1 2. Physiologisches Institut, Medizinische Fakultät, Universität des Saarlandes, D-66421 Homburg/Saar; and 2 Zoologie II, Universität Karlsruhe, D-76128 Karlsruhe, Germany
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ABSTRACT |
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Muscarinic acetylcholine receptors (mAChRs) in exocrine tissue from the avian nasal salt gland are coupled to phospholipase C and generate inositol phosphate and Ca2+ signals upon activation. An early effect of receptor activation in the secretory cells is a transient accumulation of c-Fos protein. In cooperation with constitutively expressed Jun, Fos presumably serves as a transcription factor altering gene expression during cell growth and differentiation processes in the gland associated with adaptation to osmotic stress in animals. Nothing is known, however, about the mAChR-dependent signaling pathways leading to Fos expression in these cells. By incubation of isolated nasal gland tissue in short-term culture with activators or inhibitors of signaling pathways and quantitative Western blot analysis of Fos abundance, we have now identified the sustained elevation of the intracellular Ca2+ concentration and the activation of the p38 mitogen-activated protein (MAP) kinase as intermediate signaling elements for the regulation of c-Fos by muscarinic receptor activation. It is suggested that p38 MAP kinase, rather than exclusively mediating stress responses, is involved in the regulation of cellular growth and differentiation controlled by G protein-coupled receptors.
muscarinic acetylcholine receptors; signal transduction; protooncogenes; Jun; intracellular calcium concentration; protein kinases; mitogen-activated protein kinase
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INTRODUCTION |
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MUSCARINIC RECEPTOR ACTIVATION in exocrine cells from the avian nasal gland activates phospholipase C, resulting in inositol phosphate accumulation, Ca2+ release from intracellular stores, and Ca2+ influx from the extracellular space (26, 27), as well as activation of protein kinase C (PKC) (3), presumably by elevating diacylglycerol production. Under conditions of osmotic stress in animals, these signals are involved in controlling chloride secretion and are necessary for the induction of cell growth and differentiation processes in the gland (14, 25). It is not well known, however, how a G protein-coupled receptor like the muscarinic acetylcholine receptor (mAChR) transmits signals for transcriptional or translational changes resulting in altered protein expression.
It has recently been shown (13) that osmotic stress in intact ducklings or, specifically, muscarinic activation of isolated nasal gland tissue, results in the induction of the protooncogene c-fos and a transient accumulation of Fos protein in the nuclei of secretory cells. We assume that heterodimerization of Fos with constitutively expressed Jun protein is a mechanism to control the transcription rate of growth- and differentiation-related genes in the gland as already shown for a variety of mammalian cell systems (2).
A potential target site of muscarinic receptor signaling for changes in Fos protein expression is the promoter region of the c-fos gene that contains two sensitive elements, the serum response element (SRE) (32) and the cAMP response element (CRE) (8). The SRE can be activated by a PKC-dependent signaling cascade (5), whereas both the SRE and the CRE are subject to regulation by cytosolic Ca2+ concentration (31, 33). Alternatively, mAChR-mediated signals may affect translational or posttranslational control mechanisms for the regulation of protein abundance (19).
In exocrine cells from the avian nasal salt gland, there is no c-fos mRNA or Fos protein present if tissue is being freshly isolated from "naïve" animals (Anas platyrhynchos) that are not osmotically stressed (13). Two hours after the onset of osmotic stress by feeding 1% NaCl solution to the animals, however, we already found increases in both fos mRNA and Fos protein, with maximum Fos levels occurring between 4 and 6 h. Fos accumulation seemed to depend on the activation of muscarinic receptors because organotypically cultured nasal gland tissue responded to mAChR activation with accumulation of Fos protein within 2 h. This response was sensitive to actinomycin D (13), indicating that muscarinic activation of nasal gland tissue induces transcription of the c-fos gene (and subsequent protein accumulation), rather than modulating mRNA stability and/or translational or posttranslational processes, although contributions of these mechanisms to the actual level of Fos in nasal gland tissue cannot be generally excluded.
To identify signal transduction pathways involved in Fos regulation in
muscarinically activated nasal gland tissue, we measured changes in Fos
levels at 2 h of organotypic culture in the absence or presence of the
muscarinic agonist carbachol. This stimulation period was chosen
because Fos accumulation at this time is well below maximum, indicating
that the actual level of Fos expression is more dependent on production
of mRNA and protein than on degradation. We used various activators or
inhibitors of intracellular signaling pathways (cf. Table
1) to enhance or block activities of
discrete signal transduction elements that are potential downstream
targets in signaling pathways of the muscarinic receptor. Although none of the applied agents affected Jun expression in cultured tissue samples (therefore, Jun was used as an internal control), we identified the sustained elevation in cytosolic Ca2+ levels and the
activation of p38 mitogen-activated protein (MAP) kinase as two
signaling elements that are required for Fos accumulation in
muscarinically activated nasal gland cells.
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MATERIALS AND METHODS |
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Animals and tissue culture. One-day-old ducklings (Anas platyrhynchos) were purchased from a commercial hatchery, placed in cages in a room with a 12:12-h light-dark cycle, and fed chick starter crumbs with drinking water ad libitum. All experimental animals were 6-12 days old. Ducklings were killed by decapitation, and the nasal glands were rapidly dissected out. The glands were cut into slices (250 µm) and cultured for 2 h as described previously (12). Drugs (cf. Table 1) were added directly to the culture medium as 100-1,000× stock solutions in distilled water or DMSO. The DMSO concentration never exceeded 0.1% (vol/vol) in the culture medium and was inefficient by itself in altering expression levels of Fos or Jun.
Sample preparation and electrophoresis. Slices were removed from the culture medium, frozen in liquid nitrogen, and immediately homogenized on ice in HEPES-buffered intracellular saline (13) containing 0.2 mmol/l of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Roth, Karlsruhe, Germany), 0.5% (vol/vol) of the nonionic detergent Igepal CA630 (Sigma, Deisenhofen, Germany), and 0.9 mmol/l of the phosphatase inhibitor sodium vanadate (Sigma). Homogenates were centrifuged for 5 min at 13,000 g and 4°C. Protein concentration was determined in the supernatant and aliquots were mixed with 2 vol of SDS sample buffer. Samples with identical protein contents were loaded onto 10% SDS polyacrylamide gels. Electrophoresis was carried out for 90 min at 120 V in a minigel apparatus (Bio-Rad, Munich, Germany). Proteins were blotted onto nitrocellulose membranes (Schleicher and Schüll, Dassel, Germany) using a Bio-Rad Trans-Blot cell. Blots were probed with 1:1,000 dilutions of polyclonal antisera against Fos (sc-253), Jun (sc-45), both from Santa Cruz (Heidelberg, Germany), extracellularly regulated kinase (ERK)-type MAP kinases, or p38 MAP kinase (all from New England Biolabs, Schwalbach, Germany). A horseradish peroxidase-linked donkey anti-rabbit antibody (Amersham, Freiburg, Germany) was used as the secondary antibody (dilution 1:6,000). Visualization of specific antibody binding was achieved using enhanced chemoluminescence technology (Amersham) and multiple exposure of the blots to X-ray film.
None of the drugs used in the culture media was able to induce a change in the abundance of the protooncogene product Jun in cultured tissue. Therefore, the level of Jun expression in each sample was used as an internal reference.P38 MAP kinase activity.
P38 MAP kinase was immunoprecipitated from the soluble fractions of
tissue slice homogenates with an equal protein content prepared in PBS
containing (in mmol/l) 154 NaCl, 5.4 Na2HPO4, 3.1 KH2PO4, 0.9 Na3VO4,
and 0.2 PMSF, pH 7.4. Homogenates were precleared by incubation with
protein A-agarose beads (Sigma) with a binding capacity of 1 mg IgG for
1 h at 4°C. Agarose beads were removed from the homogenate by
centrifugation at 10,000 g for 1 min. In parallel, protein
A-agarose beads with a binding capacity of 2 mg IgG were washed in PBS
and incubated with 3 µl of the original p38 MAP kinase antiserum (New
England Biolabs) for 1 h at 4°C. After washing these antibody-bound
beads in fresh PBS, the precleared tissue homogenates were added, and
the mixture was incubated at 4°C for 2 h. In preparation for the
kinase assay, the beads were washed twice in fresh PBS and finally
suspended in HEPES-buffered saline containing (in mmol/l) 25 HEPES and
10 MgCl2, pH 7.5. Kinase assays were performed according to
the manufacturer's instructions using a p38 MAP kinase assay kit
(Stratagene, La Jolla, CA) with the 22-kDa protein PHAS-I and
[-32P]ATP (New England Nuclear, Cologne,
Germany) as the substrates in the phosphorylation reaction. Reactions
were terminated by addition of SDS sample buffer and by heating to
90°C for 5 min. Samples were loaded onto 13% SDS electrophoresis
gels and run for 1 h at 120 V in a Bio-Rad minigel apparatus. Proteins
were transferred onto nitrocellulose membranes. Dried blots were
repeatedly exposed to X-ray film for different periods of time to
ensure that signals were in the linear range of the film. Western
blotting with p38 MAP kinase antisera was performed on the same blots
after the 32P radiation had decayed. This indicated that
equal amounts of p38 MAP kinase were used in parallel assay tubes in
the kinase assay.
Data processing. Western blot and kinase assay signals were analyzed using a computerized densitometer (Vilber-Lourmat, Marne la Vallee, France). The optical density of protein bands in the respective controls was used as a reference for calculating the relative density values of experimental protein bands. Statistical analysis (Student's t-test for unpaired samples and for equal or unequal variances) was performed using Prophet 5.0 software for Windows 95 (BBN Systems and Technologies, San Francisco, CA).
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RESULTS AND DISCUSSION |
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The substantial Fos accumulation elicited by activation of the
muscarinic receptor in salt gland tissue using carbachol [374 ± 58% of control (mean ± SD), n = 34, P < 0.01] was sensitive to the high-affinity muscarinic antagonist
atropine (Fig. 1; 96 ± 15%
of control, n = 12), indicating that it is
specifically the signal transduction cascade triggered by the receptor
that mediates the response. Incubation of nasal gland tissue in media with an elevated osmolality (400 mosmol/kgH2O, adjusted by
addition of NaCl) did not affect Fos abundance (data not shown).
Because the muscarinic receptor in nasal gland cells is coupled to
phospholipase C, receptor activation results in the generation of two
second messengers, D-myo-inositol
1,4,5-trisphosphate (InsP3), which releases
Ca2+ from intracellular stores, and diacylglycerol, which
activates PKC. We performed experiments to test which of these
signaling pathways is involved in mediating Fos accumulation.
Incubation of nasal gland tissue with 250 nmol/l thapsigargin, a
membrane-permeant inhibitor of intracellular Ca2+-ATPases
(29) that induces sustained elevations in intracellular Ca2+ concentration without activating phospholipase C (11),
resulted in Fos accumulation (Fig. 1; 314 ± 61%, n = 12, P < 0.01). Because various isoforms of PKC can be activated
by diacylglycerol or by elevated Ca2+ levels in cells, the
selective PKC inhibitor, bisindolylmaleimide, was added to the culture
medium together with carbachol or thapsigargin. As shown in Fig. 1, the
PKC inhibitor failed to suppress the Fos response induced by carbachol
or thapsigargin (carbachol: 298 ± 31%, n = 4, P < 0.01; thapsigargin: 284 ± 70%, n = 4, P < 0.05), indicating that PKC is not involved. This finding was confirmed by
experiments in which nasal gland tissue was incubated with the
synthetic diacylglycerol analog oleoyl-acetyl-glycerol (OAG) in
concentrations that had previously been shown to activate PKC in nasal
gland cells (3). As expected, OAG was not able to induce Fos
accumulation above the control level (Fig. 1; 104 ± 12%, n = 6). The same result was obtained when nasal gland tissue was treated
with phorbol ester for 1 h during the culture period (results not
shown). These results indicate that PKC is not involved in Fos
upregulation by the muscarinic receptor. On the other hand, both
carbachol- and thapsigargin-mediated Fos accumulation were suppressed
by the chelation of free Ca2+ in the extracellular medium
by EGTA (Fig. 1; 98 ± 11%, n = 8; 107 ± 17%, n = 8). EGTA, however, did not have any effect on Fos expression by itself
(data not shown). Chelation of free Ca2+ in the medium
prevents receptor-mediated Ca2+ entry through the plasma
membrane into the cytosol and the development of a sustained cytosolic
Ca2+ signal in the cell. These results indicate that a
sustained elevated plateau concentration of cytosolic Ca2+
is necessary and sufficient to upregulate Fos levels in these cells.
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It was therefore interesting to identify intermediate signaling
components that are involved in Ca2+-dependent steps in Fos
regulation. To test whether cellular protein kinase cascades are
involved, we treated cultured tissue with okadaic acid, an inhibitor of
protein serine and threonine phosphatases (4). The
presence of okadaic acid in the culture medium strongly enhanced Fos
accumulation (Fig. 2; 451 ± 92%,
n = 4, P < 0.01), indicating that
endogenous mAChR activation may induce Fos accumulation via activation
of serine- and threonine-specific protein kinases. To identify specific
downstream targets for Ca2+ signaling in these cells, we
cultured gland tissue in the presence of selective inhibitors for
cellular protein kinases.
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Various authors have reported that Ca2+- and calmodulin (CaM)-dependent protein kinases are involved in the induction of c-fos by different stimuli (24, 34). In cultured nasal gland tissue, however, the inhibition of Ca2+/CaM kinase II using KN-93 (28) did not result in any significant reduction in carbachol-mediated Fos accumulation (352 ± 51%, n = 4, P < 0.01). Moreover, the presence of CaM antagonists (W-7 or calmidazolium, n = 4 each) did not attenuate the mAChR-mediated Fos accumulation (Fig. 2; W-7: 341 ± 49%, n = 4, P < 0.01; calmidazolium: 374 ± 43%, n = 4, P < 0.01). This indicates that activation of CaM-dependent protein kinase may not be required for the processing of cytosolic Ca2+ signals mediating Fos accumulation.
The possible involvement of the 42/44 kDa ERK, which have been implicated in Ca2+-dependent nuclear signaling events in other cell systems (7), was studied in nasal gland tissue as well. Inhibition of ERK-activating kinase (MEK) using PD-98059 (1) in the culture medium or direct inhibition of ERK-type MAP kinases by olomoucine (18) did not suppress the carbachol-mediated Fos accumulation (Fig. 2; PD-98059: 316 ± 42%, n = 9, P < 0.01; olomoucine: 332 ± 40%, n = 4; P < 0.01). Furthermore, Western blot studies using soluble protein extracts from cultured nasal gland tissue probed with phosphospecific ERK-antisera (New England Biolabs) gave no indication of carbachol- (n = 7) or thapsigargin-mediated (n = 3) increases in the phosphorylation level of the enzyme, although this MAP kinase was readily detectable in nasal gland extracts using ERK kinase antisera in Western blot experiments (results not shown). This indicates that the ERK-type MAP kinase cascade is not involved in muscarinic receptor-mediated upregulation of Fos in these cells.
Fos expression can be induced in cells of higher organisms by exposure
to ultraviolet light or other forms of cellular stress (10, 15, 22).
This response is mediated by the p38 MAP kinase, a homolog of the yeast
HOG protein (9). As reported recently, G protein-coupled receptors are
able to activate p38 MAP kinase in certain cases (17, 20, 21).
Therefore, we tested nasal gland cells for possible effects of mAChR
activation on the p38 MAP kinase. With the use of phosphospecific
antibodies against p38 MAP kinase in Western blot experiments on
extracts of cultured nasal gland tissue, we found a significant
elevation in the level of phosphorylated p38 MAP kinase upon activation
of endogenous mAChRs (290 ± 68%, n = 6, P < 0.01),
although the total content of p38 MAP kinase did not change during the
stimulation period (Fig. 3). This response
was blocked by atropine (100 ± 16%, n = 4), indicating that
the effect of carbachol was specifically mediated by the mAChR.
Increases in p38 phosphorylation levels were also observed after
treatment of cultured tissue with thapsigargin (340 ± 55%, n = 4, P < 0.01) or with the phosphatase inhibitor okadaic acid
(Fig. 3; 328 ± 78%, n = 4, P = 0.01). Because
phosphorylation of the p38 MAP kinase is generally considered to be a
requirement for enzyme activation (23), we performed kinase assays
using immunoprecipitated p38 MAP kinase obtained from homogenates of cultured nasal gland tissue. As shown in Fig.
4, activation of nasal gland tissue in
culture by carbachol resulted in activation of the p38 MAP kinase as
indicated by the elevated level in radioactive phosphate present in the
MAP kinase substrate PHAS-I (475 ± 124%, n = 4, P < 0.01). These results indicate that muscarinic receptors may
activate the p38 MAP kinase by inducing phosphorylation of the enzyme
in a Ca2+-dependent manner.
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In other cell types, p38 MAP kinase activation mediates Fos
accumulation by transcriptional activation of the c-fos gene
(15, 22, 30). In addition, p38 may be able to affect subsequent Fos-regulatory processes at the levels of translation or protein degradation as well. Whether p38 MAP kinase activation in nasal gland
cells is involved in the control of one of these processes was studied
using a specific inhibitor for the p38 MAP kinase, SB-203580 (6).
Addition of this compound to the culture medium suppressed the
carbachol- and thapsigargin-mediated upregulation of Fos in nasal gland
tissue entirely (Fig. 5; carbachol: 99 ± 7%, n = 4; thapsigargin: 102 ± 18%, n = 4). This
indicates that p38 MAP kinase activation is a necessary signaling step
in the receptor-mediated upregulation of Fos.
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To identify possible upstream or downstream kinases involved in activation of p38 MAP kinase or in relaying p38 MAP kinase signals to the nucleus or to other effectors, we immunoprecipitated activated p38 MAP kinase from tissue homogenates and looked for potentially interacting proteins in Western blot assays (results not shown). In other cell systems, Ca2+/CaM-dependent kinase II is involved in c-fos induction (24, 34). In p38 immunoprecipitates of nasal gland cell extracts, however, we found no indication of p38 interaction with Ca2+/CaM kinase II (antibodies from Biomol, Hamburg, Germany), although this enzyme was detectable in Western blots using total nasal gland homogenates (results not shown). Together with the results mentioned in Fig. 2 that CaM antagonists were unable to suppress receptor-mediated Fos accumulation in cultured tissue, this indicates that the activation of p38 MAP kinase in nasal gland cells is mediated by other, as yet unknown Ca2+-dependent processes.
There are several downstream kinases that are activated by p38 MAP kinase-mediated protein phosphorylation. We obtained antisera against MAPKAP kinase-2 (Upstate Biotechnology, Lake Placid, NY) and against 3pK (a kind gift from S. Ludwig, Würzburg, Germany). Neither kinase could be detected in p38 immunoprecipitates from activated nasal gland tissue by Western blotting. Therefore, the question about target sites of activated p38 MAP kinase is still open as is the problem of whether Fos protein abundance is regulated via transcriptional or other mechanisms.
We have tried to provide proof for a significant suppression of c-fos transcription in carbachol-treated tissue samples in the presence of the p38 MAP kinase inhibitor SB-203580. However, the semiquantitative PCR assay we utilized for that purpose showed a substantial intrinsic variability in the amounts of amplified products. This may be partially due to the fact demonstrated in a previous paper (13) that tissue slicing and culturing procedures by themselves cause some Fos protein accumulation in salt gland tissue that is, however, restricted to cells of the connective tissue of the gland and does not occur in the exocrine cells where Fos accumulation is strictly dependent on receptor activation. This is also the reason for the observation that there is always a low level of Fos protein present in the controls of cultured tissue samples (Figs. 1, 2, and 5), which never occurs in freshly isolated tissue. Because we do not have a chance of separating exocrine from connective tissue before isolating mRNAs, the "contamination" of our mRNA isolates with connective tissue mRNA makes it extremely difficult to obtain exocrine cell-specific PCR-amplification signals in a reproducible manner. Although there seems to be a tendency in our results that SB-203580 lowers the amount of mRNA in short-term (10-20 min) carbachol-activated samples compared with samples that were treated with carbachol alone, we cannot exclude that p38 MAP kinase activation does have additional target mechanisms besides activation of transcription factors affecting c-fos gene transcription in the nucleus.
If we assume a transcriptional control of Fos accumulation in mAChR-activated nasal gland tissue, it seems possible that nasal gland cells lack intermediate kinases between p38 MAP kinase and the nuclear transcription factors, as has been shown recently for other cell systems (16, 22). In that case, further studies are needed to identify these potential nuclear targets of p38 MAP kinase. On the other hand, p38 MAP kinase signaling may have (additional) interaction sites in the posttranscriptional machinery (mRNA stability, translation, protein degradation) that remain to be identified as well.
In summary, this study presents evidence for the presence of a novel signaling cascade of a G protein-coupled receptor for the transient upregulation of the fos protooncogene product in exocrine cells. The results indicate that agonist binding to endogenous mAChRs causes an increase in the level of c-Fos protein by activating the p38 MAP kinase cascade, a process that requires the generation of a sustained cytosolic Ca2+ signal that induces p38 MAP kinase activation by a still unknown intermediate step.
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ACKNOWLEDGEMENTS |
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We thank Dr. Irene Schulz for support, Marion Schwarz for excellent technical assistance, and Dr. Frank Thevenod for comments on the manuscript.
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FOOTNOTES |
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This work was supported by Deutsche Forschungsgemeinschaft Grants Hi 448/4-1 and -2 and a Heisenberg stipend (to J.-P. Hildebrandt) at the time the experiments were performed.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J.-P. Hildebrandt, Zoologisches Institut und Museum, Ernst-Moritz-Arndt-Universitaet, Joh.-Seb.-Bach-Strasse 11/12, D-17489 Greifswald, Germany (E-mail: jph{at}mail.uni-greifswald.de).
Received 8 October 1998; accepted in final form 3 December 1999.
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