Muscarinic activation of mitogen-activated protein kinase in rat thyroid

epithelial cells

Eugenio Jimeneza,*, M. Idoia Gameza, M. Julia Bragadob, Mercedes Montiela

aDepartamento de Bioquımica y Biologıa Molecular, Facultad de Medicina, Universidad de Malaga, Campus de Teatinos s/n, 29080 Malaga, SpainbDepartamento de Bioquımica, Biologıa Molecular y Genetica, Facultad de Veterinaria, Universidad de Extremadura, Avda. de la Universidad s/n,

10071 Caceres, Spain

Received 27 July 2001; accepted 19 December 2001

Abstract

Carbachol (Cch), a muscarinic acetylcholine receptors (mAChR) agonist, produces time- and dose-dependent increases in mitogen-

activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in nondifferentiated Fischer rat thyroid (FRT)

epithelial cells. Cells pretreatment with the selective phospholipase C inhibitor U73122 resulted in a decrease of Cch-stimulated ERK1/2

phosphorylation. These data indicated that the effect of mAChR on ERK activation could be mediated through agonist-induced Ca2 +

mobilization or PKC activation. Phosphorylation of ERK1/2 was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate

acetate (PMA), but was not altered either by PKC inhibitor GF109203X or by down-regulation of PKC. Phosphorylation of ERK1/2 was

elevated by a direct [Ca2 + ]i increase caused by thapsigargin or ionophore. Additionally, Cch-induced ERK1/2 phosphorylation was

reduced after either inhibition of Ca2 + influx or intracellular Ca2 + release. Nevertheless, Cch-mediated ERK1/2 activation was genistein

sensitive, indicating the involvement of protein tyrosine kinases on the downstream signalling of mAChR. Pretreatment of the cells with

PP2 markedly decreased Cch-induced ERK1/2 phosphorylation, suggesting a role of Src family of tyrosine kinases in the signal

transduction pathway involved in ERK1/2 activation by mAChR. To test the biological consequences of ERK activation, we examined

the effect of mAChR on cell functions. Cch stimulation of FRT cells did not affect cell proliferation, but increased protein synthesis. This

effect was significantly attenuated by PD98059, a selective inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK). This

study demonstrated that muscarinic receptor-mediated increase in the ERK1/2 phosphorylation was dependent on [Ca2 + ]i but

independent of PKC and was mediated by the Src family of tyrosine kinases. Our results also supported the idea that the protein

synthesis stimulated by mAChR in polarized FRT epithelial cells was regulated by the ERK1/2 phosphorylation pathway. D 2002

Elsevier Science Inc. All rights reserved.

Keywords: Fischer rat thyroid cells; Muscarinic acetylcholine receptors; Carbachol; MAPK/ERK; protein synthesis

1. Introduction

Muscarinic acetylcholine receptors (mAChR) belong to a

class of receptors whose members possess seven transmem-

brane domains and transmit signals by coupling to hetero-

trimeric G proteins. Molecular biology approaches have

demonstrated the existence of five different mAChR forms,

termed m1, m2, m3, m4, and m5. There seems to be a general

agreement that these molecular subtypes represent the

pharmacological subtypes M1, M2, M3, M4, and M5,

respectively [1].

It has been shown that m1, m3, and m5 mAChR subtypes

stimulate the adenylyl cyclase, phospholipase C (PLC) and

phospholipase A2 (PLA2) activities, whereas m2 and m4

mAChR subtypes do not interact with PLA2, lightly activate

PLC, and inhibit the adenylyl cyclase activity [2,3]. In

addition to the regulation of these classical G-protein

effectors, it has been recently reported that mAChR sub-

types activate a variety of protein kinases and signalling

molecules. The acetylcholine analogue carbachol (Cch)

rapidly activated mitogen-activated protein kinase/extracel-

lular signal-regulated kinase (MAPK/ERK), and caused

tyrosine phosphorylation of the adapter protein Shc and

0898-6568/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved.

PII: S0898 -6568 (02 )00010 -4

* Corresponding author. Tel.: +34-952-131-536; fax: +34-952-131-

534.

E-mail address: ejg@uma.es (E. Jimenez).

www.elsevier.com/locate/cellsig

Cellular Signalling 14 (2002) 665–672

epidermal growth factor (EGF) receptors in HEK cells

stably expressing muscarinic m3 receptors [4]. Moreover,

activation of m3 mAChR subtype also evokes tyrosine

phosphorylation of the p125FAK, and its substrates paxillin

and p130cas, in rat pancreatic acini [5].

It is now known that depending upon receptors and cell

types, G protein-coupled receptors (GPCR) utilize multiple

strategies for the activation of mitogenic signalling path-

ways, and different mechanisms have been proposed to link

GPCR to MAPK/ERK [6]. One mechanism is the direct

activation of Raf-1 by protein kinase C alpha (PKCa) [7]. Asecond mechanism, Ras dependent, is mediated by trans-

activation of receptor tyrosine kinases mediated by Gbgsubunits, and subsequent recruitment of Src family of

tyrosine kinases and adaptor proteins, such as Shc and

Grb2 [4,8,9]. A third possible mechanism is mediated by

p125FAK or PYK2, and subsequent recruitment of Src

family of tyrosine kinases and adaptor proteins to focal

adhesion [10,11].

Recently, we have demonstrated the existence of m3

mAChR in plasma membrane preparations of Fischer rat

thyroid (FRT) epithelial cells [12], which are coupled to

intracellular calcium mobilization, but not to adenylyl

cyclase activity [13]. Calcium plays a critical role in signal

transduction pathways in a wide variety of cells. Intra-

cellular signalling pathways coupled to m3 mAChR in

FRT cells are poorly understood. The purpose of the present

study was to determine whether the activation of this

muscarinic receptor subtype evokes phosphorylation of

MAPK/ERK, and the intracellular pathways involved in

its regulation. This study shows, for the first time, that

muscarinic m3 receptor activation increases MAPK/ERK

phosphorylation by a calcium- and Src-dependent pathway,

which likely mediates the stimulation of protein synthesis

induced by Cch in nondifferentiated FRT cells.

2. Materials and methods

2.1. Materials

Calf serum was from Gibco (Paisley). Thapsigargin,

genistein, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyra-

zolo[3,4-d]pyrimidine (PP2), 1-[6-((17b-3-methoxyestra-

1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione

(U73122), and rapamycin were from Calbiochem-Nova-

biochem (San Diego, CA). Cch, Coon’s modified Ham’s

F12 medium, antibiotics, 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyl-tetrazolium bromide (MTT), protease inhib-

itors, bisindolylmaleimide I (GF109203X), PMA, BSA,

DMSO, and other reagents were obtained from Sigma

(St. Louis, MO). Monoclonal p-ERK1/2 and polyclonal

ERK1/2 antibodies were from Santa Cruz Biotechnology

(Santa Cruz, CA), and anti-mouse IgG–horseradish per-

oxidase conjugated, calibrated prestained standards and

the chemiluminescence kits were obtained from Pierce

(Rockford, IL). L-[3H]phenylalanine (60–90 Ci/mmol)

was from American Radiolabeled Chemicals (St. Louis,

MO). Poly(vinylidene difluoride) membranes were from

Millipore (Bedford, MA) and Hyperfilm ECL was from

Amersham (Buckinghamshire).

2.2. Cell culture conditions

FRT epithelial cells were routinely grown in Coon’s

modified Ham’s F12 medium, supplemented with 5% calf

serum, containing gentamicin (50 mg/ml) and amphotericin

B (0.25 mg/ml). The cells were maintained in a water-

saturated atmosphere of 5% CO2 and 95% air at 37 �C.Before the experiments, cells were harvested with 0.05%

trypsin–EDTA solution, plated onto plastic 100-mm culture

dishes or six-well plates for 3 days, and incubated 24 h in

serum-free fresh medium prior to the experiments.

2.3. MAPK/ERK Western blot analysis

Quiescent cultures of FRT cells were incubated at 37 �Cwith vehicle, agents, or Cch as indicated. Cells were rinsed

with ice-cold phosphate-buffered saline (PBS) containing

0.5 mM Na3VO4 and then collected in 0.5 ml lysis buffer

(50 mM Tris–HCl, pH 7.5, containing 150 mM NaCl, 1%

Triton X-100, 1% deoxycholate, 0.2 mM Na3VO4, 1 mM

EGTA, 0.4 mM EDTA, 1 mg/ml of aprotinin and leupeptin,

and 0.1 mg/ml of phenylmethylsulphonyl fluoride). Lysates

were centrifuged at 10,000� g for 15 min at 4 �C to

remove insoluble material and normalized for protein

content. Equal amounts of protein (15 mg) were separated

by 10% SDS–polyacrylamide gel electrophoresis and then

transferred electrophoretically to poly(vinylidene difluor-

ide) membranes for 90 min using a Multiphor II electro-

phoresis unit (LKB, Bromma) at 1 mA/cm2. Membranes

were incubated for 1 h at 25 �C in blocking buffer (10 mM

Tris–HCl, pH 7.5, containing 150 mM NaCl, 5% milk,

and 0.1% Tween-20), and then incubated for 90 min at

25 �C with monoclonal p-ERK1/2 antibody diluted 1:1000

in blocking buffer. Membranes were washed twice with

blocking buffer and incubated for 45 min at 25 �C with

anti-mouse IgG–horseradish peroxidase conjugated

(1:3000 in blocking buffer) as a secondary antibody.

Membranes were finally washed twice with blocking

buffer and twice with washing buffer (10 mM Tris–HCl,

pH 7.5, containing 150 mM NaCl, and 0.1% Tweeen-20),

incubated with enhanced chemiluminescence detection

reagents and exposed to Hyperfilm ECL. Approximate

molecular masses were estimated using calibrated pre-

stained standards.

2.4. Protein synthesis measurements

FRT cells maintained 24 h in serum-free medium were

incubated in the presence or absence of protein kinase

inhibitors, and were then stimulated with 100 mM Cch

E. Jimenez et al. / Cellular Signalling 14 (2002) 665–672666

for 24 h. Cells were labelled with 3[H]-phenylalanine

(2 mCi/ml) for the last 6 h of treatment, rinsed with cold

PBS and harvested with 10% trichloroacetic acid on ice.

Samples were centrifuged at 25,000� g for 5 min at 4 �C,washed with trichloroacetic acid, and recentrifuged. Pellets

were solubilized in 0.2 N NaOH (500 ml, 30 min, 70 �C)and the radioactivity was measured in a Wallac 1414

liquid scintillation counter (Perkin-Elmer, Turku). A por-

tion of each sample was analysed for total protein, and

the results were expressed as disintegrations per minute

(dpm)/microgram protein.

2.5. Cell proliferation assay

Growth studies in vitro were conducted using the MTT

spectrophotometric assay [14]. FRT cells were placed in

96-well plates in Coon’s modified Ham’s F12 medium with

5% calf serum and allowed to adhere overnight. Subcon-

fluent conditions were chosen to allow detection for max-

imal growth. The medium was changed to Coon’s modified

Ham’s F12 medium without calf serum for 24 h to induce

quiescence. Cch concentrations (1–100 mM) were added to

each well for 48 h, and then 10 ml of MTT solution (5 mg/ml)

was added to the assay plates. Cells were incubated for

another 3 h at 37 �C and the purple formazan crystals

formed were dissolved by addition of 200 ml DMSO

followed by thorough mixing. The plates were subsequently

read on a MRX spectrophotometer at 570 nm (Dinex

Technologies, Chantilly, VA).

2.6. Protein analysis

Protein contents were measured using the Coomassie

blue binding method of Bradford [15].

2.7. Data analysis

Results were expressed as mean ± S.E.M. Data were

compared by ANOVA followed by the Newman–Keuls

multiple comparison test using SigmaStat, version 2.01

(Jandel Scientific).

3. Results

3.1. Cch induced MAPK/ERK phosphorylation in FRT cells

in a time- and concentration-dependent manner

Since MAPK/ERK are activated by phosphorylation of

both threonine and tyrosine residues [16], and changes in

the phosphorylation state reflect changes in activity [17],

we first examined whether stimulation with Cch was able

to induce phosphorylation of these kinases in FRT cells.

Using a monoclonal p-ERK1/2 antibody, which specifically

recognizes only the phosphorylated forms, two immuno-

reactive bands of 42 and 44 kDa were detected in lysates

prepared from untreated FRT cells. Cch (100 mM) rapidly

and transiently stimulated ERK1/2 phosphorylation. As

shown in Fig. 1B, ERK1/2 phosphorylation reached a

maximum between 5 and 10 min after stimulation with

Cch (3.5-fold increase versus basal), and returned to

baseline in 30 min. Western blotting was also performed

using an anti-ERK1/2 antibody that recognizes both phos-

phorylated and nonphosphorylated forms of these kinases

(Fig. 1A). Results demonstrated that the expression level of

the proteins ERK1/2 remained unchanged over the ana-

lysed time after Cch stimulation.

In FRT cells expressing muscarinic m3 receptors, Cch

stimulated ERK1/2 phosphorylation in a concentration-

dependent manner (Fig. 2). A half-maximal increase in

ERK1/2 phosphorylation was induced after cell stimulation

with 1 mM Cch and the maximal stimulation was achieved at

10 mM. This time- and concentration-dependent response of

ERK1/2 activation is consistent with previous studies in

different cell types [4,18,19].

Fig. 1. Cch time-dependent effect on ERK1/2 phosphorylation. FRT cells

were stimulated with 100 mM Cch for different times (1–60 min), and cell

lysates were analysed by Western blotting with anti-total ERK1/2 antibody

(A) or anti-p-ERK antibody (B). Results shown are from a representative

experiment. Data from three to five experiments are expressed as a

percentage of unstimulated cells. *P< .05 versus control.

E. Jimenez et al. / Cellular Signalling 14 (2002) 665–672 667

3.2. Role of Ca2+ in Cch-induced MAPK/ERK

phosphorylation

Since calcium has been implicated as a potential regu-

lator of the ERK pathway in many cell types, different

approaches were used in this study to investigate the Ca2 +

requirement for Cch-induced MAPK/ERK activation in

nondifferentiated thyroid epithelial cells. It is well known

that Cch receptor occupation causes a rapid activation of

PLC, resulting in the generation of inositol phosphates and

diacylglycerol, which releases intracellular calcium and

activates PKC, respectively [20]. Our first approach was

to preincubate FRT cells with different concentrations of

U73122, a specific inhibitor of PLC. We have previously

demonstrated in these cells that the PLC inhibitor U73122

markedly reduced the calcium mobilization induced by Cch

at 1 mM and abolished it at higher concentrations such as

10 mM [13]. Results in Fig. 3 showed that 30 min pretreat-

ment of FRT cells with U73122, at concentrations that

abolished the Ca2 + response, resulted in a concentration-

dependent decrease in Cch-stimulated MAPK/ERK phos-

phorylation (Lanes 3 and 4).

To further dissect the downstream pathways of PLC, we

studied the direct effect of increasing the intracellular

calcium concentration on ERK1/2 phosphorylation. There-

fore, we have used thapsigargin, an inhibitor of endo-

plasmic reticulum Ca2 + –ATPase that caused a gradual

Fig. 3. Effect of the PLC inhibitor U73122 on Cch-stimulated ERK1/2

phosphorylation. FRT cells were pretreated with 5 or 10 mM U73122 for

20 min before stimulation with 100 mM Cch, and cell lysates were analysed

by Western blotting with anti-p-ERK1/2 antibody. Results shown in the

bottom panel are from a representative experiment. Data from three to five

experiments are expressed as a percentage of unstimulated cells. *P < .05

versus control; aP < .05 versus Cch.

Fig. 2. Cch concentration-dependent effect on ERK1/2 phosphorylation.

FRT cells were stimulated with various Cch concentrations (0.1–100 mM)

for 5 min, and cell lysates were analysed by Western blotting with anti-p-

ERK1/2 antibody. Results shown in the bottom panel are from a

representative experiment. Data from three to five experiments are

expressed as a percentage of unstimulated cells. *P < .05 versus control.

Fig. 4. Effect of intracellular Ca2 + increase on ERK phosphorylation. FRT

cells were treated for 5 min with 100 mM Cch or 1 mM thapsigargin (Tha) or

1 mM calcium ionophore A23187, and cell lysates were analysed by

Western blotting with anti-p-ERK1/2 antibody. Results shown in the bottom

panel are from a representative experiment. Data from three to five

experiments are expressed as a percentage of unstimulated cells. *P < .05

versus control.

E. Jimenez et al. / Cellular Signalling 14 (2002) 665–672668

increase in [Ca2 + ]i in FRT cells, as we previously showed

[13], and the calcium ionophore A23187. As seen in Fig. 4,

an elevation of intracellular calcium concentration, caused

by 1 mM thapsigargin or 1 mM A23187, induced a pro-

nounced increase on ERK1/2 phosphorylation (Lanes 3

and 4 respectively), similar to the increase caused by Cch

(Lane 2).

To further investigate the effect of calcium on Cch-

induced ERK1/2 phosphorylation, we have used a different

approach. We have previously shown that incubation in a

Ca2 + -free medium (with 2 mM EGTA) inhibited the Ca2 +

influx stimulated by Cch in FRT cells [13]. Results shown in

Fig. 5, Lane 3, demonstrated that phosphorylation of

ERK1/2 induced by Cch was significantly reduced in a

Ca2 + -free medium (with EGTA). To study the effect of

intracellular Ca2 + released by Cch in ERK1/2 phosphor-

ylation, we have depleted the intracellular calcium stores

using thapsigargin for 15 min in a Ca2 + -free medium (2 mM

EGTA). In similar experimental conditions (5 min), we have

previously demonstrated that the intracellular Ca2 + increase

in response to Cch was inhibited in FRT cells [13]. Pretreat-

ment of FRT cells with thapsigargin for longer time as

15 min in a Ca2 + -free medium significantly reduced the

Cch-induced ERK1/2 phosphorylation (Fig. 5, Lane 4).

3.3. Role of PKC in MAPK/ERK phosphorylation

As mentioned, Cch receptor occupation activates PLC,

resulting in the generation of inositol phosphates, which

subsequently release intracellular calcium, and diacylgly-

cerol that activates PKC. Because inhibition of PLC with

U73122 caused a significant reduction of the ERK1/2

phosphorylation, we further investigated the role of PKC,

the other downstream pathway of PLC. To determine

whether PKC activation might be involved in mediating

Cch-stimulated ERK1/2 activation, we next evaluated the

effect of an activator of PKC, the phorbol ester PMA, on

Cch stimulation of MAPK/ERK. Treatment of FRT cells

with PMA (1 mM) for 5 min caused a rapid phospho-

rylation of ERK1/2 (Fig. 6, Lane 3), comparable to the

effect obtained with Cch (Fig. 6, Lane 2). However, in

cells incubated overnight with 1 mM PMA to down-

regulate PKC, the Cch-induced MAPK/ERK activation

was not significantly affected (Fig. 6, Lane 4). We

further evaluated the role of PKC on Cch stimulation

of MAPK/ERK by using a selective inhibitor of PKC,

GF109203X. Pretreatment of FRT cells with 5 mMGF109203X demonstrated that Cch-induced ERK1/2

phosphorylation was not affected when PKC is inhibited

(Fig. 6, Lane 5).

Fig. 5. Effect of Ca2 + on Cch-stimulated ERK1/2 phosphorylation. FRT

cells were treated with 100 mM Cch for 5 min or pretreated for 2 min with

2 mM EGTA or for 15 min with thapsigargin (Tha) in a Ca2 + -free medium

(containing 2 mM EGTA) before stimulation with 100 mM Cch, and cell

lysates were analysed by Western blotting with anti-p-ERK1/2 antibody.

Results shown in the bottom panel are from a representative experiment.

Data from three to five experiments are expressed as a percentage of

unstimulated cells. *P < .05 versus control; aP < .05 versus Cch.

Fig. 6. Effect of protein kinase C on Cch-stimulated ERK1/2 phosphor-

ylation. FRT cells were treated with 100 mM Cch or 1 mM PMA for 5 min,

or pretreated overnight with 1 mM PMA or 5 mM GF109203X (GFX) for

30 min before stimulation with 100 mM Cch, and cell lysates were analysed

by Western blotting with anti-p-ERK1/2 antibody. Results shown in the

bottom panel are from a representative experiment. Data from three to five

experiments are expressed as a percentage of unstimulated cells. *P < .05

versus control.

E. Jimenez et al. / Cellular Signalling 14 (2002) 665–672 669

3.4. Role of protein tyrosine kinases in MAPK/ERK

phosphorylation

In most cases, MAPK activation by GPCR may occur by

tyrosine kinase-dependent pathways that converge at the

level of the ternary complex Shc–Grb2–Sos1 [21]. We

therefore evaluated the involvement of tyrosine kinases on

the signalling leading to ERK phosphorylation after Cch

stimulation of FRT cells. We have pretreated cells with

genistein, a general and nonspecific inhibitor of different

tyrosine kinases, and results are shown in Fig. 7. Two

different concentrations of genistein, 50 and 100 mM,

significantly attenuated the ERK phosphorylation induced

by Cch (Fig. 7, Lanes 3 and 4). Since Cch-mediated ERK

phosphorylation was sensitive to this nonspecific inhibitor

of tyrosine kinases, genistein, we next investigated the effect

of the tyrosine kinase inhibitor PP2, specific for the Src

family of tyrosine kinases. Pretreatment of FRT cells with

two different concentrations of PP2, 10–20 mM, greatly

reduced (by more than 50%) the Cch-induced ERK phos-

phoryation (Fig. 7, Lanes 5 and 6). 3.5. Cch stimulated protein synthesis in FRT cells

Since we have demonstrated that muscarinic m3 receptor

occupation in FRT cells induced intracellular signalling

pathways leading to ERK phosphorylation, our next goal

was to investigate a plausible physiological role of this Cch-

induced cascade. We first studied whether Cch stimulation

led to FRT cell proliferation and results using the MTT assay

showed that Cch did not affect cell proliferation (data not

shown). We next analysed the protein synthesis after Cch

stimulation and results are shown in Fig. 8. Cch caused a

significant increase in protein synthesis in FRT cells, with an

enhancement of 30 ± 8.5% over untreated cells. In addition,

pretreatment of the FRT cells with different concentrations

of PD98059 (5–30 mM), a specific inhibitor of ERK1/2

activation, markedly reduced in a dose-dependent manner

the L-[3H]phenylalanine incorporation into total protein

induced by Cch.

4. Discussion

It is now known that the activation of signal trans-

duction pathways by growth factors and GPCR is medi-

ated, in part, through two closely related MAPKs, ERK1

and ERK2, which are regulated by dual phosphorylation

of specific tyrosine and threonine residues mapping within

a characteristic Thr–Glu–Tyr motif. Significant hetero-

geneity exists in the signalling mechanism utilized by

GPCR agonists to stimulate MAPK/ERK in different cell

types. The predominant determinants underlying the sig-

nalling pathways used between GPCR and MAPK/ERK

activation appear to lie in the nature of receptor coupling

to associated G proteins, and in the particular complement

of signalling molecules expressed in different cell types

[22,23].

Fig. 7. Effect of protein tyrosine kinases on Cch-stimulated ERK1/2

phosphorylation. FRT cells were treated with 100 mM Cch for 5 min, or

pretreated with genistein (Gen) or PP2, for 30 min, at the indicated

concentrations, before stimulation with 100 mM Cch, and then cell lysates

were analysed by Western blotting with anti-p-ERK1/2 antibody. Results

shown in the bottom panel are from a representative experiment. Data from

three to five experiments are expressed as a percentage of unstimulated

cells. *P < .05 versus control; aP < .05 versus Cch.

Fig. 8. Inhibitors of ERK1/2 activation block Cch-mediated protein

synthesis. FRT cells were treated with 100 mM Cch for 24 h, and then

labelled with L-[3H]phenylalanine (2 mCi/ml; 6 h). Cells were pretreated

with PD98059 at the indicated concentrations for 30 min before Cch

stimulation. Results are expressed as disintegrations per minute/microgram

total protein normalized to untreated cells. *P< .05 versus control; aP < .05

versus Cch.

E. Jimenez et al. / Cellular Signalling 14 (2002) 665–672670

Both Gi- and Gq-coupled mAChR have been shown to

activate MAPK in various systems, but few data have

demonstrated endogenous MAPK activation in thyroid

cells. The major finding of this study is that mAChR

activation by Cch rapidly and transiently stimulated

ERK1/2 phosphorylation in FRT cells in a time- and

dose-dependent manner, as shown in Figs. 1 and 2. This

time- and concentration-dependent response of ERK1/2

activation is consistent with previous studies in different

cell types [4,18,19]. It is well demonstrated that Cch

receptor occupation activates PLC, resulting in the sub-

sequent generation of inositol phosphates and diacylgly-

cerol, which releases intracellular calcium and activates

PKC, respectively [20]. In nondifferentiated and polarized

FRT cells, the inhibition of PLC by using U73122, led to a

dose-dependent inhibition of Cch-induced ERK phospho-

rylation (Fig. 3). In the same FRT cells, we have previously

shown that the inhibition of PLC led to a total blockade of

the Ca2 + mobilization induced by Cch [13]. We further

confirmed the role of Ca2 + in this pathway by demonstrating

that an increase in intracellular Ca2 +, caused by two different

agents, ionophore and thapsigargin [13], is sufficient to

induce ERK phosphorylation up to levels similar to those

induced by Cch in these cells (Fig. 4). The mechanisms by

which intracellular Ca2 + stimulates the phosphorylation of

ERK1/2 are complex and appear to be dependent on the

nature of mAChR subtype coupling to heterotrimeric

G proteins. Intracellular Ca2 + can modulate the MAPK

cascade, via activation of the monomeric G-protein p21ras

[24], through two convergent mechanisms; one, calcium-

dependent tyrosine kinase (PYK2) and the other mediated by

calmodulin [19]. In T84 colonic epithelial cells, which

express endogenous m3 mAChR subtypes [25], the eleva-

tions in [Ca2 + ]i in response to Cch activate signalling

mechanisms involving calmodulin-, PYK2-, and p60src-

mediated transactivation of the EGF receptor [26].

Additionally, we have demonstrated in this study that

when the extracellular Ca2 + is removed, the ERK phos-

phorylation response induced by Cch is significantly

reduced by 40% (Fig. 5). When we used at the same time

a Ca2 + -free medium (EGTA) plus an inhibitor of the intra-

cellular Ca2 + mobilization in response to Cch, thapsigargin,

the effect on ERK phosphorylation was also significantly

reduced (Fig. 4). These results supported the idea that Cch-

induced ERK phosphorylation in FRT cells is at least

partially dependent of intracellular Ca2 + mobilization.

Besides Ca2 + , the other downstream pathway induced

after PLC activation is the PKC transduction cascade. This

study demonstrated that the direct activation of PKC, by

the phorbol ester PMA, was sufficient to increase the

phosphorylation of ERK1/2 reaching levels similar to those

induced by Cch in FRT cells (Fig. 6). However, the Cch-

induced ERK1/2 phosphorylation was not mediated by

PKC as shown by two different experimental approaches,

with the inhibitor of PKC, GF109203X, and by down-

regulation of the PKC (Fig. 6). None of these approaches

affected the Cch-induced ERK1/2 phosphorylation. The

conclusion of these results is that Cch-stimulated ERK

phosphorylation in FRT cells is mediated by Ca2 + -depend-

ent but PKC-independent mechanism(s).

Although our data indicated that intracellular Ca2 + is

partly mediating the activation of MAPK family members

ERK1 and ERK2, other intracellular signalling pathways

should also be involved in the MAPK/ERK activation in

these nondifferentiated thyroid cells. It is well known that

activation of MAPK by GPCR, including mAChR,

involves tyrosine phosphorylation of one or more proteins,

such as p125FAK, p130cas, or paxillin [27,28]. Src family of

protein tyrosine kinases has been implicated in mAChR-

induced ERK activation in different cell lines [29]. Further

studies were performed to determine the possible relation-

ship between the activation of these protein tyrosine

kinases and the downstream effects of mAChR after G

protein activation.

Since FRT cells treatment with genistein significantly

decreased MAPK/ERK phosphorylation, our results suggest

that Cch effects on ERK1 and ERK2 phosphorylation were

probably mediated through the activation of protein tyrosine

kinases (Fig. 7). The present study demonstrated that Cch-

induced ERK activation was dependent on the activity of

cytoplasmatic Src-like tyrosine kinase family, since phar-

macological inhibition of the Src family of tyrosine kinases

with specific PP2 effectively blocked the Cch-induced

MAPK/ERK activation in FRT cells (Fig. 7). The Src family

of tyrosine kinases has been implicated in the ERK activa-

tion by various agonists for GPCR. Recent information

suggests that activation of Src tyrosine kinases may lead

to the phosphorylation of the adaptor protein Shc and the

recruitment of Grb/Sos complex to the plasma membrane,

resulting in the activation of ERK pathway [4,8,9].

Since ERK family is implicated in the regulation of

various cell functions as cell proliferation, migration, dif-

ferentiation, and survival [30,31], the biological consequen-

ces of MAPK/ERK activation were investigated in FRT

cells. Activation of mAChR did not affect cell proliferation

in our experimental conditions, although caused a slight but

significant increase in protein synthesis in these nondiffer-

entiated thyroid cells (Fig. 8). Cch-stimulated protein syn-

thesis was inhibited by pretreatment of FRT cells with

PD98059, a highly selective inhibitor of ERK1 and ERK2

phosphorylation, suggesting that MAPK/ERK phosphoryla-

tion might play a physiological role in regulating protein

synthesis in FRT cells.

In summary, our results demonstrated that mAChR

activation in nondifferentiated FRT cells led to an increase

on MAPK/ERK phosphorylation via a PLC, intracellular

Ca2 + mobilization, and Src-like tyrosine kinase family-

dependent mechanisms, but is PKC independent. The intra-

cellular transduction pathway of ERK1/2 phosphorylation

induced after mAChR activation might be involved in the

regulation of the protein synthesis in nondifferentiated

FRT cells.

E. Jimenez et al. / Cellular Signalling 14 (2002) 665–672 671

Acknowledgments

Fischer rat thyroid epithelial cells were a generous gift

from Dr. Di Jeso (Centro di Endocrinologia ed Oncologia

Sperimentale ‘‘G. Salvatore,’’ CNR, Italy). This work was

supported by Grant PM98-0221 from the Direccion General

de Ensenanza Superior e Investigacion Cientıfica, Madrid.

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