Journal of Neurochemistry, 2001, 79, 931±940The I1-imidazoline receptor in PC12 pheochromocytoma cellsactivates protein ki...
932 L. Edwards et al.I1-imidazoline receptors have been identi®ed in neural and        that are divided into three classes...
I1-Imidazoline, PKC and MAPK 933centrifugation at 2000 g for 5 min at 48C. Cell pellets were            Tris-HCl pH 7.4,10...
934 L. Edwards et al.vehicle-treated controls run in parallel. Because identical results      Resultswere obtained for ERK...
I1-Imidazoline, PKC and MAPK 935                                                                        Fig. 3 Time course...
936 L. Edwards et al.Fig. 5 Dose dependence of the activation of ERK by moxonidinetreatment. PC12 cells were treated with ...
I1-Imidazoline, PKC and MAPK 937                                                                        Fig. 10 Concentrat...
938 L. Edwards et al.ester. In addition, aPKCz showed clear subcellular relocal-          nearly 125% upon exposure to mox...
I1-Imidazoline, PKC and MAPK 939   In the present study, a small but persistent and dose-                       of MAP kin...
940 L. Edwards et al.Marshall C. J. (1995) Speci®city of receptor tyrosine kinase signaling:        Activation of phosphat...
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The I1 Imidazoline Receptor In Pc12 Pheochromocytoma Cells

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The I1 Imidazoline Receptor In Pc12 Pheochromocytoma Cells

  1. 1. Journal of Neurochemistry, 2001, 79, 931±940The I1-imidazoline receptor in PC12 pheochromocytoma cellsactivates protein kinases C, extracellular signal-regulated kinase(ERK) and c-jun N-terminal kinase (JNK)Lincoln Edwards,* Daniel Fishman,* Peleg Horowitz,* Nicole Bourbon,² Mark Kester² andPaul Ernsberger**Departments of Nutrition, Medicine, Pharmacology, and Neuroscience, Case Western Reserve University School of Medicine,Cleveland, Ohio, USA²Department of Pharmacology, Pennsylvania State University, Hershey, Pennsylvania, USAAbstract and JNK followed similar time courses with peaks at 90 min.We sought to further elucidate signal transduction pathways The action of moxonidine on ERK activation was blocked byfor the I1-imidazoline receptor in PC12 cells by testing the I1-receptor antagonist efaroxan and by D609, an inhibitorinvolvement of protein kinase C (PKC) isoforms (bII, 1, z), of phosphatidylcholine-selective phospholipase C (PC-PLC),and the mitogen-activated protein kinases (MAPK) ERK and previously implicated as the initial event in I1-receptorJNK. Stimulation of I1-imidazoline receptor with moxonidine signaling. Inhibition or depletion of PKC blocked activation ofincreased enzymatic activity of the classical bII isoform in ERK by moxonidine. Two-day treatment of PC12 cells with themembranes by about 75% and redistributed the atypical I1/a2-agonist clonidine increased cell number by up to 50% in aisoform into membranes (40% increase in membrane-bound dose related manner. These data suggest that ERK and JNK,activity), but the novel isoform of PKC was unaffected. along with PKC, are signaling components of the I1-receptorMoxonidine and clonidine also increased by greater than pathway, and that this receptor may play a role in cell growth.two-fold the proportion of ERK-1 and ERK-2 in the phos- Keywords: arachidonic acid metabolism, imidazoline, PC12phorylated active form. In addition, JNK enzymatic activity cells, pheochromocytoma, phospholipases C, receptors.was increased by exposure to moxonidine. Activation of ERK J. Neurochem. (2001) 79, 931±940.The existence of a novel imidazoline receptor was ®rst 2000). The encoded protein contains motifs commonlyproposed to account for differential responses to imidazoline associated with cytokine receptors, including leucine-richand phenylethylamine a2-adrenergic agonists (Bousquet et al. repeats and serine-rich regions. When the gene is expressed1984). Subsequently, binding sites speci®c for imidazolines in Chinese hamster ovary (CHO) cells, high-af®nitywere characterized (Ernsberger et al. 1987). It is now binding sites for imidazolines are induced that showaccepted that there are at least two subtypes of imidazoline nanomolar af®nity for clonidine and moxonidine. Functionalreceptors, the I1- and I2-subtypes, and possibly a third I3-subtype (Eglen et al. 1998). The I1-subtypes are character- Resubmitted manuscript received September 5, 2001; acceptedized by a high af®nity for a group of agents which act in the September 6, 2001.brainstem to lower blood pressure, including clonidine, Address correspondence and reprint requests to Dr Paul Ernsberger,rilmenidine and moxonidine (Ernsberger et al. 1995, 1997; Department of Nutrition, Case Western Reserve University School of Medicine, Cleveland, OH 44106±4906, USA.Regunathan and Reis 1996). The I2-subtype shows lower E-mail: pre@po.cwru.eduaf®nity for these antihypertensives with a central nervous Abbreviations used: DAG, diacylglycerides; DMSO, dimethylsystem site of action but higher af®nity for other imidazo- sulfoxide; ERK, extracellular signal-regulated kinase; JNK, c-junlines and guanidines, and represents a novel recognition site N-terminal kinase; MAPK, mitogen-activated protein kinases; MTS,on mitochondrial monoamine oxidase (Limon-Boulez et al. [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo- phenyl)-2H-tetrazolium] inner salt; NGF, nerve growth factor; PC121996). cells, PC12 pheochromocytoma cell line; PC-PLC, phosphatidyl- A gene encoding an imidazoline binding protein has choline-selective phospholipase C; PKC, protein kinase C; SDS±been cloned from a human brain cDNA library (Piletz et al. PAGE, sodium dodecyl sulfate±polyacrylamide gel electrophoresis.q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940 931
  2. 2. 932 L. Edwards et al.I1-imidazoline receptors have been identi®ed in neural and that are divided into three classes, namely the extracellularepithelial cells, including the rostral ventrolateral medulla regulated protein kinase (ERK), c-jun kinase or JNK (alsooblongata (RVLM) region which mediates sympatholytic known as stress-activated protein kinase or SAPK) and theactions of imidazoline agonists (Ernsberger and Haxhiu p38 family. Activated MAPKs phosphorylate several sub-1997; Ernsberger et al. 1997), in the eye where they regulate strates in PC12 cells including various transcription factorsocular pressure (Campbell and Potter 1994), and in the (Cowley et al. 1994). In the present study, we sought tokidney where they promote urinary sodium excretion determine whether activation of the I1-imidazoline receptor(Smyth and Penner 1999). Many ligands active at imidazo- by moxonidine leads to activation of one or more PKCline receptors also bind to a2-adrenergic receptors. There- isoforms or MAPK species, and further whether an increasefore, functional studies are typically carried out with prior in cellular proliferation might therefore result from stimula-blockade of a2-adrenergic receptors. Cellular responses to tion of I1-imidazoline receptors.I1-imidazoline receptor activation, such as effects onproliferation, have not been described previously. The predominant cellular model for investigation of Materials and methodsI1-imidazoline receptor signaling pathways has been PC12 Materialspheochromocytoma cells. These adrenal tumor cells express RPMI medium and horse serum were obtained from GIBCOI1-imidazoline receptors but lack a2-adrenergic receptors, as (Gaithersburg, MD, USA). Fetal bovine serum, rat tail collagen andshown by radioligand binding as well as molecular approaches anti-ERK af®nity puri®ed antibodies were obtained from Upstate(Separovic et al. 1996). Stimulation of the I1-imidazoline Biotechnology (Lake Placid, NY, USA). Moxonidine was kindlyreceptor in PC12 cells with the agonist moxonidine leads to provided by Kali-Chemie (Hannover, Germany). Efaroxan andactivation of phosphatidylcholine selective phospholipase C clonidine were purchased from Research Biochemicals Inter-(PC-PLC) (Separovic et al. 1996, 1997; Ernsberger 1999). national (Natick, MA, USA). The enzyme inhibitors D609 and H-7 were purchased from Biomol (Plymouth Meeting, PA).Activation of PC-PLC is characteristic of the signaling nPKC1, nPKCz, cPKCb11 and JNK goat polyclonal af®nitypathways coupled to certain cytokine receptors, including puri®ed antibodies were obtained from Santa Cruz Biotechnologysome of the interleukins receptors (Cobb et al. 1996; Ho (Santa Cruz, CA, USA). Anti-active ERK antibody and donkeyet al. 1994), and also mediates some of the actions of anti-rabbit horseradish peroxidase antibody were purchased fromthromboxanes in astrocytes (Kobayashi et al. 2000). Activa- Promega (Madison, WI, USA). Nerve growth factor (NGF) wastion of PC-PLC by imidazoline agonists results in increased obtained from Austral Biologicals (San Ramon, CA, USA). Proteinformation of the second messenger diacylglyceride (DAG) assay reagents and the colorimetric PKC assay kit were obtainedfrom phosphatidylcholine, and the release of phospho- from Pierce (Rockford, IL, USA). All other chemicals were fromcholine. These effects can be blocked by both efaroxan, an Sigma Chemical Co. (St Louis, MO, USA) or Fisher (Pittsburgh,I1-imidazoline receptor antagonist, and by D609, an inhibi- PA, USA) and were of analytical grade.tor of PC-PLC. Cell signaling steps subsequent to the PC12 cell cultureaccumulation of DAG have not been characterized for PC12 cells were cultured as previously reported (Separovic et al.I1-imidazoline receptor signaling, but DAG commonly 1996). Brie¯y, PC12 cells were grown on 75 cm2 ¯asks coated withactivates several isoforms of PKC. rat tail collagen at 5% CO2 in RPMI 1640 supplemented with 10% At least 11 isoforms comprise the PKC family (Liu and (v/v) heat-inactivated horse serum, 5% (v/v) fetal bovine serumHeckman 1998) and these differ according to structure, (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (com-substrate speci®city, cofactor requirement and subcellular plete medium). Cells were subcultured at a plating density of 1 : 6localization. The PKC isoforms can be classi®ed as classi- once per week and medium was refreshed every two days. Because previous studies showed that the response to I1-imidazolinecal, novel and atypical. The classical PKC isoforms (cPKC, receptor stimulation was enhanced following differentiation ofa, b1, b11, g) are calcium-dependent and activated by DAG PC12 cells with NGF, for most experiments PC12 cells werederived from phosphatidylinositol or phosphatidylcholine. treated with NGF (50 ng/mL) in RPMI 1640 medium supplementedThe novel PKC isoforms (nPKC, d, 1, h, u) are also with 1% FBS for 2 days in order to initiate neuronal differentiation.sensitive to DAG but are calcium independent owing to theabsence of a calcium binding domain. Finally, the atypical Preparation of cell fractions for assay of PKC activityPKC isoforms (aPKC, i, l, z) are insensitive to DAG or PC12 cells were pre-incubated in RPMI 1640 medium with 10 ng/ mL NGF for 30 min. Cells were then exposed to the followingcalcium and may be activated by other cellular signals. treatments for 10 min: 1.0 mm moxonidine, or 200 nm phorbol-12-Because I1-imidazoline receptors trigger the accumulation myristate-13-acetate (PMA), or 0.02% DMSO as vehicle control.of DAG, we hypothesized that classical and novel PKC All treatments were made up in RPMI medium supplemented withisoforms might be activated by imidazoline agonists. 10 ng/mL NGF. After treatment, cells were washed with ice-cold Possible downstream targets for PKC in PC12 cells are RPMI containing 5 mm EGTA, and then removed from the ¯ask bythe family of mitogen-activated protein kinases (MAPKs) scraping. All subsequent steps were carried out at 48C, and each(Cowley et al. 1994). MAPKs are intracellular mediators ¯ask of cells was processed separately. Cells were pelleted by q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940
  3. 3. I1-Imidazoline, PKC and MAPK 933centrifugation at 2000 g for 5 min at 48C. Cell pellets were Tris-HCl pH 7.4,10 mm MgCl2, 2 mm ATP, 0.1 mm CaCl2,homogenized with a polytron (Tekmar Tissumizer; setting 6 for 0.002% Triton X-100 detergent, and 0.2 mg/mL phosphatidyl-l-30 s) in 1.0 mL of homogenization buffer containing Tris-HCl, serine. Negative controls were treated identically, but containedpH 7.4, 50 mm NaF, 0.2 mm Na3VO4, 2.1 mm EDTA, 6.0 mm 10 mL of Tris-HCl buffer at pH 7.4 containing 50% glycerol in2-mercaptoethanol, 2 mm EGTA, and a cocktail of protease the place of cell fraction. Antibodies and agarose were included ininhibitors (0.06 mg/mL anti-pain-HCl, 0.01 mg/mL bestatin, the negative controls. The assay mixture also contained 200 nm0.02 mg/mL chymostatin, 0.06 mg/mL E-64 {N-[N-(l-3-trans- phorbol myristate acetate, except for assays of preactivated PKCcarboxirane-2-carbonyl-l-leucyl]agmatine}, 0.01 mg/mL leupeptin, where this was omitted. After the incubation, a 20 mL aliquot was0.01 mg/mL pepstatin, 0.06 mg/mL phosphoramidon, 0.4 mg/mL applied to a ferrite af®nity ®lter (Toomik et al. 1993) and washedpefabloc, and 0.01 mg/mL aprotinin). The homogenate was with three times by vacuum ®ltration with 250 mL of wash buffer,centrifuged at 106 000 g for 1 h. The resulting supernatant was consisting of 0.5 m NaCl and 0.1 m sodium acetate at pH 5.0.retained as the cytosolic fraction. Membrane fractions were Phosphopeptide was eluted with 15% formic acid. Absorbance ofobtained by homogenizing the particulate fraction (setting 6 for the eluate was measured at 570 nm in a Rainbow plate reader with30 s) in 1.5 mL of solublization buffer (homogenization buffer rhodamine-chromagranin as standard. Protein was assayed by thecontaining 1% Triton X-100), bath sonication on ice for 15 min, bicinchoninic acid method (Smith et al. 1985). A signi®cantmixing by slow rotation for 30 min, and then centrifugation at increase in the phosphorylation of rhodamine-chromagranin15 000 g for 10 min. The resulting supernatant was kept as the substrate, relative to blanks containing buffer and immunocomplexmembrane fraction. alone, was found for each of the three immunoprecipitated PKC isoforms.Immunoprecipitation and assay of PKC activityImmunoprecipitation was carried out on the cytosolic and mem- Assay of ERK activationbrane fractions as previously described (Mandal et al. 1997). Differentiated PC12 cells in 75 cm2 culture ¯asks were treated withAliquots of each fraction (15 mL containing 2±5 mg of protein) various doses of moxonidine (0.1 nm21 mm) or clonidine (100 nm)were treated with 10 mL of the appropriate isozyme speci®c for 0±180 min. In some experiments, cells were pretreated withantibody (cPKCb11, nPKC1, aPKCz) then incubated with mixing inhibitors (efaroxan, D609 or H-7) or vehicle (0.1 mm acetic acidfor 18 h at 48C. The immunoprecipitates were captured by adding in RPMI) alone for 10 min before the addition of moxonidine. In25 mL of agarose conjugated to donkey anti-rabbit secondary other experiments, cells were pretreated with 200 nm phorbolantibodies to each sample, followed by overnight incubation. myristate acetate for 20 h to deplete PKC. After treatment, cellsPrecipitates were isolated by centrifugation at 2000 g for 5 min, were washed with ice-cold calcium-free Hanks buffer, removedwashed twice by resuspension and centrifugation with homo- from the ¯ask by scraping, and then collected by centrifugation.genization buffer and ®nally resuspended in 100 mL of Tris-HCl Cells were subsequently homogenized in lysis buffer (1% Tritonbuffer at pH 7.4 containing 50% glycerol. X-100, 0.5% NP-40, 150 mm NaCl, 10 mm Tris pH 7.4, 1 mm The ef®ciency of immunoprecipitation was determined by Western EDTA, 1 mm EGTA pH 8.0, 0.2 mm sodium ortho-vanadate,blot analysis of the supernatant and immunoprecipitated fractions. 0.2 mm PMSF, and protease inhibitor cocktail (BoehringerThe immunoprecipitating antibody was used as the primary anti- Mannheim GmbH, Mannheim, Germany) with a polytron (Tecmarbody for western blot analysis. Following immunoprecipitation of Tissuemizer, 15 s at setting 60) followed by centrifugationeither cytosol or membrane fractions with the cPKCb11 antibody, (16 000 g, 48C) for 10 min. Equal amounts of protein (20 mg)the supernatants contained immunoreactivity for nPKC1 and from the resulting supernatants were subjected to SDS±PAGE on aaPKCz, but cPKCb11 could not be detected. Similar results were 10% gel and proteins were electrophoretically transferred to aobtained following immunoprecipitation of cytosol and membrane nitrocellulose membrane for immunodetection with anti-Activefractions with nPKC1 and aPKCz antibodies. Thus, the ef®ciency MAPK and anti-MAPK antibodies. with a polytron (Tecmarof immunoprecipitation by each PKC isozyme antibody approached Tissuemizer, 15 s at setting 6) followed by centrifugation at100%, within the limits of detection of western blot methods. 97 000 g at 48C for 1 h. Aliquots (10 mg protein as assayed by Immunoprecipitated PKC activity in both membrane and the bicinchoninic acid method) from the resulting supernatantscytosolic fractions were assayed using a Pierce PKC Colorimetric were subjected to SDS±PAGE on a 10% acrylamide gel andAssay Kit employing the eight-well strip format. Dye-coupled proteins were electrophoretically transferred to a nitrocellulosechromagranin (Lissamine Rhodamine B at the N-terminal) was membrane for immunodetection with anti-active ERK andused as the substrate because this chromaf®n granule protein is an anti-ERK antibodies.endogenous PKC substrate in PC12 cells. The assay was carried out A dual antibody method was used to quantitate activation ofaccording to the manufacturers instructions with two exceptions. ERK as the ratio of active to total ERK. The anti-active antibodyFirst, the incubation period was lengthened from 30 to 120 min as recognizes the dually phosphorylated activated forms of ERK-1 andpilot experiments with both cytosolic and membrane fractions ERK-2, whereas anti-ERK recognizes all forms of ERK-1 andindicated that four times more reaction product was obtained with a ERK-2. A donkey anti-rabbit secondary antibody coupled to horse-120-min incubation compared to 30 min. Second, an additional radish peroxidase was utilized to visualize protein bands by chemi-wash step was added prior to the ®nal elution of phosphopeptide luminescence using Hyper ®lm ECL (Amersham, Buckinghamshire,with formic acid to reduce background absorbance at 570 nm. UK). Film images were quanti®ed by using a scanning densito-Aliquots (10 mL) of PC12 cell membranes or cytosol were incu- meter (United States Biochemical, Cleveland, OH, USA). Resultsbated 120 min at 378C in a total volume of 25 mL of assay were expressed as a ratio of arbitrary density times area unitsbuffer containing 5 mm rhodamine-chomagranin substrate, 20 mm between anti-active and anti-ERK blots and then normalized toq 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940
  4. 4. 934 L. Edwards et al.vehicle-treated controls run in parallel. Because identical results Resultswere obtained for ERK-1 and -2, the data presented here representthe combined ERK-1 and -2 bands. Effect of moxonidine and phorbol myristate acetate on the activity of three PKC isoforms We ®rst determined whether the selected PKC isoformsAssay of c-jun kinase activityAssay of immunoprecipitated c-jun kinase (JNK) was conducted as could be detected in PC12 cells using dye-coupled chromo-previously described (Coroneos et al. 1996). Cell lysates were granin substrate. The absolute activities for cPKCbII inimmunoprecipitated with rabbit polyclonal IgG directed against untreated control PC12 cells were: cytosol 1.2 ^ 0.2, andJNK overnight at 48C, and the resulting immunocomplexes were membrane 0.86 ^ 0.1 mg of phosphorylated substrate percaptured with goat anti-rabbit IgG agarose for 8 h at 48C. The ¯ask. For nPKC absolute activities were: cytosol 0.59 ^ 0.1,agarose complexes were collected by centrifugation and washed and membrane 1.0 ^ 0.1 mg per ¯ask. The activity of aPKCztwice with PBS. The pellets were then incubated at 378C for 20 min was: cytosol 0.72 ^ 0.1, and membrane 1.0 ^ 0.2 mg ofwith 1 mg rat c-jun, 3 mL ATP (cold, ®nal concentrated 25 mm), phosphorylated substrate per ¯ask. Thus, membrane-bound1 mL [32P]ATP (speci®c activity . 4500 Ci/mmol) in a kinase PKC activity was comparable for the three isoforms, inbuffer (25 mL) as previously described (Coroneos et al. 1996). The agreement with previous reports (Wooten et al. 1994).samples were then boiled with Laemmli buffer for 2 min followed The effect on PKC activity of treatment with eitherby SDS±PAGE. After transfer to nitrocellulose, the blots wereexposed to Kodak OMAT ®lm for 24 h at 2808C. Protein bands moxonidine or phorbol myristate acetate is shown in Fig. 1.were quanti®ed by scanning densitometry as described for ERK. Data are expressed as a net increase above untreated control values determined in parallel. In response to 10 min of treatment with 1.0 mm moxonidine, immunoprecipitatedCell proliferation assays cPKCbII showed increased activity in solubilized membraneCell proliferation was measured by using the Cell Titer system ( p , 0.05, paired t-test), whereas cytosolic activity was(Promega; Madison, WI, USA) as speci®ed in the manufacturers unchanged (Fig. 1). Treatment with 200 nm phorbol myri-instructions. PC12 cells were plated at one-quarter of their normal state acetate for 10 min induced a nearly identical response.density in 96-well plates in low-serum medium (1% horse serum In contrast, nPKC showed no signi®cant response to eitherand 0.5% fetal calf serum). Cells were treated with increasing doses treatment in membrane or cytosolic fractions. The activity ofof clonidine or with 0.1% DMSO vehicle for 48 h. The number of the atypical isoform, aPKC1 showed translocation from theviable cells was estimated by incubating the cells for 2 h at 37 8C cytosol to the membrane, as indicated by a decrease in thewith the metabolic dye [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy- former and an increase in the latter (both p , 0.05, pairedmethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] inner salt (MTS; t-test). As expected, there was no in¯uence of phorbolOwens reagent) (Cory et al. 1991). Metabolically oxidizedformazan product was read from an absorbance plate reader at myristate acetate on the activity of aPKCz, a DAG-490 nm, with the absorbance in cell-free control wells subtracted. insensitive isoform.Results were expressed as corrected absorbance relative to vehicle-treated controls run on the same plate. Pilot experiments indicated that moxonidine had signi®cant Effect of moxonidine on ERK activation in extracts fromproliferative action only when added every 12 h, consistent with differentiated PC12 cellsthe short half life of this compound in vivo (Ernsberger et al. 1993). The activation of ERK-1 and ERK-2 was determined as theClonidine, an analog with similar I1-imidazoline receptor af®nity, ratio of the amount of dually phosphorylated active form towas found to be effective when added once for up to 48 h, so total ERK immunoreactivity. A representative blot is shownsubsequent experiments were carried out with clonidine. This agent in Fig. 2, illustrating the time course of the response tohas a greater activity at a1- and a2-adrenergic receptors than 100 nm moxonidine. An increase in the amount of immuno-moxonidine, but this was not thought relevant because PC12 cells reactivity to the anti-active antibody is apparent at the laterlack both a1- and a2-adrenergic receptors (Jinsi-Parimoo and Deth time points. The lower blot shows that the amount of ERK-21997; Berts et al. 1999). Indeed, PC12 cells have been used for immunoreactivity was constant between lanes, indicatingtransfection studies of these receptors speci®cally because they lack equal loading. Mean data from four experiments showedendogenous expression. that moxonidine treatment of PC12 cells increased ERK activation by about 160% relative to vehicle-treated controls (Fig. 3). Signi®cant activation of ERK ( p , 0.05 Newman±Data analysis Keuls test) was detected at 30 min, and the peak activation ofStatistical comparisons were performed by t-test for two groups oranalysis of variance for multiple comparisons, with Newman± ERK occurred at 90 min, with a decline towards baselineKeuls post hoc tests. Dose±responses data were ®tted to logistic after 2 h.equations (Motulsky and Ransnas 1987) using the Prism data The dose-dependence for the action of moxonidine onanalysis package (GraphPad software, San Diego, CA, USA) to ERK is illustrated in Fig. 4. The immunoreactivity to theobtain EC50 values. anti-active antibody increased with the concentration of q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940
  5. 5. I1-Imidazoline, PKC and MAPK 935 Fig. 3 Time course of ERK and JNK activation in PC12 cells follow- ing moxonidine (100 nM) treatment. The relative activation of ERK-1 and ERK-2 is de®ned by the ratio of total enzyme to the dually phos- phorylated form, as illustrated in Fig. 2. JNK activity was measured as immunoprecipitated kinase activity. For both kinases, the data were expressed relative to vehicle treated controls run in parallel. Data are presented as mean percentage change ^ SE from four separate experiments run in duplicate. moxonidine tested, whereas the total amount of ERK-2 protein was constant. Summary data from four separate experiments show that moxonidines effect on ERK was dose-dependent up to 100 nm, with an EC50 of 1.3 nmFig. 1 Activity of PKC isoforms in fractions from PC12 cells treated (Fig. 5). A higher concentration of moxonidine, 1.0 mm,with moxonidine or phorbol myristate acetate. Shown are relative activated ERK to a lesser extent than 100 nm. Comparablerates of dye-labeled substrate phosphorylation activity of immuno- biphasic dose±response relationships have been reported forprecipitated PKC isoforms. Three representative PKC isozymes DAG accumulation (Separovic et al. 1996).expressed in PC12 cells were isolated: cPKCbII, nPKC1, and In order to test whether the effect of moxonidine on ERKaPKCz. The effects of phorbol myristate acetate and moxonidine are stimulation was mediated by the I1-imidazoline receptor andrepresented by their percentage change ^ SE relative to controlsrun in parallel in the same experiment. Data represent the through its known transmembrane signaling pathways, wemean ^ SE from 12 75-cm2 ¯asks of cells. Asterisks mark statisti- treated the cells with efaroxan, a selective I1-imidazolinecally signi®cant increases ( p , 0.05, paired t-test). receptor antagonist, or with D609, an inhibitor of phospha- tidylcholine-selective phospholipase C and I1-imidazoline receptor signaling in PC12 cells (Fig. 6). Efaroxan (10 mm) abolished ERK activation by 100 nm moxonidine treatment, but had no signi®cant effect when given alone. The PC-PLC inhibitor D609 (1.0 mm) also effectively abolished the effect of moxonidine.Fig. 2 Western blot illustrating the time course of ERK-2 activationby moxonidine. The band labeled `phospho-ERK-2 MAPK was froma blot labeled with anti-active ERK antibody. The band labeled `pan-ERK-2 MAPK was stained for total ERK immunoreactivity. Eachlane was obtained from different ¯asks of PC12 cells incubated with Fig. 4 Western blot illustrating the dose-dependence of ERK-2 acti-100 nM moxonidine for increasing amounts of time. Data were ana- vation by moxonidine. Bands are labeled as in Fig. 2. Each lane waslyzed by determining the ratio of optical density between the ®rst obtained from different ¯asks of PC12 cells incubated with increas-and second blot. ing concentrations of moxonidine for 90 min.q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940
  6. 6. 936 L. Edwards et al.Fig. 5 Dose dependence of the activation of ERK by moxonidinetreatment. PC12 cells were treated with increasing concentrations ofmoxonidine for 90 min and then analyzed for total and activatedERK as illustrated in Fig. 4. Data are presented as mean percentage Fig. 7 Western blot showing abrogation of the ERK activationchange ^ SE from four separate experiments run in duplicate. response to moxonidine by PKC depletion or inhibition. Bands are labeled as in Fig. 2. Each lane was obtained from different ¯asks of We next sought to test whether the activation of ERK PC12 cells incubated with various for 20 h or 90 min prior to har-was mediated through PKC (Figs 7 and 8). Treatment with vesting. First and last lanes are from vehicle-treated control cells. The second lane shows the response to 100 nM moxonidine relativethe non-selective PKC inhibitor H-7 [1-(5-isoquinoline- to the vehicle control lane. The third lane is from a ¯ask of PC12sulfonyl)-2-methylpiperazine] blocked the action of moxon- cells that was processed in parallel but was pretreated with 200 nMidine. Treatment with H-7 alone had no effect on ERK phorbol-12-myristate-13-acetate overnight to deplete PKC. The fourthactivation. In order to down-regulate DAG-sensitive iso- lane shows the response to short-term treatment with phorbol ester.forms of PKC, we pretreated PC12 cells with 200 nm The ®fth lane shows that results treatment with the PKC inhibitorphorbol myristate acetate for 20 h prior to exposure to H-7 during the 10 min pre-incubation and during moxonidine treat-either phorbol or moxonidine for 90 min. Depletion of PKC ment, while the next lane illustrates the lack of effect of H-7 alone.by prolonged treatment with phorbol myristate acetate The seventh lane shows that the response to phorbol ester is lostabolished the response to short-term phorbol, con®rming after 20 h exposure to 200 nM phorbol-12-myristate-13-acetate.that the prolonged treatment eliminated responsiveness ofERK to PKC. In this series of experiments, treatmentwith 200 nm moxonidine for 90 min roughly tripled the proportion of ERK in the active dually phosphorylated state (Fig. 8). This action of moxonidine was eliminated by depletion of PKC by chronic treatment with phorbol myristate acetate. We next sought to determine whether another I1-imidazo- line agonist, clonidine, would elicit similar effects as moxo- nidine. Flasks of PC12 cells were treated in parallel for 90 min with 100 nm moxonidine, 100 nm clonidine, or vehicle. The ratio of activated ERK was 272 ^ 36% of control in cells treated with moxonidine and 273 ^ 35% of control in cells treated with clonidine. Thus, moxonidine and clonidine induced similar activation of ERK, consistent with their similar binding af®nities for the I1-imidazoline receptor in PC12 cells (Separovic et al. 1996).Fig. 6 Effects of a receptor blocker and an enzyme inhibitor onERK activation. PC12 cells were incubated with or without moxoni-dine (100 nM) in the presence or absence of the I1-imidazoline Effect of moxonidine on JNK activity in PC12 cellantagonist efaroxan (10 mM) or the PC-PLC inhibitor D609 (10 mM) extractsfor 90 min. Efaroxan or D609 were also present during a 10-min In addition to the ERKs, an independently regulatedpre-incubation. ERK activation was then determined as describedabove. Values are expressed as a percentage of vehicle treated kinase cascade in PC12 cells involves JNKs. Moxonidinecontrols. Each value represents the mean ^ SE of at least nine dose-dependently increased cellular activity of JNK up toseparate experiments. The effect of moxonidine alone was signi®- two-fold (Fig. 9). Peak effects were observed at 300 nmcant ( p , 0.01, paired t-test) but no other treatment or combination moxonidine. In the presence of 10 mm efaroxan, 100 nmof treatments had any signi®cant effect ( p . 0.10, paired t-test). moxonidine did not increase JNK activity (data not shown). q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940
  7. 7. I1-Imidazoline, PKC and MAPK 937 Fig. 10 Concentration-dependent increase in PC12 cell proliferationFig. 8 Effects of PKC depletion or inhibition on ERK activation by by clonidine. PC12 cells were grown in low-serum medium in themoxonidine. PC12 cells were incubated with vehicle alone, moxoni- presence of increasing concentrations of clonidine for 48 h, and thendine (100 nM) alone, moxonidine in the presence of the PKC inhibitor the density of viable metabolically active cells was determined byH-7 (1.0 mM), H-7 alone, moxonidine following overnight exposure to using the MTT metabolic dye. Values are expressed as a percen-200 nM phorbol-12-myristate-13-acetate in order to deplete PKC, or tage of vehicle treated controls. Each value represents the mean ^the response to short-term treatment with phorbol ester with and SEM of 18 separate wells.without overnight exposure to phorbol-12-myristate-13-acetate. ERKactivation was determined as described above. Values areexpressed as a percentage of vehicle treated controls. Each value Proliferative response of PC12 cells to imidazolinerepresents the mean ^ SE of at least six separate experiments. The agonistseffect of moxonidine alone and phorbol-12-myristate-13-acetatealone were signi®cant ( p , 0.01, paired t-test), but no other treat- The activity of ERK and possibly JNK as well is linked toment or combination of treatments had any signi®cant effect cell proliferation, particularly in transformed cell lines such( p . 0.10, paired t-test). as PC12 cells (Cowley et al. 1994). Therefore, we tested the effect of an I1-imidazoline receptor agonist on PC12 cell number during 2 day treatment (Fig. 10). We used clonidine The time course of JNK activation is indicated in Fig. 3 rather than moxonidine because of its longer metabolic half-(squares). The increase in JNK activity tended to parallel life in vivo (Ernsberger et al. 1993) and because these twothe activation of ERK, with both peaking around 90 min I1-agonists showed similar activation of ERK (see above).and declining by 120 min. The increase in JNK activity PC12 cells were seeded at one-fourth normal density in 96was evident earlier, and reached signi®cance at 15 min well plates in low-serum medium in order to reduce( p , 0.05, Newman±Keuls test), whereas ERK was not background levels of proliferation. The ®nal number ofincreased until 30 min of moxonidine treatment. viable PC12 cells after 2 days of treatment, as determined with a metabolic dye, was increased by about 20% at the lowest dose tested (0.1 nm), and by 50% at the highest dose (1.0 mm), as shown in Fig. 10. Thus, stimulation of I1-imidazoline receptors appears to induce a small but consistent increase in PC12 cell number, suggesting an increase in the number of proliferating cells. Discussion The present study identi®es several downstream cell signaling events that are coupled to the stimulation of I1-imidazoline receptors in PC12 rat pheochromocytomaFig. 9 Concentration-dependent stimulation of JNK activity by cells. A common and an atypical isoform of PKC eachmoxonidine. PC12 cells were incubated with or without varying showed increased enzymatic activity (cPKCbII and aPKCz),doses of moxonidine (0.1 nM21 mM) or vehicle for 90 min and then whereas nPKC1 was not affected. The stimulation oflysates were assayed for JNK phosphorylation. Each value repre- cPKCbII by the I1-imidazoline agonist moxonidine wassents the mean ^ SEM of at least four experiments. comparable to that induced by treatment with a phorbolq 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940
  8. 8. 938 L. Edwards et al.ester. In addition, aPKCz showed clear subcellular relocal- nearly 125% upon exposure to moxonidine. The possibleization, with activity in the cytosol decreasing and that in the modulation of PC12 cell neuronal differentiation bymembrane fraction increasing. Two members of the MAPK I1-imidazoline receptors remains to be determined.family of kinase cascades were also activated in response to Cellular DAG are known to regulate the activity ofmoxonidine: ERK and JNK. These kinases showed roughly cPKCbII. Stimulation of I1-imidazoline receptors in PC12parallel activation with a peak effect occurring around cells with moxonidine elevates total cellular mass of DAG90 min of treatment. In PC12 cells stimulated with the (Separovic et al. 1996). Moreover, in the present study, theI1-imidazoline agonist moxonidine, the proportion of ERK effects of moxonidine closely resembled those of phorbolin its active dually phosphorylated form was increased ester, a diglyceride analog. Thus, the activation of cPKCbII150%, whereas JNK activity was elevated nearly two-fold. by moxonidine might plausibly be the result of increasedThe activation of both kinases was dose-dependent, and diglyceride levels. The mechanisms behind the activation ofin the case of ERK the EC50 for moxonidine was in aPKCz are not as clear. Arachidonic acid activates atypicalclose agreement with the binding af®nity of the drug PKC isoforms in isolated brain membranes (Huang et al.for I1-imidazoline receptors [Ki ˆ 7.8 nm; (Separovic et al. 1993).1996)]. Finally, a modest but concentration-dependent The ERK family of MAPK were also activated inincrease in cell number was elicited by 2-day treatment of response to I1-imidazoline receptor stimulation. The MAPKPC12 cell cultures with the I1-imidazoline agonist clonidine. family members, including ERK and JNK, typically mediateThis result implies a weak mitogenic action of I1-imidazo- responses to mitogenic stimuli and promote cell prolifera-line receptors, consistent with their apparent activation of tion (Marshall 1995). Sustained activation of the MAPKMAPK cascades. signaling pathway is reportedly both necessary and suf®- In the present study, the activation of ERK was apparently cient to induce neuronal differentiation of PC12 cellsreceptor-mediated, because it could be blocked by cotreat- (Cowley et al. 1994). We have reported a two-fold increasement with the I1-imidazoline antagonist efaroxan. Moreover, in ERK-activation by the I1-imidazoline agonists moxon-the concentration range wherein moxonidine was effective idine and clonidine which can be blocked by efaroxan, anin activating ERK and JNK was consistent with its binding I1-imidazoline receptor antagonist, and by D609, an inhibi-af®nity for I1-imidazoline receptors, and the dose±response tor of PC-PLC. These data imply that activation of MAPK iscurves for ERK and JNK activation closely resembled pre- receptor-mediated and is downstream from phospholipidviously reported dose±response relationships for I1-imidazo- hydrolysis pathways associated with the I1-imidazolineline receptor activation of arachidonic acid release (Ernsberger receptor. Efaroxan can also acts as an a2-adrenergic antag-1998), prostaglandin production (Ernsberger et al. 1995), onist in some cells in the dose range used in the presentand DAG accumulation (Separovic et al. 1996). study, but these receptors are not present in PC12 cells. The I1-imidazoline receptor has been previously shown to Efaroxan has negligible af®nity for the mitochondrialbe coupled to activation of PC-PLC in PC12 cells, which I2-imidazoline subtype (Lione et al. 1996) which are presentleads to formation of DAG from phosphatidylcholine and an in these cells. The activation of ERK and JNK by moxo-increased total cellular mass of this second messenger nidine was not sustained, but rather peaked around 90 min(Separovic et al. 1996). We therefore hypothesized that PKC and declined substantially within 120 min. This patternmay be activated by I1-imidazoline receptor stimulation. resembles the response to epidermal growth factor and otherThe PKC multigene family of enzymes is involved in the agonists that activate ERK in PC12 cells, but stands incontrol of many biological events and is a major transducer contrast to NGF-activation of ERK, which is sustained forof receptor-mediated stimuli. In PC12 cells the I1-imidazo- many hours (Marshall 1995).line receptor agonist moxonidine has been shown to activate The activation of ERK in response to imidazoline agonistsat least two isoforms of PKC (cPKCbII and aPKCz). PC12 appears to be downstream of PKC activation. Thus, deple-cells are known to express the following PKC isotypes: a, tion of classical and novel isoforms of PKC by prolongedbI, bII, d, 1, h and z (Hundle et al. 1995) and differentiation exposure to a phorbol ester blocked the response to moxon-of PC12 cells to a neuronal phenotype by treatment with idine. Furthermore, a non-selective PKC inhibitor blockedNGF induces increased expression of bII, d, and z, and the the response to moxonidine as well. These results implicateappearance of PKC 1 and h within the nucleus (Borgatti the stimulation of PKC in the activation of ERK byet al. 1996). This suggests that the I1-imidazoline receptor imidazoline agonists. The subtype of PKC responsible maymight modulate cell proliferation or neuronal differentiation be the bII isoform, as this form was activated in responsethrough activation of key PKC isoforms. In addition, the to moxonidine. While the aPKCz isoform was alsoatypical z-PKC is required for neuronal differentiation activated, this atypical subtype of PKC is not known toand neurite outgrowth of PC12 cells in response to NGF be depleted by prolonged stimulation with phorbol esters.(Coleman and Wooten 1994), and thus the activity of this The isoform may participate in other downstream signalingisoform in PC12 cell membrane fractions was increased by events. q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 931±940
  9. 9. I1-Imidazoline, PKC and MAPK 939 In the present study, a small but persistent and dose- of MAP kinase kinase is necessary and suf®cient for PC12dependent increase in the number of viable cells was differentiation and for transformation of NIH 3T3 cells. Cell 77, 841±852.noted in cultures of serum-starved PC12 cells treated with Eglen R. M., Hudson A. L., Kendall D. A., Nutt D. J., Morgan N. G.,I1-imidazoline agonist clonidine. An increase in total cell Wilson V. G. and Dillon M. P. (1998) `Seeing through a glassnumber is a strong indicator that increased cell proliferation darkly: casting light on imidazoline `I sites. Trends Pharmacol.has occurred, although the number of actively dividing cells Sci. 19, 381±390.was not measured. The activation of MAPK cascades may Ernsberger P. (1998) Arachidonic acid release from PC12 pheochromo- cytoma cells is regulated by I1-imidazoline receptors. J. Auton.have been too transient to induce a dramatic proliferative Nerv. Syst. 72, 147±154.response. The mitogenic effect was not altered when serum Ernsberger P. (1999) The I1-imidazoline receptor and its cellularlevels in the media were increased or the initial seeding signaling pathways. Ann. NY Acad. Sci. 881, 35±53.density of the cells were changed systematically (data not Ernsberger P. and Haxhiu M. A. (1997) The I1-imidazoline-binding siteshown), implying that the effect of clonidine was not is a functional receptor mediating vasodepression via the ventralstrongly dependent upon culture conditions. medulla. Am. J. Physiol. 273, R1572±R1579. Ernsberger P., Meeley M. P., Mann J. J. and Reis D. J. (1987) Clonidine In conclusion, we have extended our model of the signal- binds to imidazole binding sites as well as a2-adrenoceptors in theing pathway for I1-imidazoline receptor in PC12 cells to ventrolateral medulla. Eur. J. Pharmacol. 134, 1±13.include coupling to PKC and MAPK. One possible function Ernsberger P., Elliott H. L., Weimann H.-J., Raap A., Haxhiu M. A.,of these intermediates may be in promoting cell proliferation È È Hofferber E., Low-Kroger A., Reid J. L. and Mest H.-J. (1993)or possibly the modulation of neuronal differentiation. Moxonidine: a second-generation central antihypertensive agent. Cardiovasc. Drug Rev. 11, 411±431. Ernsberger P., Graves M. E., Graff L. M., Zakieh N., Nguyen P., CollinsAcknowledgements L. A., Westbrooks K. L. and Johnson G. G. (1995) I1-imidazoline receptors: De®nition, characterization, distribution and transmem-This work was supported by HL44514 (to PE and MK) and brane signaling. Ann. NY Acad. Sci. 763, 22±42.DK53715 (to MK) from the National Institutes of Health. We Ernsberger P., Friedman J. E. and Koletsky R. J. (1997) Theacknowledge the technical assistance of Kathryn Zalovcik, BS I1-imidazoline receptor: from binding site to therapeutic targetand David Bedol, BS. in cardiovascular disease. J. Hypertens. 15, S9±S23. Ho J. 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