro5 cell line, confirming the specificity of sauvagine-induced phosphorylation of CREB in CRF-1 and CRF-2{alpha} cultures (Fig. 2Go, e and f). Treatment of CHO-CRF-1 and CHO-CRF-2{alpha} cultures with various concentrations of sauvagine for 15 min caused a dose-dependent increase in phosphorylation of CREB (Fig. 3Go, a and b). Analysis of the dose-dependent increase in pCREB-ir from three independent experiments revealed EC50 values of 0.106 ± 079 and 0.04 ± 0.073 nM for CRF-1 and CRF-2{alpha} receptors, respectively (Fig. 3Go, c and d). These were comparable to the EC50 values for sauvagine stimulation of cAMP production of 1.27 nM (range, 0.69–2.85 nM) for CHO-CRF-1 and 0.16 nM (range, 0.14–0.23 nM) for CHO-CRF-2{alpha} (n = 3 independent experiments; data not shown). The stimulatory effect of sauvagine (1 µM) was not apparent in the CHO-Pro5 parental line from which the CHO-CRF-1 and CHO-CRF-2{alpha} cultures were derived (Fig. 4aGo). However, forskolin (1 µM), 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM), and ionomycin (1 µM) were all able to increase significantly the phosphorylation of CREB in the CHO-Pro5 line, confirming the ability of CREB to undergo phosphorylation in this cell type (Fig. 4aGo). In addition to sauvagine, TPA (100 nM), forskolin (1 µM), and ionomycin (1 µM) all significantly increased phosphorylation of CREB in CHO-CRF1 and CHO-CRF-2{alpha} cultures (Fig. 4Go, b and c, respectively).



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  		Figure 2. Western blot analysis of CHO-CRF-1 (a and c), CHO-CRF-2{alpha} cultures (b and d), and CHO-Pro5 cultures (e and f) treated with sauvagine (1 µM) for varying time intervals and immunostained using antiserum specific for either total CREB protein (CREB) or the Ser133-phosphorylated form of CREB (pCREB). C, Unstimulated cultures; -ve, unstimulated NEB-CREB kit control; +ve, agonist-treated NEB-CREB kit control. Cells were starved of serum for 30 min before incubation with 1 µM sauvagine for varying time intervals. Immunostaining using CREB-specific antiserum (c–e) revealed no change in the absolute level of CREB protein (43 kDa) in any cell line and confirmed the identity of the 43-kDa band observed in Western blots using pCREB-specific antisera (a, b, and f) as the phosphorylated form of CREB. Blots presented are representative of three independent experiments.

 


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  		Figure 3. Levels of pCREB-ir in CHO-CRF-1 (a) and CHO-CRF-2{alpha} cultures (b) treated with various concentrations of sauvagine. Cultures were starved of serum for 30 min, then treated for a 15-min period with agonist before extraction of cellular proteins. The relative amounts of pCREB-ir were analyzed and plotted as ROD values against the log (M) of the sauvagine concentration [CHO-CRF-1 (c) and CHO-CRF-2{alpha} (d)]. Figures presented are single representatives of three independent experiments.

 


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  		Figure 4. Effects of treatment with sauvagine (S; 1 µM), forskolin (F; 1 µM), TPA (T; 100 nM), or ionomycin (I; 1 µM) on the pCREB-ir of CHO-Pro5 (a), CHO-CRF-1 (b), or CHO-CRF-2{alpha} (c) cultures. Cells were starved of serum for 30 min and then either extracted directly, (unstimulated control, C) or treated with the indicated agonists for a total period of 10 min. d and e show the effects of astressin (+A; 1 µM) and PD171729 (+P; 1 µM) on forskolin (10 µM)- and sauvagine (1 µM)-stimulated pCREB-ir in CHO-CRF-1 (d) and CHO-CRF-2{alpha} (e) cultures. Cells were starved of serum for 30 min before treatment. Blots presented are representative of three independent experiments.

 
The stimulatory effects of sauvagine, but not those of forskolin, on pCREB-ir in CHO-CRF-1 and CHO-CRF-2{alpha} cultures were blocked by the CRF receptor antagonist astressin at a concentration (1 µM) known to fully inhibit the binding of [125I]sauvagine to both receptor subtypes (Fig. 4Go, d and e). The CRF-1 receptor antagonist PD171729 fully inhibited the stimulatory actions of sauvagine, but not those of forskolin, in the CHO-CRF-1 cell line. However, PD171729 was without effect on sauvagine-stimulated phosphorylation of CREB in CHO-CRF-2{alpha} cultures, confirming the receptor subtype-specific nature of its action.

Effects of sauvagine on the MAP kinase signaling pathways
Activation of the MAP kinase signal transduction pathways is a significant route by which phosphorylation of CREB can occur (27). Sauvagine (1 µM) stimulation of CHO-CRF-1 cultures caused a time-dependent increase in phospho-MAP kinase p42/p44-ir in two bands, with p42 appearing to be the predominant form. However, no increase was apparent in the total amount of p42/p44 MAPK-ir identified using antiserum that recognized both nonphosphorylated and phosphorylated forms of p42/p44 MAP kinase (Fig. 5Go, a and b). Results similar to these were observed in CHO-CRF-2{alpha} cultures (Fig. 5Go, c and d). Sauvagine was without effect on phosphorylation of p38 and SAP-JNK kinases in CHO-CRF-1 and CHO-CRF-2{alpha} cultures (data not shown). Increased phospho-p42/p44-ir was apparent 1 min after stimulation, became maximal between 5–10 min, and fell to unstimulated levels by 60 min of treatment (Fig. 5Go, b and d). Dose-dependent increases in phosphorylation of MAP kinase p42/p44 were observed in CHO-CRF-1 and CHO-CRF-2{alpha} cultures stimulated with sauvagine (Fig. 5Go, e and f, respectively).



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  		Figure 5. Effect of treatment with sauvagine (1 µM) for varying time intervals on the total amount of p44/42 MAP kinase protein (a) and on the amount of the phosphorylated form of p44/42 MAP kinase (b) in CHO-CRF-1 cultures. The effects of treatment with sauvagine (1 µM) for varying time intervals on the total amount of p44/42 MAP kinase protein (c) and on the amount of the phosphorylated form of p44/42 MAP kinase (d) in CHO-CRF-2{alpha} cultures are shown. Nonphosphorylated p42 MAP kinase (20 ng; -ve) and phosphorylated p42 MAP kinase (20 ng; +ve) samples were run as Western blot controls. The effects of varying concentrations of sauvagine on the phospho-p44/42 MAP kinase-ir of CHO-CRF-1 (e) and CHO-CRF-2{alpha} (f) cultures after a 10-min stimulation period are shown. Blots presented are representative of three independent experiments.

 
The effects of the CRF receptor antagonist astressin (1 µM), the MEK inhibitor PD98059 (50 µM), and the CRF receptor antagonist PD171729 (1 µM) on phosphorylation of MAP kinase p42/p44 were investigated (Fig. 6aGo). Both astressin and PD98059 were able to significantly inhibit the stimulatory actions of sauvagine in both cell lines at a concentration (1 µM) previously shown to strongly stimulate phosphorylation of p44/p42 MAP kinase (Fig. 5Go, e and f). The CRF1 antagonist PD171729 fully inhibited the stimulatory actions of sauvagine in CHO-CRF-1 cultures, but was without effect in CHO-CRF-2{alpha} cultures. Simultaneous analysis of the experimental samples for the phosphorylated form of CREB confirmed the effects of astressin and PD171729 observed previously (Fig. 4Go). However, the MEK inhibitor PD98059 was not able to modulate phosphorylation of CREB at a dose that fully inhibited the phosphorylation of MAP kinase (Fig. 6bGo).



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  		Figure 6. Western blot showing the effect of astressin (+A; 1 µM), PD171729 (+P; 1 µM), and PD98059 (+M; 50 µM) on sauvagine-stimulated (1 µM, 10 min) phospho-p44/42 MAP kinase-ir of both CHO-CRF-1 and CHO-CRF-2{alpha} cultures (a) and on pCREB-ir of similarly treated cultures (b). The blots presented are representative of three independent experiments.

 
Treatment of the parental CHO-Pro5 cultures with either sauvagine or forskolin was without effect on phosphorylation of MAP kinase p42/p44 (Fig. 7aGo). However treatment with TPA or ionomycin increased phosphorylation of MAP kinase, confirming the existence of functional MAP kinase signaling pathways in the parental line. Sauvagine, TPA, and ionomycin were all found to increase phosphorylation of MAP kinase p42/p44 CREB in CHO-CRF1 and CHO-CRF-2{alpha} cultures (Fig. 7Go, b and c). The ability of sauvagine to stimulate MAP kinase p42/p44 activity was confirmed using an immunoprecipitation Western blot assay (New England Biolabs, Inc.), which uses the ability of immunoprecipitated phospho-MAP kinase p42/p44 to phosphorylate Elk1 fusion protein in vitro (see Materials and Methods; Fig. 7Go, d and e). The nonspecific higher molecular mass band represents IgG peptide (~55 kDa) derived from the immunoprecipitation reaction (Fig. 7Go, d and e). Treatment with forskolin was without effect on phospho-Elk1 levels. However, treatment with TPA (100 nM), ionomycin (1 µM), or sauvagine (1 µM) strongly increased phosphorylation of Elk1, confirming that phosphorylation of p44/p42 MAP kinase correlates with activation.



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  		Figure 7. Effects of treatment for a 10-min period with no agonist (C), sauvagine (S; 1 µM), forskolin (F; 10 µM), TPA (T; 100 nM), or ionomycin (I; 1 µM) on the phosphorylated form of p44/42 MAP kinase in CHO-Pro5 (a), CHO-CRF-1 (b), and CHO-CRF-2{alpha} (c) cultures. Effects of treatment for a 10-min period with no agonist (C), sauvagine (S; 1 µM), forskolin (F; 10 µM), TPA (T; 100 nM), or ionomycin (I; 1 µM) on the phosphorylation of the transcription factor Elk1 induced by MAP kinase immunoprecipitated from CHO-CRF-1 cultures (d) and from CHO-CRF-2{alpha} cultures (e) are shown. Controls for the immunoprecipitation and phosphorylation assay steps were provided using 20 ng active MAP kinase added to nonstimulated cell extracts (+M). Blots presented are representative of three independent experiments.

 
Effects of sauvagine on [Ca2+]i
CHO-CRF-1 and CHO-CRF-2{alpha} cells loaded with the calcium-sensitive dye fura-2 (2 µM) and treated with sauvagine at concentrations of 100 nM and 1 µM, but not 10 nM, gave a transient increase in R340:380, indicative of an increase in [Ca2+]i (Fig. 8Go, a and b). Sauvagine at a concentration of 1 µM was without effect on the R340:380 ratio of the parental CHO-Pro5 line (Fig. 8cGo). Experiments were undertaken in the absence of extracellular calcium (nominally calcium-free EM with 1 mM EGTA added) to investigate whether the increase in intracellular calcium elicited by sauvagine occurred as a consequence of mobilization from intracellular stores and/or of calcium entry across the plasma membrane in CHO-CRF-1 cultures (Fig. 8dGo). Exchanging normal EM for calcium-free EM (with 1 mM EGTA) caused a reduction in [Ca2+]i shown by R340:380 values (Fig. 8dGo). Stimulation of CHO-CRF-1 cells with sauvagine (1 µM) after a 5-min period in calcium-free medium caused an increase in R340:380 above basal values, with a reduction in peak width characteristic of a loss of a secondary calcium entry component. Reintroduction of calcium to the perfusate led to an increase in basal R340:380 values (Fig. 8dGo). A third sauvagine treatment period in the presence of extracellular calcium gave a characteristic broad peak indicative of both a mobilization and an entry component contributing to the observed increase in intracellular calcium (Fig. 8dGo). To confirm that treatment of CHO-CRF-1 cultures with sauvagine caused mobilization of intracellular calcium stores, experiments were undertaken using U73122 (10 µM), a potent inhibitor of phospholipase C. Treatment of cultures with sauvagine alone (1 µM) increased R340:380 (Fig. 8eGo). However, in cells treated with sauvagine in the presence of U73122, no increase in R340:380 values was observed (Fig. 8eGo). No differences were observed between the patterns of responses observed for CHO-CRF-1 cultures and those observed for CHO-CRF-2{alpha} cultures (data not shown).



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  		Figure 8. Effects of treatment with varying concentrations of sauvagine or with ATP (500 nM) for a 1-min period on the average change in the 340/380 nm ratio levels of CHO-CRF-1 and CHO-CRF-2{alpha} cultures (a; n = 78 CHO-CRF-1 and n = 87 CHO-CRF-2{alpha} cells for each treatment group). A representative trace shows the concentration effect of sauvagine (10 nM, 100 nM, and 1 µM) on the 340/380 nm ratio of a single CHO-CRF-1 cell (b). The effect of treatment with sauvagine (1 µM) or ATP (500 nM) on the 340/380 nm ratio level of a single CHO-Pro5 cell (c; n = 48) is shown. The effect of the removal of extracellular calcium (nominally Ca2+-free medium containing 1 mM EGTA) on the response of a representative CHO-CRF-1 cell to sauvagine (1 µM; d) is indicated. The effect of the PLC inhibitor U73122 (10 µM) on the response of a CHO-CRF-1 cell to treatment with sauvagine (1 µM; e) is shown. For b–e: black bar, sauvagine treatment period; white bar, 500 µM ATP treatment period; striped bar, 2 µM ionomycin (1 µM) treatment period. Data are presented as the 340/380 nm ratio values against time (seconds) unless stated. All experiments were repeated at least 4 times with a minimum of 3 coverslips for each experiment, and more than 70 cells were analyzed in total for each group.

 
Effect of kinase modulators on phosphorylation of CREB
The effects of H89, an inhibitor of PKA, on phosphorylation of CREB were investigated in CHO-CRF-1 and CHO-CRF-2{alpha} cultures treated with sauvagine (0.1 nM, 1 nM) or forskolin (10 µM; Fig. 9Go, a and b, respectively). Cells were pretreated with H89 for a total of 90 min before stimulation with agonist. Sauvagine at concentrations of 0.1 and 1 nM and forskolin at 10 µM all increased phosphorylation of CREB in untreated cells. The stimulatory effects of both sauvagine and forskolin were inhibited fully by pretreatment with H89 (10 µM, 1.5 h). Inhibition of PKA by H89 was without effect on the total level of CREB protein (data not shown).



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  		Figure 9. The effect of treatment with the PKA inhibitor H89 on both sauvagine- and forskolin-stimulated phosphorylation of CREB in CHO-CRF-1 (a) or CHO-CRF-2{alpha} (b) cultures. Cells were incubated for 1 h in culture medium containing H89 (H; 10 µM), then for an additional 30 min in serum-free medium containing H89 (10 µM). Parallel cultures were prepared in the absence of H89. Cultures were treated for a 10-min period with sauvagine (S; 0.1 nM, 1 nM) or forskolin (F; 10 µM). The effects of treatment of CHO-CRF-1 (c) or CHO-CRF-2{alpha} (d) cultures with the PKA inhibitor H89 (H, incubated as above), U73122 (U, 10 µM for 10 min before agonist treatment), and LY294002 (L, 10 µM for 10 min before agonist treatment) on sauvagine-stimulated (100 nM for 10 min) phosphorylation of p44/42 MAP kinase are shown. Blots presented are representative of three independent experiments.

 
Effect of signaling modulators on phosphorylation of MAP kinase
The effects of H89, LY294002, and U73122 [inhibitors of PKA, phosphoinositide 3-kinase (PI3 kinase), and PI-specific PLC, respectively] on phosphorylation of p44/42 MAP kinase were investigated in CHO-CRF-1 (Fig. 9cGo) and CHO-CRF-2{alpha} (Fig. 9dGo) cultures treated with sauvagine (100 nM for 10 min). Sauvagine increased phosphorylation of p44/42 MAP kinase above control levels in all experiments. Inhibition of PKA by H89 at a concentration (10 µM) that fully inhibited phosphorylation of CREB was without effect on the total level of phospho-p44/p42 MAP kinase. Inhibition of PI-specific PLC with U73122 at a concentration (10 µM) that fully inhibited sauvagine-mediated mobilization of intracellular calcium caused a slight, but consistent, inhibition of MAP kinase phosphorylation. Inhibition of PI3 kinase (LY294002, 10 µM) also partially inhibited sauvagine-stimulated phosphorylation of MAP kinase in both CHO-CRF-1 and CHO-CRF-2{alpha} cultures.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we investigated signaling pathways used by cloned CRF receptors in two cell lines derived from the CHO-pro5 parental cell line, CHO-CRF-1 and CHO-CRF-2{alpha}, expressing the rat CRF type 1 and the rat CRF type 2{alpha} receptors, respectively. Treatment with sauvagine caused a time- and dose- dependent increase in phosphorylation of nuclear CREB and of p42/p44 MAP kinase in both CHO-CRF-1 and CHO-CRF-2{alpha} cultures. Sauvagine was without effect on the parental CHO-Pro5 cell line. Phosphorylation was blocked by the CRF receptor antagonist astressin, demonstrating the requirement for activation of the CRF receptor for phosphorylation to occur. The EC50 values for sauvagine stimulation of cAMP and for phosphorylation of CREB are consistent with phosphorylation occurring as a consequence of activation of adenylyl cyclase. Inhibition of PKA fully blocked the stimulatory effects of sauvagine on phosphorylation of CREB; however, inhibition of MEK did not affect CREB, demonstrating that phosphorylation of CREB requires activation of PKA, but not of MAP kinase. Inhibition of PI3 kinase or of PI-specific PLC reduced phosphorylation of MAP kinase. Treatment of cultures with sauvagine increased intracellular levels of Ca2+ through both mobilization of intracellular stores and influx across the plasma membrane. In human epidermoid A-431 cells, sauvagine (EC50 of 1.4 fM) increases [Ca2+]i by calcium influx through G protein-coupled channels and by mobilization from IP3-sensitive stores (20). However, the increase in [Ca2+]i observed in the present study was not apparent when cultures were treated with 10 nM sauvagine, a dose that caused maximal stimulation of CREB phosphorylation, and it is possible that the stimulatory effect of sauvagine on [Ca2+]i may occur as a consequence of promiscuous G protein coupling. These results demonstrate for the first time CRF receptor-mediated phosphorylation of CREB and activation of MAP kinase signaling pathways.

The data presented in the present study indicate that activation of either CRF-1 or CRF-2{alpha} receptor type leads to phosphorylation and activation of p42/p44 MAP kinase. The mechanisms by which sauvagine causes activation of MAP kinase in CHO-CRF1 and CHO-CRF-2{alpha} cultures are not fully understood. However, forskolin was without effect on p42/p44 MAP kinase activity in the present study, and inhibition of PKA did not modulate sauvagine stimulation of MAP kinase phosphorylation. Therefore, sauvagine does not activate MAP kinase through increased intracellular levels of cAMP, and it is unlikely that sauvagine-stimulated activation of PKA inhibits MAP kinase as could have been expected from the observations of Crespo et al. (39). In COS-7 cells, activation of MAP kinase is mediated by the ß{gamma}-subunits of heterotrimeric G proteins through interaction with Ras-dependent pathways (35, 36). Additional studies have demonstrated the ability of ß{gamma}-subunits to increase MAP kinase activity in both Rat-1 fibroblasts stimulated with insulin-like growth factor I (37) and in signaling pathways mediated by activation of the ß-adrenergic receptor (38, 39), confirming the general significance of ß{gamma}-subunit modulation of MAP kinase activity (reviewed in Ref. 40). One mechanism by which ß{gamma}-subunit modulation of MAP kinase activity may occur is through PI3 kinase activation of cellular protein tyrosine kinases. The ability of the PI3 kinase inhibitor LY294002 to partially inhibit sauvagine-stimulated phosphorylation of MAP kinase in CRF-1 and CRF-2{alpha} cultures demonstrates that PI3 kinase may provide a link between CRF receptor activation and MAP kinase phosphorylation in our cells. In addition, U73122 was found to inhibit sauvagine-stimulated phosphorylation of MAP kinase in CRF-1 and CRF-2{alpha} cultures, potentially through inhibition of calcium-sensitive Pyk-2-mediated activation of protein tyrosine kinases. It is possible that PI3 kinase and calcium activate Pyk-2 in concert to stimulate MAP kinase activation in response to sauvagine. Activation of the small G protein Ras by ß{gamma} causes recruitment and activation of Raf, which phosphorylates MEK, the p42/p44 MAP kinase kinase (reviewed in Refs. 39, 41). Phosphorylation of p42/p44 by MEK leads to activation and translocation to the cell nucleus. Activated p42/p44 MAP kinase phosphorylates RSK2, leading to phosphorylation and activation of CREB (27), providing a mechanism by which MAP kinase can modulate gene expression through the CRE. In addition, MAP kinase activates the transcription factor Elk1, which binds to the serum response element (SRE) together with the serum response factor protein to cause increased transcription of immediate early genes containing the SRE. These pathways provide mechanisms by which activation of either CRF-1 or CRF-2{alpha} receptor type can alter gene transcription.

Three signaling pathways known to cause phosphorylation of CREB, those sensitive to cAMP, Ca2+, and MAP kinase, are stimulated by treatment of CHO-CRF-1 and CHO-CRF-2{alpha} cultures with sauvagine. The major pathway by which sauvagine regulates phosphorylation of CREB is not through calcium-mediated activation of calmodulin- dependent kinase, as changes in intracellular calcium are not apparent after treatment with sauvagine at a concentration (10 nM) that causes maximal phosphorylation of CREB in both cell types. The MEK inhibitor PD98059 at a concentration (10 µM) that fully inhibited the phosphorylation of MAP kinase was without effect on phosphorylation of CREB, raising the possibility that phosphorylation was mediated entirely by cAMP-dependent pathways. In support of this, treatment with the PKA inhibitor H89 (10 µM) fully inhibited the effects of sauvagine on CREB at a concentration (1 nM) known to maximally phosphorylate CREB. This demonstrates that cAMP-, PKA-dependent signaling pathways are responsible for sauvagine-stimulated phosphorylation of CREB. However, cooperative or synergistic effects of p42/p44 MAP kinase signaling through activation of the transcription factor Elk1, and PKA-dependent signaling through activation of CREB may be apparent at the level of the control of gene expression. One example of this may be control of transcription of the immediate early gene c-fos, which under certain circumstances can be modulated by Elk1 acting at the SRE site and CREB acting at the CRE site (27).

The molecular mechanisms by which CRF exerts control on the expression of the POMC gene encoding ACTH in the anterior pituitary are currently under investigation. CRF has been shown to stimulate POMC promoter activity by 3.5-fold (42), and this effect was dependent on PKA activity. However, activation of PKA, although required for POMC gene expression, may not be sufficient to cause maximal stimulation, and other PKA- and protein kinase C-independent signaling pathways may be required (43). One intermediate between kinase activation and increased POMC gene expression may be activation of the immediate early gene c-fos (44, 45). However, at least one novel transcription factor, PCRH-REB-1, has been identified that responds to CRF and increases activation of the POMC promoter (46), emphasizing the complexity of control of the POMC promoter. Although the POMC human gene does not possess the classical CRE, it has been shown to have a novel POMC-CRE and that CREB proteins can bind to this POMC-CRE site (47). This raises the possibility that phosphorylated activated CREB may directly modulate POMC gene expression. In addition to a direct effect of CREB on POMC gene expression, CRF may, through modulation of MAP kinase and Elk1 activity, increase nuclear levels of c-Fos protein and thereby increase POMC gene expression.

The current study suggests that PKA-mediated phosphorylation of the transcription factor CREB may be an important intermediary step in the transduction pathways arising from activation of CRF-1 and CRF-2{alpha} receptors and leading to modulation of gene transcription within the nucleus of target cells. In addition, receptor stimulation causes increased activation of p44/42 MAP kinase independently of cAMP and phosphorylation of CREB. These observations raise the possibility that PKA and MAP kinase may act in concert to control gene transcription in CRF-responsive cells. Therefore, it is possible that both phosphorylation of CREB and activation of MAP kinase signaling pathways modulate POMC gene expression and adaptive responses to stress in vivo in response to increased CRF levels.


   Acknowledgments
 
We acknowledge Michael Crouch, David Winstrow, Pauline Carnell, Jim Murray, Elizabeth Hammond, Steve Waterman, Ken Young, Ruth Franks, and Louise Webdale for invaluable help in the preparation and culture of the CHO cell lines and for assistance in the preparation of this manuscript.

Received December 19, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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