Transcription factor CREB and its stimulus-dependent phosphorylation ...

Cell Tissue Res (2002) 308:131–142 DOI 10.1007/s00441-002-0522-2

REGULAR ARTICLE

S. Schöniger · E. Maronde · M.D.A. Kopp H.-W. Korf · F. Nürnberger

Transcription factor CREB and its stimulus-dependent phosphorylation in cell and explant cultures of the bovine subcommissural organ Received: 23 July 2001 / Accepted: 8 January 2002 / Published online: 8 March 2002 © Springer-Verlag 2002

Abstract The subcommissural organ (SCO) is an ependymal brain gland that synthesizes and secretes glycoproteins. Very little is known about the signal transduction cascades operating in this organ and their impact on gene expression. An important transcription factor that regulates gene expression in glial cells and neurons is the cyclic-AMP-responsive element binding protein (CREB), which is activated by phosphorylation of the serine residue 133. Here, we analyzed the presence of CREB in bovine SCO cells and its phosphorylation by drugs that activate cyclic-AMP-dependent or calcium-dependent signal transduction pathways. We also investigated the effects of three natural signaling molecules, serotonin (5HT), substance P (SP) and ATP, on CREB phosphorylation and on the second messengers cyclic AMP and calcium. Investigations were performed with cell and explant cultures by using immunocytochemistry, immunoblot, enzyme-linked immunosorbent assay, and the Fura-2 technique. A strong immunosignal for total (phosphorylated and unphosphorylated) CREB was found in virtually all SCO cells. Total CREB levels did not change upon stimulation. Phosphorylated (p)CREB levels were low in unstimulated cells and significantly elevated by drugs that increase the levels of cyclic AMP or free calcium ions. pCREB was also induced by SP and ATP; both substances increased the intracellular calcium concentration but did not affect the formation of intracellular cyclic This work was supported by the Volkswagen-Stiftung (grant no. I/74062) Drs. S. Schöniger and E. Maronde should be considered equally as first authors S. Schöniger · E. Maronde · H.-W. Korf · F. Nürnberger (✉) Dr. Senckenbergische Anatomie (Anatomie II), Fachbereich Medizin, J.W. Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany e-mail: [email protected] Tel.: +49-69-63016906, Fax: +49-69-63013835 M.D.A. Kopp MPI f. Hirnforschung, Deutschordensstr. 46, 60528 Frankfurt, Germany

AMP. 5HT did not influence the phosphorylation of CREB, the intracellular calcium concentration, or the formation of cyclic AMP. Our data identify CREB as an SCO transcription factor that can be activated by the second messengers cAMP and calcium. SP and ATP stimulate the phosphorylation of CREB apparently via a calcium-dependent mechanism and are thus involved in the control of gene expression in the bovine SCO. Keywords Adenosine · ATP · Cyclic AMP · Forskolin · Serotonin · Substance P · Bovine

Introduction The subcommissural organ (SCO) is an ependymal brain gland that belongs to the circumventricular organs and is located beneath the posterior commissure. SCO cells secrete various glycoproteins into the cerebrospinal fluid (CSF) that aggregate to form Reissner’s fiber (RF; Oksche 1961; Rodríguez et al. 1984, 1992; Oksche et al. 1993). Some of these glycoproteins, in particular SCOspondin, are potent activitors of neuritic outgrowth (Monnerie et al. 1995, 1996, 1997, 1998; Gobron et al. 1996, 2000); it has been suggested that they are involved in the formation of the posterior commissure and the differentiation of the spinal cord during ontogeny. Although these glycoproteins are partially characterized (Hein et al. 1993; Meiniel et al. 1993; Pérez et al. 1995, 1996; Gobron et al. 1996, 2000; Creveaux et al. 1998; Didier et al. 2000; for a review, see Rodríguez et al. 1992), to date very little is known about the regulation of their secretion. Some preliminary data indicate that the secretory activity of the SCO may be controlled at the transcriptional level (H. Richter and E. M. Rodríguez, personal communication) and thus may involve transcription factors. An important transcription factor is the Ca2+/cAMP response element binding protein (CREB), which is ubiquitously distributed in the central nervous system (CNS) and regulates transcription in glial cells and in neurons by

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acting upon a Ca2+/cAMP response element (CRE) present in the promotor of many genes (for a review, see Herdegen and Leah 1998). CREB is activated by phosphorylation of a specific amino acid residue, serine-133 (ser-133). Initially, CREB phosphorylation was described as being induced by stimuli that raise intracellular levels of cAMP and activate protein kinase A (PKA; Gonzalez and Montminy 1989; Montminy et al. 1990). Subsequently, rises in the intracellular concentrations of Ca2+ followed by activation of various Ca2+-dependent protein kinases, i.e., protein kinase C (PKC), Ca2+/calmodulindependent protein kinase II or IV (CaM Kinase II, IV), mitogen-activated protein kinase (MAPK), have also been found to induce CREB phosphorylation (Sheng et al. 1991; Pende et al. 1997). Thus, CREB respresents a molecular interface at which distinct signaling pathways converge. In the present study, we have investigated whether CREB is present in the SCO, and whether CREB phosphorylation is regulated by drugs that increase the intracellular concentration of either Ca2+ or cyclic AMP. Furthermore, we have studied the effects on CREB phosphorylation elicited by serotonin (5HT) and substance P (SP), which have previously been shown to be present in nerve fibers innervating the SCO (Ljungdahl et al. 1978; Møllgård and Wiklund 1979; Léger et al. 1983; Bouchaud 1993; Knigge and Schock 1993; Mikkelsen et al. 1997; Schöniger et al. 2001) or by ATP, an important neurotransmitter in the CNS and peripheral nervous system, shown to activate neurons and glial cells (for a review, see Zimmermann 1994) including SCO cells (Schöniger et al. 2002). The experiments have been performed with explant and dissociated cell cultures of the SCO from the bovine, because the SCO secretory glycoproteins of this species have been extensively characterized in previous studies (Rodríguez et al. 1987; Meiniel et al. 1988; Nualart et al. 1991; Hein et al. 1993), and the bovine SCO can be readily isolated and cultured (Nürnberger and Schöniger 2001; Schöniger et al. 2001, 2002).

Materials and methods Explant culture of bovine SCOs SCO explant cultures were prepared according to the original protocol of Lehmann et al. (1993; cf. Schöniger et al. 2001). They were cultured in an incubator at 37°C in an atmosphere of 7% CO2 and 93% air for 4–6 weeks. The culture medium was DMEM/F12 supplemented with 10 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml ascorbic acid, 10% fetal calf serum, and 50 nM cytosine β-D-arabino-furanoside. Some experiments were performed in serum-free supplemented DMEM/F 12 (for details, see Schöniger et al. 2001, 2002; Nürnberger and Schöniger 2001). Dispersed cell culture of bovine SCOs Bovine SCO explant cultures were dissociated by papain digestion and repeated pipetting (Schöniger et al. 2001, 2002; cf. Schaad et al. 1993; Schomerus et al. 1995, 1996; Kopp et al. 1999). The dis-

sociated cells were either plated onto poly-L-lysine-coated coverslips (for immunocytochemical analysis and calcium imaging) or maintained as suspension cultures (for measurement of cAMP). Stimulation experiments Drugs, SP, and ATP were applied in concentrations known to affect the intracellular levels of either cyclic AMP or free Ca2+. For 5HT, a wide range of concentrations was used to verify the negative results. All stimulation experiments were performed at least in triplicate. For immunocytochemistry or immunoblot, explant cultures (≥25 explants for each exposition to a particular stimulant) and dispersed immobilized cells (≥25 coverslips for each individual experiment) were transferred to supplemented serum-free culture medium 1 day before the experiments started. Stimulation was performed with 5HT (10 µM), SP (100 nM), or ATP (10 µM) in serum-free culture medium for 30 min in explant cultures and in dispersed cell cultures. In addition, dispersed immobilized cells were treated with the following drugs: (1) forskolin (10 nM– 100 µM, 15 min–6 h), an activator of adenylyl cyclase; (2) SpcDBiMPS (1 µM, 30 min) plus N6-PHE-cAMP (1 µM, 30 min), agonists of the cAMP-dependent protein kinase; (3) thapsigargin (2 µM, 30 min), which stimulates calcium release from IP3-sensitive stores by inhibiting the microsomal Ca-ATPase (Thastrup et al. 1990); (4) ionomycin (5 µM, 30 min), a calcium ionophore that increases the influx of extracellular calcium; (5) caffeine (10 µM, 30 min), which mobilizes calcium from ryanodine-sensitive stores. In order to measure cyclic AMP by enzyme-linked immunosorbent assay (ELISA), cells were cultured in suspension and stimulated with forskolin (10 µM, 3–30 min), forskolin (10 µM) plus 5HT (1–100 µM, 10 min), 5HT (1–100 µM, 10 min), SP (100 nM, 10 min), ATP (10 µM, 10 min), and adenosine (10 µM, 10 min). For calcium imaging, dispersed immobilized cells were stimulated with 5HT (10 µM), SP (100 nM), ATP (10 µM) in artificial cerebrospinal fluid (aCSF) for approximately 2 min (for details, see below). In control experiments, explant and dispersed cell cultures were treated with vehicle only. Immunocytochemistry Total CREB and pCREB The ABC-method was used for immunocytochemical demonstration of pCREB (phosphorylated at ser-133) and total (i.e., phosphorylated and nonphosphorylated) CREB in dispersed cell cultures and explant cultures. Stimulated and unstimulated cell cultures plated onto poly-L-lysine-coated coverslips were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; 15 min) and washed with PBS. Stimulated and unstimulated explant cultures were fixed in 4% PFA (30 min), cryoprotected by successive incubation in 10%, 20%, and 30% sucrose (dissolved in PBS) and snap-frozen. Cryostat sections (10 µm) were prepared, mounted onto poly-L-lysine-coated slides and air-dried for 1 h before use. Endogenous peroxidase in both explants and dispersed cells was blocked by treatment with methanol containing 0.45% peroxide (10 min). The preparations were incubated with the primary polyclonal antibodies against ser-133 pCREB (1:500; New England Biolabs, Beverley, Mass.) or total CREB (1: 1000; New England Biolabs, overnight) followed by biotin-conjugated anti-rabbit IgG (1:100, 1 h; Sigma, Deisenhofen, Germany), horseradish-peroxidase (HRP)-conjugated streptavidin (1:100, 1 h; Sigma) and the chromogen 3,3′diaminobenzidine (0.05% DAB, plus 0.02% H2O2, 10 min). Between the incubation steps, the preparations were thoroughly washed with PBS (3×10 min). The primary antibodies against pCREB and total CREB were diluted in PBS containing 1% bovine serum albumin and 0.3% Triton X-100. The second and third antibodies were diluted in PBS containing 0.3% Triton X-100. The preparations were finally mounted in “Kaiser’s Glyceringelatine”.

133 Marker AFRU To identify SCO cells, the dispersed cell cultures were immunostained with the specific marker AFRU (Rodríguez et al. 1984) according to the peroxidase-antiperoxidase (PAP) method. The preparations were fixed for 15 min in ice-cold methanol, washed with TBS (8.4 mM Na2HPO4, 3.5 mM KH2PO4, 120 mM NaCl, 41 mM TRIS-base, pH 7.7) and incubated with the AFRU antiserum (1:1500, overnight), with swine anti-rabbit immunoglobulins (1:80, 30 min; Dako, Hamburg, Germany) and PAP complexes developed in rabbit (1:50, 30 min; Dako). The antisera were diluted in TBS containing 0.7% carageenan (Sigma) and 0.3% Triton X-100 (Sigma). Washes between the incubation steps (3×10 min) were performed with TBS. The immunoreaction was visualized by the use of DAB (0.05% DAB, 0.02% H2O2) as coloring reagent, and the preparations were finally mounted in “Kaiser’s Glyceringelatine”. To verify the colocalization of AFRU and pCREB in identical SCO cells, ex-vivo slices (vibratome slices, ~150 µm thick) were prepared and treated with forskolin under the culture conditions described for explant and dispersed cell cultures. After the preparation of cryostat sections (15 µm thick), serial sections were stained for AFRU (immunofluorescence) or pCREB (the ABC method). Controls For specificity tests, the primary antisera were omitted and replaced by buffer. Furthermore, some preparations were treated with primary antisera preabsorbed with the respective antigen, i.e., CREB, pCREB, or urea-extracted bovine Reissner’s fiber material). None of the controls exhibited positive immunolabeling.

rescent calcium indicator Fura-2 pentacetoxymethylester (Fura-2/ AM; 3 µM, Molecular Probes, Eugene, Ore.) in supplemented DMEM/F12 for 20 min in the incubator and then rinsed with aCSF containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose. Thereafter, coverslips were transferred to a perfusion chamber (volume: 600 µl) on the heatable stage of an inverted microscope (Axiovert 100, Zeiss, Jena, Germany) and superfused with aCSF at a constant flow rate and constant temperature of 37°C. Calcium responses were analyzed by use of this type of inverted microscope equipped with an Attofluor illumination and photomultiplier-camera system (Atto Instruments, Rockville, Md.) and a personal computer with appropriate software (Attoflour Ratio Vision, Atto Instruments) to control the optic equipment and to record, analyze, and store the images and data (for details, see Schomerus et al. 1995; Kroeber et al. 1997; Kopp et al. 1999). All substances tested were stored frozen as stock solutions and diluted with pre-warmed aCSF immediately before use. For stimulation, the perfusion was stopped, 500 µl aCSF was removed from the perfusion chamber, and the drugs (diluted in 500 µl aCSF) were added directly into the chamber with a micropipette. The drugs were washed out by starting the superfusion again, and complete washout of the chamber was achieved within 30 s. All data are presented in a semiquantitative manner as 334/380 nm emission ratios. A positive response to a stimulation was defined by an increase in this value of more than 0.15. Following the fluorometric measurements, cells were fixed and immunostained for AFRU. Immunocytochemical and functional data were correlated at the level of single cells; the location of the cells was determined by means of a grid on the coverslip (Schöniger et al. 2001, 2002; Schomerus et al. 1995; Kroeber et al. 1997; Kopp et al. 1999). Statistics

Immunoblot analysis Explants (protein content: 8–10 µg) were directly lysed in 50 µl sample buffer (Rittenhouse and Marcus 1983), sonicated, boiled for 10 min, and chilled on ice. Electrophoresis and blotting were performed as previously described (Rittenhouse and Markus 1983; Szewcyk and Kozloff 1985). After a blocking step with a synthetic blocking buffer (Rotiblock; Carl Roth, Karlsruhe, Germany) for 1 h at room temperature (RT), the membranes were incubated with the polyclonal antibodies pCREB (1: 2500, 12 h, 4°C; New England Biolabs) or CREB (1: 2500, 12 h, 4°C; New England Biolabs). HRP-conjugated anti-rabbit IgG (1:50,000, 1 h, RT; New England Biolabs) was used as the secondary antibody. Signals were detected by chemoluminescence (UltraSignal; Pierce, Rockford, Ill.) on autoradiographic film (CIXposure; Pierce). To confirm the equal loading of the lanes, immunoblots were stained with India ink (Pelikan, Hannover, Germany) after chemoluminescence detection. cAMP assay The formation of cAMP in SCO cells cultured in suspension was measured by use of a commercially available ELISA kit (Institut für Hormon- und Fortpflanzungsforschung, Hamburg, Germany). Intracellular cAMP was extracted with 80% ethanol, separated from the cellular fragments by centrifugation (1000g, 4°C, 5 min), and concentrated by lyophilization. The lyophilized cAMP was dissolved in serum-free culture medium, and the ELISA was performed according to the protocol of the supplier. The protein content of each pellet was determined and used to normalize the cyclic AMP content (Bradford 1976). Calcium imaging Dispersed cells were cultured on coverslips with an alphanumeric grid. For the analysis of [Ca2+]i, cells were loaded with the fluo-

All data are presented as means ± SEM. Statistic analysis was performed with the unpaired Student’s t-test. Cell cultures immunostained for pCREB and total CREB were analyzed semiquantitatively by computer-assisted image analysis (Wicht et al. 1999). The integrated density was measured as SUMDENSCORR values (product of the mean inverted gray value of a signal and the area covered by the signal corrected for the total area covered by the cells). The intensity of immunosignals in the immunoblots was analyzed semiquantitatively by measuring the integrated optical density of immunoreactive areas (SUMDENS values; Wicht et al. 1999). To compare separate experiments, the ratio between the SUMDENS values for pCREB immunoreactivity (pCREBir) and CREB immunoreactivity (CREBir) on each membrane (pCREBir/ CREBir ratio) was determined. Materials Drugs and chemicals were obtained from the following sources: NaCl, KCl, CaCl2, MgCl2, Triton X-100, KH2PO4, glucose, EGTA, and “Kaiser’s Glyceringelatine” from Merck, Darmstadt, Germany; EBSS, DMEM/F12, fetal calf serum, penicillin, streptomycin, glutamine, and HEPES from Gibco Life Technologies, Eggenstein, Germany; papain, ATP, ADP, AMP, adenosine-5′-O(3-thiotriphosphate) tetralithium salt (ATPγS), and uridine-5′-triphosphate (UTP) from Boehringer Mannheim, Germany; N6-phenyladenosine 3′5′-cyclic monophosphate (N6-Phe-cAMP), the axial diastereoisomer of 5,6-dichlorobenzimidazole-1-β-D-ribofuranosyl 3′5′-cyclic monophosphothioate (Sp-cDBiMPS), from BioLog Lifescience Institute, Bremen, Germany; SP and forskolin from Calbiochem-Novabiochem, Bad Soden, Germany. All other drugs and chemicals were supplied by Sigma. Coverslips with a coordinate grid etched onto the glass were obtained from Eppendorf (Hamburg, Germany).

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Results Effects of drugs elevating cyclic AMP and/or [Ca2+]i

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Immunocytochemical investigations with AFRU, a SCOcell-specific marker, showed an intense cytoplasmic immunolabel in the majority of the dispersed immobilized cells (Fig. 1a) and in slices (Fig. 2a). Virtually all of these cells displayed a strong nuclear immunoreaction for total CREB, irrespective of whether they were stimulated (Fig. 1b). The immunoreaction for pCREB was very low in untreated control preparations (Fig. 1c) but was strongly induced when the cells or slices were treated for 30 min with forskolin (10 µM), which activates adenylate cyclase (Fig. 1d, Fig. 2b), with Sp-cDBiMPS (1 µM) plus N6-PHE-cAMP (1 µM), agonists of the cAMP-dependent protein kinase (data not shown), with thapsigargin (2 µM), which stimulates calcium release from IP3-sensitive stores by inhibiting the microsomal Ca-ATPase (Thastrup et al. 1990; Fig. 1e), and with ionomycin (5 µM), a calcium ionophore that increases the influx of extracellular calcium (Fig. 1f). The induced pCREB immunoreaction was exclusively located in the nucleus. Caffeine (10µM), which mobilizes calcium from ryanodine-sensitive stores, did not influence the phosphorylation of CREB (data not shown). Semiquantitive analyses of immunocytochemical preparations revealed that forskolin, Sp-cDBiMPS plus N6-PHE-cAMP, thapsigargin, and ionomycin caused a 7-fold to 8-fold increase in pCREB immunoreaction (Fig. 3) compared with controls. The treatment with forskolin was shown to induce CREB phosphorylation in a dose- and time-dependent manner. A gradual increase in pCREB immunoreaction was observed when forskolin was applied in concentrations ranging from 100 nM to 100 µM, with 100 µM being the most effective concentration. No significant difference in pCREB immunoreaction was observed between control cultures and cultures treated with 10 nM forskolin (Fig. 4a). Time-course experiments with 10 µM forskolin showed an induction of pCREB after 15 min; this reached its maximum after 30 min. pCREB levels were still elevated after 6 h of stimulation (Fig. 4b). The forskolin-dependent induction of pCREB was preceded by increases in the intracellular concentration of cyclic AMP. An approximately 7-fold increase in cAMP levels was observed after 3, 5, and 10 min of stimulation. Fig. 1a–f Immunocytochemical demonstration of total CREB and ser-133-phosphorylated (p)CREB in cultured dispersed cells obtained from bovine SCO explants after stimulation with drugs activating cyclic AMP- or Ca2+-signal transduction pathways. a SCO cells identified by strong cytoplasmic immunostaining for AFRU (AFRU specific antiserum to glycoproteins secreted by SCO cells). b Strong nuclear immunoreactivity for total CREB (tCREB) in all SCO cells (the micrograph shows non-stimulated cells; however, the reactivity is virtually identical in stimulated cells). c Little nuclear immunostaining for pCREB in controls treated with vehicle (Co). d–f Strong pCREB immunostaining observed after application of (d) forskolin (Fk, 10 µM, 30 min), (e) thapsigargin (Tg, 2 µM, 30 min), or (f) ionomycin (Iono, 5 µM, 30 min). Bar 25 µM

Fig. 2a, b Comparison of the occurrence of AFRU and pCREB in serial sections of the bovine SCO (CP posterior commissure, III entrance into the mesencephalic aqueduct). a Immunofluorescence staining (Alexa 488) for AFRU (white arrows). Staining is located predominantly at the apical pole of the SCO cells. The cell nuclei are generally free of label. b Immunostaining (ABC) for pCREB (black arrows) in an adjacent serial section. Only the cell nuclei contain label. Bar 100 µM

Fig. 3 Semiquantitive analysis of pCREB immunoreactivity in cultured dispersed cells after stimulation (30 min) with various drugs activating cyclic AMP- and/or Ca2+-signal transduction pathways (Caff stimulation with 10 µM caffeine, cAMP-Analoga stimulation with 1 µM Sp-cDBiMPS plus 1 µM N6-PHE-cAMP, Co control, application of vehicle, corrSUMDENS product of inverted gray value and the area of immunostained structures divided by the respective sample area, Fk stimulation with 10 µM forskolin, Tg stimulation with 2 µM thapsigargin, Iono stimulation with 5 µM ionomycin, ***P≤0.001 vs control, error bars SEM)

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Thereafter, the cyclic AMP levels dropped but were still elevated 4-fold compared with controls after 30 min of stimulation (Fig. 4c). Basal cAMP levels differed from 0.6 to 5 pMol/100 µg protein; maximal concentrations ranged from 3.96 to 92.14 pMol/100 µg protein (data not shown). Effects of neuroactive substances

Fig. 4a–c Effects of forskolin on the formation of pCREB and cyclic AMP in cultured bovine SCO cells (Co controls treated with vehicle, corrSUMDENS product of inverted grey value and the area of immunostained structures divided by the respective sample area, Fk forskolin, Fk conc.(log M) logarithmic scale of molar forskolin concentration, *P≤0.05 vs control, **P≤0.01(vs control, *** P≤0.001 vs control, error bars SEM). a Dose-response curve of forskolin stimulation on CREB phosphorylation. b Time dependency of forskolin stimulation (10 µM) on CREB phosphorylation. c Time dependency of forskolin stimulation (10 µM) on cyclic AMP formation

We next tested the effects of 5HT, SP, and ATP on the induction of CREB phosphorylation and the second messengers cyclic AMP or Ca2+ in dispersed cells. Stimulation with 5HT (10 µM, 30 min) did not increase the pCREB immunoreaction above control levels (Fig. 5a, b). On the other hand, application of SP (100 nM, 30 min) or ATP (10 µM, 30 min) induced pCREB immunoreactions in 17% or 74% of the analyzed cells, respectively (Fig. 5c, d). Similar results were obtained from stimulation experiments with undissociated SCO explant cultures (Fig. 6). The induced pCREB immunoreaction was exclusively located in the nucleus in both explant and dispersed cell cultures. Semiquantitative analysis of the pCREB immunoreaction in dispersed cell cultures confirmed that 5HT did not influence pCREB, and that the pCREB immunoreaction was increased 2-fold after stimulation with SP and 7-fold after stimulation with ATP (Fig. 7a). The intensity of the total CREB immunoreaction did not vary among preparations that were stimulated with 5HT, SP, or ATP and untreated control cells (Fig. 7a). These results obtained from immunocytochemical preparations agreed with findings from immunoblotting of explant cultures. Stimulation with 5HT (10 µM, 30 min) did not change the pCREB immunoreaction compared with the control (Fig. 7b, c), but treatment with SP (100 nM, 30 min) or ATP (10 µM, 30 min) increased the pCREB immunoreaction significantly above the control level, with ATP being more effective than SP (Fig. 7b, c). Under all conditions, the signals for total CREB were stronger than those for pCREB, and their intensity did not vary between stimulated and unstimulated preparations (Fig. 7b). Therefore, the ratios of the intensities of the pCREB immunoreaction and the total CREB immunoreaction appeared as an appropriate and reliable marker to assess the changes in the amount of pCREB in a semiquantitative manner (Fig. 7c). Stimulation with SP (100 nM), ATP (10 µM), or adenosine (10 µM), a possible enzymatic cleavage product of ATP, did not raise the intracellular cyclic AMP concentrations. The principal responsiveness of the cells was shown by treatment with forskolin (10 µM), which caused an 8-fold increase over the control level (Fig. 8a). Stimulation with 1–100 µM 5HT also caused no rise in cyclic AMP over the control levels and had no effect on the forskolin-induced cyclic AMP formation (Fig. 8b). As demonstrated by the Fura-2 technique, SP (100 nM; n=1576) or ATP (10 µM; ATP: n=2020) elicited rises in [Ca2+]i in approximately 30% or 85% of the

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Fig. 5a–d Immunocytochemical demonstration of pCREB in cultured dispersed SCO cells after stimulation with neuroactive substances for 30 min. a Control treatment with medium only (Co). b Stimulation with serotonin (5HT; 10 µM). c Stimulation with substance P (SP; 100 nM). d Stimulation with adenosine triphosphate (ATP; 10 µM). Bar 25 µM

analyzed cells (Fig. 9a), whereas no calcium responses were obtained after application of aCSF (Co; n=200) or 5HT (10 µM; n=287). Figure 9b presents the correlation between the number of cells showing an induction of pCREB and a calcium response after stimulation with SP and ATP. After stimulation with SP (100 nM) or ATP (10 µM), 17% or 74% of the analyzed cells (SP: n=524, ATP: n=384) were pCREB immunoreactive and 30% (n=1576) or 85% (n=2020) of the analyzed cells displayed an increase in [Ca2+], respectively.

Discussion By immunocytochemistry, immunoblotting, ELISA, and calcium-imaging, we have demonstrated that (1) CREB

is present in the bovine SCO, (2) the amount of total CREB is constant irrespective of the stimulation of the cells, (3) CREB is phosphorylated at ser-133 by rises in the intracellular concentration of cyclic AMP or calcium ions, and (4) SP and ATP cause CREB phosphorylation at ser-133 and increases in [Ca2+]i. Total CREB, which represents both phosphorylated and unphosphorylated CREB, is readily detectable in unstimulated SCO cells, and its levels are not affected by stimulation. Thus, CREB appears to be constitutively expressed in the bovine SCO as has been shown for other cell types of the CNS, e.g., pinealocytes (Schomerus et al. 1996). As demonstrated by semiquantitative analyses, the immunosignal of total CREB, even in stimulated preparations, is significantly stronger than that of pCREB. After treatment with SP or ATP, we have found the relative share of pCREB to be approximately 30% or 50%, respectively. This ratio suggests that only a fraction of the total amount of CREB molecules is phosphorylated upon stimulation with naturally occurring neuroactive substances. The large pool of unphosphorylated CREB may ensure a broad dynamic range for CREB

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Fig. 6a–d Immunocytochemical demonstration of pCREB in explant cultures of the bovine SCO after stimulation with neuroactive substances for 30 min. a Control treatment with medium only (Co). b Stimulation with serotonin (5HT; 10 µM). c Stimulation with substance P (SP; 100 nM). d Stimulation with adenosine triphosphate (ATP; 10 µM). Bar 25 µM

phosphorylation in the SCO, as has also been discussed for rat pinealocytes (Maronde et al. 1999). By stimulation of the cells with an activator of the adenylyl cyclase (i.e., forskolin) or different activators of the PKA (i.e., Sp-cDBiMPS, N6-PHE-cyclic AMP, Schwede et al. 2000), we have observed that the phosphorylation of CREB in the bovine SCO can be mediated by the cyclic AMP–PKA pathway. Treatment with forskolin elicits maximal intracellular concentrations of cyclic AMP within 3–10 min and maximal pCREB levels after 30 min. A similar time course of CREB phosphorylation has been found in rat pinealocytes (Tamotsu et al. 1995). The natural neuroactive substances that induce CREB phosphorylation in the bovine SCO via the elevation of cyclic AMP levels have not yet been identified, since none of the natural neuroactive substances tested in our study causes an increase in cAMP levels in bovine SCO cells (see below).

By stimulation of the SCO cells with drugs that raise the intracellular concentration of free calcium ions (i.e., ionomycin, thapsigargin, caffeine), we have found that the activation of calcium signal transduction cascades can also induce phosphorylation of CREB in the bovine SCO. CREB phosphorylation is observed after treatment of the cells with ionomycin, which increases the influx of Ca2+ from the extracellular space, or with thapsigargin, which depletes thapsigargin-sensitive intracellular stores. In contrast, caffeine, which mobilizes calcium from ryanodinesensitive intracellular stores, does not induce CREB phospohorylation. These findings are in agreement with a previous study showing that treatment with ionomycin and thapsigargin resulted in increases in [Ca2+]i, whereas stimulation with caffeine does not change [Ca2+]i in cultured dispersed cells of the bovine SCO (Schöniger et al. 2002). Thus, the lack of effect of caffeine on CREB phosphorylation in bovine SCO cells may be attributable to the absence of ryanodine-sensitive calcium stores from these cells. Having established that CREB phosphorylation in bovine SCO cells can be achieved by increases in both cyclic AMP levels and [Ca2+]i,we investigated the potential effects of the natural neurotransmitters 5HT, SP, and ATP on the phosphorylation of CREB. The three substances were selected for our investigations, since they were identified as messengers of important input systems of the

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Fig. 8a, b Influence of neuroactive substances and forskolin on the formation of cyclic AMP in suspended cell cultures of the bovine SCO as analyzed by the ELISA technique. Strong activation of the cyclic AMP formation was caused by forskolin, the neuroactive substances had no effect on the formation of cyclic AMP (Ads stimulation with 10 µM adenosine, the enzymatic cleavage product of ATP, ATP stimulation with 10 µM adenosine-triphosphate, Co control treatment with medium only, Fk stimulation with 10 µM forskolin, SP stimulation with 100 nM substance P, 5HT stimulation with 10 µM serotonin, **P≤0.005 vs control). a Relative increase of cyclic AMP content in SCO cells after respective stimulations (10 min). b Dose-dependency of serotonin or serotonin plus forskolin on cyclic AMP formation Fig. 7a, b Relationship of pCREB and total CREB in bovine SCO cell and explant cultures analyzed by immunocytochemistry and immunoblot technique after stimulation with neuroactive substances for 30 min (ATP stimulation with 10 µM adenosine triphosphate, Co control stimulation with medium only, SP stimulation with 100 nM substance P, corrSUMDENS product of inverted grey value and the area of immunostained structures divided by the respective sample area, 5HT stimulation with 10 µM serotonin, **P≤0.01 vs control). a Histogram of semiquantitative analysis of the content of pCREB and total CREB in immunocytochemical preparations (cf. Fig. 4). b Images of the immunoblots stained for pCREB and total CREB; the total CREB signal remains unchanged, whereas the pCREB signal changes after stimulation with SP or ATP. c Relative content of pCREB compared with total CREB

bovine SCO (cf. Nürnberger and Schöniger 2001). Despite the finding that serotonin is present in a dense nerve fiber plexus innervating the bovine SCO (Nürnberger and Schöniger 2001; Jimenez et al. 2001), stimulation with 5HT completely failed to induce phosphorylation of CREB. In contrast, SP and ATP raised the level of pCREB significantly. ATP induced the pCREB immunoreaction in approximately 75% of the SCO cells, whereas SP elicited this response in only approximately 17% of the cells. The results were virtually identical in both dispersed cell cultures and explant cultures, suggesting that the responses to 5HT, SP, and ATP may be organotypic. The difference in the numbers of cells that respond to SP and ATP by CREB phosphorylation may be ex-

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Fig. 9a, b Effects of stimulation with neuroactive substances on intracellular free calcium concentration ([Ca2+]i) as recorded with the Fura-2/AM technique and number of pCREB-immunoreactive cells in cultured dispersed cells. a Original recordings of the [Ca2+]i (estimated by the ratio of emitted light at excitation of 334 nm/ 380 nm). b Comparative histogram showing the percentage of cells responding with an increase of [Ca2+]i and with phosphorylation of CREB to the respective stimulations (ATP stimulation with 10 µM adenosine triphosphate, Co control stimulation with medium only, SP stimulation with 100 nM substance P, 5HT stimulation with 10 µM serotonin, ***P≤0.001 vs control, bar duration of stimulation)

plained by the finding that receptors for ATP are more widely distributed in the SCO than those for SP. In principle, SP and ATP can act upon different receptor subtypes that can activate either the cyclic AMP-dependent and/or the Ca2+-dependent signal transduction cascades (Watling 1998; Krause et al. 1994; Quartara and Maggi 1997; Linden 1994; Zimmermann 1994; Communi and Boeynaems 1997; Post et al. 1998). In the present study, we have shown that SP, ATP, and adenosine, a possible enzymatic cleavage product of ATP, do not elevate the intracellular concentration of cyclic AMP. This supports the notion that the SP- or ATP-induced CREB phoshorylation in bovine SCO cells is not mediated via cyclic AMP-dependent mechanisms.

To analyze a possible involvement of calcium in SPor ATP-induced CREB phosphorylation, we investigated the effects of these two substances on [Ca2+]i in bovine SCO cells by the Fura-2 technique. SP and ATP were found to increase [Ca2+]i in 30% or 85% of bovine SCO cells, respectively. Similar numbers of responding cells were also found in our previous investigations on bovine SCO cells (Schöniger et al. 2002). The latter study demonstrated that the effects of SP and ATP on [Ca2+]i were dose-dependent, involved NK3 and P2Y2 receptors linked to G-protein and phospholipase C activation, and could not be mimicked by forskolin or 8-bromo-cAMP. Calcium responses to ATP were uniform and consisted of an initial Ca2+ release from thapsigargin-sensitive intracellular stores followed by the opening of L-type voltage-gated calcium channels (VGCCs) of the plasma membrane and a subsequent influx of Ca2+ from the extracellular space (Schöniger et al. 2002). SP stimulation elicited two different response patterns. In half of the SPsensitive cells, the increase in [Ca2+]i comprised Ca2+ release from thapsigargin-sensitive intracellular stores and the influx of extracellular calcium via PKC-induced opening of VGCCs. In the remaining half of the SP-sensitive cells, the increase in [Ca2+]i was caused exclusively by the influx of extracellular calcium via VGCCs (Schöniger et al. 2002). Correlation of our results on [Ca2+]i and CREB phosphorylation indicates that ATP affects the two parameters in a similar number of cells. Moreover, this correlation suggests that ATP induces CREB phosphorylation in bovine SCO cells via calcium-dependent mechanisms involving thapsigargin-sensitive intracellular calcium stores and the subsequent opening of VGCCs. With regard to the SP-sensitive SCO cells, only half of the cells responding with increases in [Ca2+]i show induction of pCREB. These may represent the cells in which SP stimulates a similar pathway as does ATP, i.e., the release of calcium from thapsigargin-sensitive stores followed by the opening of VGCCs. It may be inferred from morphological studies that 5HT is an important regulator for SCO functions, since this neurotransmitter is present in a dense nerve fiber plexus innervating the SCO of many mammalian species including bovine and rat (Møllgård and Wiklund 1979; Léger et al. 1983; Bouchaud 1993; Mikkelsen et al. 1997). Furthermore, in experimental studies with neurotoxins against 5HT in the rat, it has been shown that 5HT suppresses the secretory activity of the SCO (Léger et al. 1983). It was thus surprising that 5 HT did not affect any of the functional parameters tested in our present and previous studies. 5HT did not stimulate CREB phosphorylation in bovine SCO cells; it neither elevated cyclic AMP levels nor did it inhibit the forskolin-induced formation of cyclic AMP. Furthermore, 5HT had no effect on [Ca2+]i. Thus, the mechanisms through which this transmitter exerts its effects on SCO cells remain to be established. They may involve intracellular signal transduction cascades not investigated in our study. It is also possible that 5HT acts upon presynaptic nerve terminals

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in the SCO that are absent from our in vitro preparations (Gonzalez et al. 1999). The lack of particular 5HT receptors caused by culture in medium free of indolamine may be another reason for the absence of serotonergic effects of SCO cells. In summary, our studies demonstrate the presence of the transcription factor CREB in the SCO; this factor can be phosphorylated by pharmacological activation of the second messengers cAMP and calcium. The natural neuroactive substances that stimulate phosphorylation of CREB via the cyclic AMP-signal transduction pathway are still unknown; however, SP and ATP have been identified as neurotransmitters that mediate CREB phosphorylation via calcium-dependent mechanisms. Further studies now need to clarify the target genes of pCREB in SCO cells. It will be of special interest to analyze whether the genes encoding for the secretory glycoproteins of the SCO bear a CRE, and whether the synthesis of these glycoproteins is regulated at the transcriptional level. Acknowledgement The authors are grateful to Dr. H. Wicht, Dr. F. Dehghani, R. Kühn, E. Laedtke, and S. Leslie for expert technical assistance. Special thanks go to Dr. E. M. Rodríguez, Valdivia, Chile, for kindly donating the AFRU antisera.

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