1 Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114; and 2 Department of Ophthalmology, Asahikawa Medical College, Asahikawa 078-8510, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The
purpose of this study was to determine the role of p42/p44
mitogen-activated protein kinase (MAPK) in
1-adrenergically and cholinergically stimulated protein
secretion in rat lacrimal gland acinar cells and the pathways used by
these agonists to activate MAPK. Acini were isolated by collagenase
digestion and incubated with the
1-adrenergic agonist
phenylephrine or the cholinergic agonist carbachol, and activation of
MAPK and protein secretion were then measured. Phenylephrine and
carbachol activated MAPK in a time- and concentration-dependent manner.
Inhibition of MAPK significantly increased phenylephrine- and
carbachol-induced protein secretion. Inhibition of EGF receptor (EGFR)
with AG1478, an inhibitor of the EGFR tyrosine kinase activity,
significantly increased phenylephrine- but not carbachol-induced
protein secretion. Whereas phenylephrine-induced activation of MAPK was
completely inhibited by AG1478, activation of MAPK by carbachol was
not. Phenylephrine stimulated tyrosine phosphorylation of the EGFR, whereas carbachol stimulated p60Src, and possibly Pyk2, to
activate MAPK. We conclude that, in the lacrimal gland, activation of
MAPK plays an inhibitory role in
1-adrenergically and
cholinergically stimulated protein secretion and that these agonists
use different signaling mechanisms to activate MAPK.
signal transduction; Pyk2; Src
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE LACRIMAL GLAND is the major contributor to the aqueous portion of the tear film (2). Its function is to secrete protein, water, and electrolytes onto the ocular surface to maintain, nourish, and protect the cornea and conjunctiva. The lacrimal gland is composed of several cell types including acinar cells, the major cell type comprising ~80% of the gland, myoepithelial cells, and ductal cells. Acinar cells are highly polarized and joined by tight junctions at the luminal membrane, thus creating distinct basolateral and apical membranes (2). The lacrimal gland is highly innervated, and these nerves release their neurotransmitters, which interact with receptors on the basolateral membrane of the acinar cells. This initiates the signaling pathways necessary for secretion of proteins, electrolytes, and water across the apical membrane.
We have previously shown that lacrimal gland secretion is under
neural control with cholinergic agonists, released from parasympathetic nerves, and 1-adrenergic agonists, released from
sympathetic nerves, being the major regulators of lacrimal gland
protein secretion (1, 7). We have also previously shown
that these agonists activate separate signal transduction pathways to
stimulate protein secretion in lacrimal gland acinar cells
(8). Cholinergic agonists interact with muscarinic
M3 receptors through a G
q/11 G protein to
activate phospholipase C (PLC) (19). Activation of PLC
generates the Ca2+-mobilizing second messenger inositol
1,4,5-trisphosphate and the protein kinase C (PKC) activator
diacylglycerol (DAG) (19). Ca2+ either alone
or with Ca2+/calmodulin-dependent protein kinases
phosphorylates specific substrates, which lead to protein secretion.
DAG activates the PKC isoforms
,
, and
, which play a direct
role in stimulating secretion (26). We have also shown
that cholinergic agonists activate phospholipase D (PLD) to generate
phosphatidic acid, which can be converted to DAG (25).
Unlike those in other tissues, lacrimal gland
1-adrenergic receptors are not coupled to activation of
PLC or PLD (8, 25). There are, in fact, few details
regarding the pathway these receptors activate in lacrimal gland acinar
cells. The subtypes of
1-adrenergic receptors present in
these cells and the effector enzyme(s) activated by these receptors are
unidentified. It is known that PKC
plays a major role in protein
secretion stimulated by phenylephrine [an
1-adrenergic
agonist in the lacrimal gland (1)], whereas PKC
and
-
play inhibitory roles (26).
Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine protein kinases. These members include MAPKs [also known as extracellular signal-regulated kinases (ERKs) or p42/p44 MAPK], the p38 MAPKs, and the c-Jun NH2-terminal kinases (JNKs) (24). The p42/p44 MAPK (hereafter called MAPK) pathway is best known for its involvement in the signaling pathway of many growth factors, including epidermal growth factor (EGF) (23). This pathway involves autophosphorylation of the EGF receptor (EGFR), which then acts as a scaffold for the recruitment of the adaptor proteins Shc and Grb2. Shc is tyrosine phosphorylated, resulting in recruitment of the adapter protein Grb2 and the guanine nucleotide exchange factor Sos (20). Sos then activates Ras (15), which in turn initiates the activation of a cascade of protein kinases, namely Raf (MAPK kinase kinase), MEK (MAPK kinase), and MAPK. Traditionally, activation of MAPK is thought to regulate long-term cellular processes such as cell proliferation and differentiation (13).
G protein-coupled receptors (GPCRs) have been traditionally
thought of as regulators of short-term processes such as protein secretion. It is now widely documented that GPCRs can also activate the
MAPK pathway (14, 16), linking these receptors to cell proliferation and differentiation. The mechanisms by which GPCRs activate MAPK are not completely understood. One hypothesized mechanism
involves the release of heparin-binding EGF (HB-EGF) through cleavage
of its precursor molecule, proHB-EGF, by metalloproteinases (22). This process, termed shedding, occurs at or near the
cell surface and converts the membrane-bound form (proHB-EGF) to the soluble HB-EGF (17). Pierce et al. (21) have
shown that this shedding is also dependent on the G subunit and
Src, implying that G
subunit activates Src, which in turn
activates one or more metalloproteinases, leading to cleavage of
proHB-EGF. The released HB-EGF can then stimulate the EGFR in an
autocrine or paracrine fashion, stimulating the MAPK cascade. In
addition, GPCRs can activate MAPK without transactivating the EGFR. PKC has been shown to directly activate Raf, whereas p60Src has
been shown to be activated by the Ca2+-dependent
non-receptor tyrosine kinase Pyk2, leading directly to activation of
Raf (13). Phosphoinositide 3-kinase, which can be a target
of G
, also activates Src (13).
In the present study we show that inhibition of MAPK increases
1-adrenergically and cholinergically stimulated protein
secretion in the lacrimal gland.
1-Adrenergic agonists
transactivate the EGFR, leading to the tyrosine phosphorylation of Shc
and recruitment of Grb2, two early events mediating MAPK activation.
Conversely, cholinergic agonists do not transactivate the EGFR to
activate MAPK. They do however, activate p60Src, and
possibly Pyk2, to activate MAPK. We conclude that activation of MAPK in
freshly isolated lacrimal gland epithelial cells by
1-adrenergic and cholinergic agonists occurs through
different mechanisms to inhibit protein secretion.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. EGF and recombinant protein A-agarose were obtained from Upstate Biotechnology (Lake Placid, NY). Collagenase type III was from Worthington Biochemical (Freehold, NJ). AG1478 [4-(3-chloroanilino)-6,7-dimethoxyquinazoline], an inhibitor of the tyrosine phosphorylation activity of the EGFR, was from Calbiochem (La Jolla, CA). Amplex Red was from Molecular Probes (Eugene, OR). U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene], an inhibitor of MEK, was from Cell Signaling Technology (Beverly, MA). PP1 {4-amino-5-(4-methylphenyl)- 7-(t-butyl)pyrazolo[3,4-d]pyrimidine}, a Src inhibitor, was purchased from Biomol (Plymouth Meeting, PA). Other chemicals were obtained from Sigma Chemical (St. Louis, MO).
Antibodies. Antibody against phosphorylated p42/44 MAPK that detects phosphorylated tyrosine 204 of p42/44 MAPK was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A monoclonal antibody against total p42 MAPK was from Santa Cruz Biotechnology. A polyclonal antibody against Shc [amino acids (aa) 359-473 in the SH2 domain] was purchased from Transduction Laboratories (Lexington, KY); and a monoclonal antibody against Shc (aa 366-473) was from Santa Cruz Biotechnology. A monoclonal anti-phosphotyrosine antibody (clone 4G10) was from Upstate Biotechnology. Polyclonal antibodies against Grb2, total Pyk2, and the EGFR were from Santa Cruz Biotechnology. Antibody against total p60Src was purchased from Biosource International.
Preparation of lacrimal gland acini. All experiments conformed to the U.S. Department of Agriculture Animal Welfare Act (1985) and were approved by the Schepens Eye Research Institute Animal Care and Use Committee. Both exorbital lacrimal glands were removed from male Sprague-Dawley rats that had been anesthetized with CO2 for 1 min and then decapitated. Lacrimal glands were trimmed of fatty and connective tissue and fragmented into small pieces 2-3 mm in diameter. The pieces were then washed at 37°C in Krebs-Ringer bicarbonate (KRB) buffer (in mM: 119 NaCl, 4.8 KCl, 1 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 25 NaHCO3) supplemented with 10 mM HEPES, 5.5 mM glucose, and 0.5% BSA (KRB-HEPES), pH 7.4. Lacrimal gland acini were prepared by incubating tissue pieces with collagenase (CLS III; 150 U/ml) in 10 ml of KRB-HEPES buffer for 30 min at 37°C under a stream of 95% O2-5% CO2. Lacrimal gland lobules were subjected to gentle pipetting ten times at regular time intervals through tips of decreasing diameter. The preparation was then filtered through nylon mesh (150-µm pore size), and the acini were pelleted with a 2-min centrifugation at 50 g. The pellet was washed twice by centrifugation (50 g, 2 min) through a 4% BSA solution made in KRB-HEPES buffer. The dispersed acini were allowed to recover for 30 min in 5 ml of fresh KRB-HEPES buffer containing 0.5% BSA. Cell viability was monitored with trypan blue (Sigma).
Detection of MAPK activation by Western blotting.
Lacrimal gland acini were incubated for the indicated time period with
phenylephrine (104 M), EGF (10
7 M), or
carbachol (10
4 M). For inhibition experiments, acini were
preincubated with either the MEK inhibitor U0126
(10
8-10
6 M) (4) or the
p60Src inhibitor PP1 (10
5 M) (6)
for 30 min or with the EGFR inhibitor AG1478 (10
7 M)
(12) for 15 min. To terminate incubation, the acini were centrifuged, the supernatant was discarded, 300 µl of ice-cold RIPA
buffer (10 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 100 µg/ml PMSF,
30 µl/ml aprotinin, and 1 mM Na3VO3),
supplemented with protease and phosphatase inhibitors, were then added,
and the acini were sonicated. The homogenate was centrifuged at 3,000 rpm for 30 min at 4°C, and proteins in the supernatant were separated by SDS-PAGE (10% acrylamide gels) and transferred to nitrocellulose membranes. Activated MAPK was detected with antibodies that
specifically recognize the phosphorylated (activated) pools of enzymes.
Films were scanned, and data were analyzed using NIH Image. Values for phosphorylated enzymes (amounts for p42 and p44 MAPK were added together) were normalized to the amount of total enzyme by using antibodies to nonphosphorylated enzyme and were expressed as multiples of increase above the control value, which was set as 1.
Immunoprecipitation experiments.
Lacrimal gland acini were incubated for the indicated time period with
carbachol (104), phenylephrine (10
4 M), or
EGF (10
7 M). To terminate incubation, the acini were
centrifuged, the supernatant was discarded, and 1 ml of ice-cold RIPA
buffer was added. The homogenate was centrifuged at 3,000 rpm for 30 min at 4°C. The supernatant (cell lysate) was incubated overnight at
4°C on a rocker platform in the presence of the specified
immunoprecipitating antibody. After the addition of 100 µl of protein
A-agarose for 2 h at 4°C, the immunoprecipitate was collected by
brief centrifugation. After the pellet had been washed four times with
RIPA buffer, the immunoprecipitate was resuspended in Laemmli sample
buffer and boiled for 5 min. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Immunoreactive bands were
visualized using the enhanced chemiluminescence method. The films were
scanned and analyzed using NIH Image. Western blotting was also
performed, using the immunoprecipitating antibody to normalize for the
amount of cells in each condition.
Measurement of protein secretion.
Lacrimal gland acini were incubated for 20 min with the
1-adrenergic agonists phenylephrine (10
4
M) or EGF (10
7 M) or the cholinergic agonist carbachol
(10
4 M). Either U0126
(10
8-10
6 M) or PP1 (10
5
M) was added to acini 30 min before addition of the agonists. AG1478
(10
7 M) was added 15 min before addition of the agonists.
To terminate the reaction, acini were centrifuged at 500 g
for 30 s, and the supernatant was removed. The acini (pellet) were
then homogenized in 10 mM Tris · HCl, pH 8.0. The amount of
peroxidase, our marker for protein secretion, was measured in the
supernatant and pellet spectrophotometrically using Amplex Red,
according to the manufacturer's protocol (Molecular Probes). In short,
in the presence of peroxidase and hydrogen peroxide, Amplex Red reacts
to form a highly fluorescent molecule. Fluorescence is read at an
excitation wavelength of 560 nm and an emission wavelength of 590 nm on
a fluorescent microplate reader (Bio-Tek, Winooski, VT). The amount of
peroxidase in the supernatant (secreted) was expressed as a percentage
of the total peroxidase (amount of peroxidase in the supernatant plus
amount of peroxidase in the pellet).
Data presentation. Data are expressed as means ± SE and were analyzed using Student's t-test with P < 0.05 considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of 1-adrenergic and cholinergic agonists on
activation of MAPK in freshly isolated lacrimal gland acinar cells.
To determine whether MAPK is activated by
1-adrenergic
and cholinergic agonists, we incubated lacrimal acinar cells in the presence of phenylephrine (10
4 M), a selective
1-adrenergic agonist in the lacrimal gland
(1), or carbachol (10
4 M). Activation of
MAPK was measured by Western blotting techniques using antibodies that
selectively recognize the phosphorylated (active) form of MAPK as
described in MATERIALS AND METHODS. As shown in Fig.
1A, phenylephrine induced a
time-dependent activation of MAPK. Cirazoline, another
1-adrenergic agonist, gave similar results (data not
shown). Activation of cholinergic receptors with carbachol and EGF
(10
7 M), used as a positive control, also induced a
time-dependent activation of MAPK (Fig. 1A). When the blots
from three independent experiments were analyzed by densitometry, the
combined results showed that the kinetics of phenylephrine-,
carbachol-, and EGF-induced activation of MAPK were similar with a
statistically significant effect reached after 5 min and returning to
basal by 30 min (Fig. 1B).
|
|
Effect of U0126 on MAPK activation.
To determine whether the MEK inhibitor U0126 inhibited MAPK in the
lacrimal gland, we preincubated acini with U0126
(108-10
6 M) for 30 min before
stimulation with the cholinergic agonist carbachol (10
4
M) for 10 min. The cells were homogenized and subjected to Western blot
analysis with the antibodies for phosphorylated MAPK and total MAPK as
described earlier. As shown in Fig.
3A, carbachol increased MAPK
activity, which was blocked by U0126 in a concentration-dependent manner. U0126 had little effect on basal activity of MAPK. When the
blots from three independent experiments were analyzed by densitometry,
the combined results showed that carbachol-stimulated MAPK was
inhibited 10, 15, 78, 91, and 96% with U0126 at 10
8,
10
7, 3 × 10
7, 6 × 10
7, and 10
6 M, respectively (data not
shown). These results indicate that U0126 inhibits MAPK activation in
the lacrimal gland.
|
Effect of inhibition of MAPK on 1-adrenergic and
cholinergic-stimulated protein secretion.
To determine the role of MAPK in phenylephrine- and
carbachol-stimulated peroxidase secretion, we preincubated lacrimal
gland acini for 30 min with the MEK inhibitor U0126 at concentrations of 10
7, 3 × 10
7, and 6 × 10
7 M, which we had previously shown to inhibit
carbachol-stimulated MAPK activation (Fig. 3A), before
stimulation with either phenylephrine (10
4 M) or
carbachol (10
4 M). We did not use U0126 at
10
6 M because it increased basal secretion in these cells
(data not shown). U0126 significantly increased phenylephrine-induced
peroxidase secretion to 135 ± 11 and 134 ± 10% at 3 × 10
7 and 6 × 10
7 M, respectively
(Fig. 3B). Carbachol-stimulated peroxidase secretion was
significantly increased to 201 ± 40% at 3 × 10
7 M U0126 and to 255 ± 50% at 6 × 10
7 M U0126 (Fig. 3C). Secretion stimulated by
either agonist was not significantly affected by U0126 at
10
7 M. The two concentrations of U0126 that were most
effective at inhibiting carbachol-stimulated MAPK activation were also
the most effective at increasing phenylephrine- and carbachol-induced protein secretion. These results indicate that, in the lacrimal gland,
activation of MAPK by
1-adrenergic and cholinergic
agonists plays an inhibitory role in stimulated peroxidase secretion.
Effect of inhibition of the EGFR on 1-adrenergic-
and cholinergic-activation of MAPK.
We next determined the ability of
1-adrenergic and
cholinergic agonists to transactivate the EGFR. Thus we tested the
effect of AG1478, a selective inhibitor of the EGFR intrinsic tyrosine kinase (12), on agonist-induced activation of MAPK. As
shown in Fig. 4A, AG1478
(10
7 M) inhibited EGF- and phenylephrine-induced MAPK
activation, whereas there was little effect on carbachol-induced MAPK
activation. Because these experiments were performed separately, both
the controls are shown. When the blots from independent experiments were analyzed by densitometry, the combined results showed that AG1478
inhibited EGF-induced MAPK activation by 81% and phenylephrine-induced MAPK activation by 93% (note that the control value was set to 1). In
contrast, AG1478 did not inhibit carbachol-induced MAPK activation
(Fig. 4B). To show that AG1478, at the concentration used,
inhibited phosphorylation of the EGFR, we measured both basal and
EGF-induced tyrosine phosphorylation of the EGFR by immunoprecipitation
and Western blotting techniques. As shown in Fig. 4C, AG1478
(10
7 M) completely inhibited EGF-stimulated
phosphorylation of the EGFR. These results suggest that
1-adrenergic, but not cholinergic, agonists activate
MAPK through transactivation of the EGFR in lacrimal gland acinar
cells.
|
Effect of inhibition of the EGFR on 1-adrenergic-
and cholinergic-induced protein secretion.
To determine the role of EGFR in MAPK-mediated inhibition of protein
secretion, we incubated acini with AG1478 (10
7 M) and
measured peroxidase secretion. As shown in Fig.
5, peroxidase secretion was statistically
significantly stimulated above basal secretion in the presence of the
each agonist alone, whereas AG1478 alone had no effect on basal protein
secretion. Inhibition of the EGFR with AG1478 statistically
significantly increased phenylephrine-induced peroxidase secretion
in a concentration-dependent manner. In contrast, AG1478 had no
significant effect on carbachol-stimulated peroxidase secretion (Fig.
5). These results again suggest that
1-adrenergic, but
not cholinergic, agonists transactivate the EGFR to activate MAPK,
which in turn inhibits protein secretion in lacrimal gland acinar
cells.
|
-Adrenergic agonists stimulate tyrosine phosphorylation of Shc
and recruitment of Grb2.
To determine whether
1-adrenergic agonist-induced
activation of the EGFR results in tyrosine phosphorylation of Shc and
recruitment of Grb2, we performed immunoprecipitation experiments in
which acinar cells were stimulated with phenylephrine, EGF, or
carbachol and immunoprecipitated with an anti-Shc antibody and then
performed Western blot analysis with antibodies against
phosphotyrosine, Grb2, or Shc. As shown in Fig.
6A, when
acinar cells were stimulated with phenylephrine (10
4 M)
and the cell lysates were immunoprecipitated with an anti-Shc antibody,
there was a time-dependent increase in the amount of tyrosine
phosphorylated p52 Shc. EGF (10
7 M), used as a positive
control, also increased the amount of tyrosine-phosphorylated p52 Shc
(Fig. 6A). Importantly, when the Shc immunoprecipitates were
blotted with an anti-Grb2 antibody, phenylephrine also increased the
amount of Grb2 associated with Shc in a time-dependent manner (Fig.
6A). EGF also increased the amount of Grb2 associated with
Shc. When four independent experiments were analyzed densitometrically,
results showed that phenylephrine caused a significant increase in the
amount of tyrosine phosphorylation of Shc after 30 s (Fig.
6B) and also increased the association of Grb2 with Shc
(Fig. 6C). EGF also significantly increased the tyrosine
phosphorylation of Shc after 5 min as well as its association with Grb2
(n = 3). It is worth noting that, similar to MAPK,
there is a substantial amount of tyrosine phosphorylated Shc under
resting conditions. In contrast, when three independent experiments
were analyzed, results showed that activation of cholinergic receptors did not increase the amount of tyrosine-phosphorylated p52 Shc (Fig.
6D). These results show that
1-adrenergic
agonists, but not cholinergic agonists, stimulate tyrosine
phosphorylation of p52 Shc and its association with the adapter protein
Grb2.
|
-Adrenergic agonists transactivate the EGFR.
To confirm that
1-adrenergic agonists transactivate the
EGFR and confirm the results shown in Figs. 4-6, we stimulated
acinar cells with EGF (10
7 M) as a positive control,
phenylephrine (10
4 M), or carbachol (10
4
M). Immunoprecipitation experiments were performed either with an EGFR
antibody and the amount of tyrosine phosphorylation quantitated by
immunoblotting or with a phosphotyrosine antibody and the amount of
EGFR quantitated by immunoblotting. Figure
7A is a representative blot
showing that stimulation with EGF (10
7 M) and
phenylephrine (10
4 M) increased the amount of tyrosine
phosphorylation of the EGFR after 5 min, whereas carbachol did not. As
shown in Fig. 7B, when the results of three to four
experiments were summed, they showed that EGF induced a significant
increase (3.8-fold) over basal in the amount of tyrosine phosphorylated
EGFR. Importantly, phenylephrine also significantly increased the
amount of tyrosine phosphorylated EGFR (2.2-fold) over basal. Carbachol
did not increase the amount of tyrosine phosphorylation of the EGFR. It
is worth noting that we often found a substantial amount of tyrosine
phosphorylated EGFR under basal conditions, similar to phosphorylated
Shc and MAPK (Fig. 7A). These results show that
1-adrenergic agonists transactivate the EGFR. They also
suggest that transactivation of the EGFR mediates
1-adrenergic agonist-induced MAPK activation.
|
Cholinergic agonists stimulate tyrosine phosphorylation of Pyk2 and
p60Src.
Another possible pathway to activate MAPK, independent of the EGFR,
involves activation of Pyk2, which in turn can activate p60Src. To determine whether 1-adrenergic
and cholinergic agonists also activate Pyk2 and p60Src to
activate MAPK, we stimulated acini with phenylephrine
(10
4 M) or carbachol (10
4 M) for 5 min and
performed immunoprecipitation experiments using an antibody against
phosphotyrosine. Western blot analysis was then performed with
antibodies against Pyk2 and Src. When four independent experiments were
analyzed densitometrically, the combined results showed that carbachol
significantly increased tyrosine phosphorylation of Pyk2 2.4-fold above
basal, whereas phenylephrine and EGF had no effect on tyrosine
phosphorylation of Pyk2 (Fig. 8A). Similarly, carbachol
increased the tyrosine phosphorylation of p60Src 1.5-fold
above basal, whereas phenylephrine and EGF did not (Fig. 8B). These results suggest that cholinergic agonists, but
not
1-adrenergic agonists, activate Pyk2 and
p60Src.
|
Effect of inhibition of p60Src on cholinergic and
1-adrenergic agonist-stimulated MAPK activation and
protein secretion.
Because carbachol activates p60Src, we investigated the
role of this enzyme in carbachol- and phenylephrine-stimulated MAPK
activation and peroxidase secretion using the specific
p60Src inhibitor PP1 (6). Acini were
preincubated with PP1 (10
5 M) for 30 min and stimulated
with carbachol (10
4 M) or phenylephrine
(10
4 M) for either 5 min (MAPK activation) or 20 min
(peroxidase secretion). A representative blot, shown in Fig.
9A, indicates that
preincubation of acini with PP1 inhibited carbachol activation of MAPK.
It is important to note that the amount of total MAPK in the cells
stimulated with phenylephrine and phenylephrine plus PP1 is
substantially less than the amount in the other conditions. These
variations are a result of the fact that the lacrimal gland acini used
in these studies were freshly isolated for each experiment. When the
blots from three independent experiments were densitometrically scanned, results showed that PP1 statistically significantly inhibited carbachol-stimulated MAPK activation by 43 ± 8% while having no effect on phenylephrine-induced activation of MAPK (Fig.
9B). Values for the amount of phosphorylated MAPK were
corrected for the amount of total MAPK. Conversely, PP1 statistically
significantly increased carbachol-stimulated peroxidase secretion over
carbachol stimulation alone and had no effect on phenylephrine-induced
protein secretion (Fig. 9C). PP1 had no effect on basal
secretion (data not shown). These results imply that p60Src
activation is required for carbachol-stimulated MAPK activation and
inhibition of peroxidase secretion.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, we have shown that the MAPK pathway is
involved in the negative regulation of protein secretion from the
lacrimal gland as inhibition of MAPK increases
1-adrenergically and cholinergically induced protein
secretion.
1-Adrenergic agonists activate MAPK through
transactivation of the EGFR and recruitment of Shc and Grb2. These
agonists do not use Pyk2 or p60Src to activate the EGFR.
Cholinergically induced activation of MAPK does not occur through
transactivation of the EGFR and involves activation of
p60Src, and possibly Pyk2, which activates MAPK distal to
the recruitment of Shc and Grb2. Classically, MAPK is involved in
long-term events such as cell growth and differentiation. This study
demonstrates the involvement of MAPK in the short-term process, protein
secretion. Keely et al. (9, 10) have shown that carbachol
activates MAPK, which is involved in the negative regulation of
chloride secretion from T84 cells. This is similar to the lacrimal
gland in that GPCR-induced MAPK activation also leads to a negative regulation of the secretory response. However, in contrast to the
lacrimal gland, in which carbachol activates Pyk2 and
p60Src but not the EGFR, in T84 cells carbachol
transactivates the EGFR to activate MAPK, an effect mediated by
intracellular Ca2+, Pyk2, and p60Src. Also in
contrast to the lacrimal gland, MAPK in T84 cells mediates carbachol
inhibition of secretion, whereas in the lacrimal gland MAPK inhibits
carbachol stimulation of secretion. Thus, although similar signaling
components are used in different cell types, the effects are cell
specific. It is possible that different anchoring proteins could
recruit the components in various configurations depending on the cell
type and, thus, have varying effects.
The main function of the lacrimal gland is to synthesize proteins and
secrete them onto the ocular surface, with cholinergic and
1-adrenergic agonists being potent stimuli of lacrimal
gland secretion (2). Many of these proteins possess
antimicrobial activities or are growth factors and are crucial for the
homeostasis of the ocular surface. As a result, the regulation
of protein secretion must be highly regulated, and the net secretion of
1-adrenergic and cholinergic agonists must be the result
of a tight balance of stimulatory and inhibitory pathways. Therefore,
it is of interest that cholinergic and
1-adrenergic
agonists activate both stimulatory and inhibitory pathways. We have
identified two such inhibitory pathways for
1-adrenergic
agonists. We have previously shown that whereas
1-adrenergic agonists activate PKC
, -
, and -
, activation of PKC
and -
inhibits
1-adrenergically
stimulated protein secretion and PKC
stimulates this secretion.
Similarly,
1-adrenergic agonists and cholinergic
agonists activate MAPK, which also inhibits protein secretion in the
lacrimal gland. It is tempting to speculate that PKC
and -
are a
part of the pathway involved in the activation of MAPK leading to
inhibition of
1-adrenergically stimulated protein
secretion, although there is no evidence to corroborate this
hypothesis. It is also possible that
1-adrenergic agonists activate two independent pathways, both of which inhibit protein secretion. It is not known why these agonists would activate both stimulatory and inhibitory pathways. It is possible that this
inhibition acts as a braking system to slow protein secretion, eventually bringing secretion back to basal levels.
In all other cells studied, cholinergic and 1-adrenergic
agonists activate a common pathway, namely the Ca2+/PKC
pathway, which occurs through activation of PLC and/or PLD (5). Unique to the lacrimal gland acinar cells, we
previously found (8, 25) that
1-adrenergic
receptors are not coupled to the activation of PLC or PLD, in contrast
to cholinergic receptors. Moreover, we showed (26) that
even though both cholinergic and
1-adrenergic agonists
activate PKC, they each activate distinct isoforms of PKC to stimulate
secretion, further supporting the fact that cholinergic and
1-adrenergic receptors operate through separate pathways
to control lacrimal gland function. The results reported in the present
study expand on this premise. Both the phosphorylation of the EGFR and
the recruitment of Shc and Grb2 indicate that
1-adrenergic agonists transactivate the EGFR to activate
MAPK, whereas cholinergic agonists do not. Cholinergic, but not
1-adrenergic, agonists activated p60Src,
leading to MAPK activation and inhibition of protein secretion. Although we have shown that cholinergic agonists also activate Pyk2, we
have no direct evidence for its role in activation of MAPK. The fact
that the p60Src inhibitor PP1 only partially inhibited
carbachol-stimulated MAPK activation could imply that there are
multiple pathways within the lacrimal gland used by cholinergic
agonists to activate MAPK. It is interesting to note that even a
partial inhibition of MAPK by PP1 still leads to a significant increase
in carbachol-stimulated protein secretion.
The mechanisms involved in GPCR-induced transactivation of the EGFR are
complicated, and many aspects are still not clearly understood. It is
known that G proteins, metalloproteinases, and PKC play pivotal roles
in activation of MAPK. Prenzel et al. (22) showed that
transactivation of the EGFR by GPCR requires cleavage of proHB-EGF by
metalloproteinases. Luttrell et al. (21) showed that the
G subunits activate Src, which subsequently activates metalloproteinases, leading to phosphorylation of the EGFR with subsequent recruitment of adapter proteins Shc and Grb2 and activation of MAPK. The EGFR can also be completely bypassed, because PKC can
directly activate Raf and Pyk2 to activate MAPK (11). In addition, Pyk2 is known to be activated by Ca2+ as well
(3). The involvement of metalloproteinases, PKC, or Ca2+ in
1-adrenergic and cholinergic
agonist-induced MAPK activation in the lacrimal gland and its
regulation of protein secretion is not understood. It is unlikely that
metalloproteinases are involved in cholinergic activation of MAPK
because cholinergic agonists do not transactivate the EGFR. We
hypothesize that cholinergic agonists activate Pyk2 and
p60Src, which in turn directly activate Raf, leading to
activation of MAPK, though the exact mechanisms by which Pyk2 and
p60Src activate MAPK are not known. It is not known whether
1-adrenergic agonists activate metalloproteinases, since
these agonists do not activate Pyk2 or p60Src. Thus the
exact mechanisms by which
1-adrenergic agonists
transactivate the EGFR to activate MAPK are not known.
During our investigations, we consistently found substantial amounts of tyrosine phosphorylated Shc associated with Grb2, tyrosine phosphorylated EGFR, and active MAPK under basal conditions. Protein secretion in the lacrimal gland occurs through two distinct pathways: a constitutive pathway in which proteins are constantly synthesized and secreted, and a regulated pathway in which synthesized proteins are stored in secretory granules awaiting external stimuli to be secreted. It is possible that the basal level of activated MAPK is necessary for regulation of protein synthesis for the constitutive secretory pathway in the lacrimal gland as well as the regulatory pathway. This is supported by the observations that inhibition of MAPK with PD-980059 and relatively high concentrations of U0126 significantly increased secretion in the absence of exogenous addition of stimuli.
It is worth noting that 1-adrenergic agonists
consistently gave a smaller response in the activation of the EGFR than
EGF. This might occur if
1-adrenergic agonists stimulate
two separate pathways, one part leading to activation of PKC and the
other part activating the EGFR leading to activation of MAPK, whereas EGF only activates the EGFR. It is also noteworthy that variations in
the amount of MAPK activation with the same agonists were observed. It
is likely that these variations are a result of the fact that the
lacrimal gland acini used in these studies were freshly isolated for
each experiment and reflect variations in responses from different individuals.
In summary, our results show that both cholinergic and
1-adrenergic agonists activate MAPK in freshly isolated
lacrimal gland epithelial cells. We have shown, using an inhibitor of
the EGFR and measuring its phosphorylation, that
1-adrenergic agonists transactivate the EGFR to activate
MAPK. Transactivation of the EGFR by
1-adrenergic
agonists results in tyrosine phosphorylation of Shc and recruitment of
Grb2, two early events in the biochemical cascade leading to activation
of MAPK. In contrast, cholinergic agonists do not transactivate the
EGFR but do activate Pyk2 and p60Src, though the role of
Pyk2 in the activation of MAPK is not completely clear. Activation of
MAPK, by either mechanism, leads to inhibition of lacrimal gland
protein secretion.
![]() |
FOOTNOTES |
---|
* I. Ota and D. Zoukhri contributed equally to this work.
Address for reprint requests and other correspondence: D. A. Dartt, Schepens Eye Research Institute, 20 Staniford St., Boston, MA 02114 (E-mail: dartt{at}vision.eri.harvard.edu).
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. Section 1734 solely to indicate this fact.
First published September 11, 2002;10.1152/ajpcell.00151.2002
Received 5 April 2002; accepted in final form 4 September 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Dartt, DA.
Regulation of inositol phosphates, calcium and protein kinase C in the lacrimal gland.
Prog Retin Eye Res
13:
443-478,
1994[ISI].
2.
Dartt, DA.
Signal transduction and control of lacrimal gland protein secretion: a review.
Curr Eye Res
8:
619-636,
1989[ISI][Medline].
3.
Dikic, I,
Tokiwa G,
Lev S,
Courtneidges SA,
and
Schlessinger J.
A role for Pyk2 and src in linking G-protein coupled receptors with MAP kinase activation.
Nature
383:
547-550,
1996[ISI][Medline].
4.
Favata, MF,
Horiuchi KY,
Manos EJ,
Daulerio AJ,
Stradley DA,
Feeser WS,
Van Dyk DE,
Pitts WJ,
Earl RA,
Hobbs F,
Copeland RA,
Magolda RL,
Scherle PA,
and
Trzaskos JM.
Identification of a novel inhibitor of mitogen-activated protein kinase kinase.
J Biol Chem
273:
18623-18632,
1998
5.
Graham, RM,
Perez DM,
Piascik MT,
Rieck RP,
and
Hwa J.
Characterization of 1-adrenergic receptor subtypes.
Pharmacol Commun
6:
15-22,
1995.
6.
Hanke, JH,
Gardner JP,
Dow RL,
Changelian PS,
Brissette WH,
Weringer EJ,
Pollok BA,
and
Connelly PA.
Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation.
J Biol Chem
271:
695-701,
1996
7.
Herman, G,
Busson S,
Ovtracht L,
Maurs C,
and
Rossignol B.
Regulation of protein discharge in two exocrine glands: rat parotid and exorbital lacrimal glands.
Biol Cell
31:
255-264,
1978.
8.
Hodges, RR,
Dicker DM,
Rose P,
and
Dartt DA.
1-Adrenergic and cholinergic agonists use separate signal transduction pathways in lacrimal gland.
Am J Physiol Gastrointest Liver Physiol
262:
G1087-G1096,
1992
9.
Keely, SJ,
Calandrella SO,
and
Barrett KE.
Carbachol-stimulated transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T84 cells is mediated by intracellular Ca2+, PYK-2, and p60src.
J Biol Chem
275:
12619-12625,
2000
10.
Keely, SJ,
Uribe JM,
and
Barrett KE.
Carbachol stimulates transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T84 cells. Implications for carbachol-stimulated chloride secretion.
J Biol Chem
273:
27111-27117,
1998
11.
Kodama, H,
Fukuda K,
Takahashi T,
Sano M,
Kato T,
Tahara S,
Hakuno D,
Sato T,
Manabe T,
Konishi F,
and
Ogawa S.
Role of EGF receptor and Pyk2 in endothelin-1-induced ERK activation in rat cardiomyocytes.
J Mol Cell Cardiol
34:
139-150,
2002[ISI][Medline].
12.
Levitzki, A,
and
Gazit A.
Tyrosine kinase inhibition: an approach to drug development.
Science
267:
1782-1788,
1995[ISI][Medline].
13.
Liebmann, C.
Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity.
Cell Signal
13:
777-785,
2001[ISI][Medline].
14.
Lopez-Ilasaca, M.
Signaling from G-protein-coupled receptors to mitogen-activated protein (MAP)-kinase cascades.
Biochem Pharmacol
56:
269-277,
1998[ISI][Medline].
15.
Lopez-Ilasaca, M,
Crespo P,
Pellici PG,
Gutkind JS,
and
Wetzker R.
Linkage of G protein-coupled receptors to the MAPK signaling pathway through PI 3-kinase .
Science
275:
394-397,
1997
16.
Luttrell, LM,
Daaka Y,
and
Lefkowitz RJ.
Regulation of tyrosine kinase cascades by G-protein-coupled receptors.
Curr Opin Cell Biol
11:
177-183,
1999[ISI][Medline].
17.
Massague, J,
and
Pandiella A.
Membrane-anchored growth factors.
Annu Rev Biochem
62:
515-541,
1993[ISI][Medline].
18.
Mauduit, P,
Herman G,
and
Rossignol B.
Protein secretion in lacrimal gland: 1-
-adrenergic synergism.
Am J Physiol Cell Physiol
250:
C704-C712,
1986
19.
Mauduit, P,
Jammes H,
and
Rossignol B.
M3 muscarinic acetylcholine receptor coupling to PLC in rat exorbital lacrimal acinar cells.
Am J Physiol Cell Physiol
264:
C1550-C1560,
1993
20.
Pelicci, G,
Lanfrancone L,
Grignani F,
McGlade J,
Cavallo F,
Forni G,
Nicoletti I,
Grignani F,
Pawson T,
and
Pelicci PG.
A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction.
Cell
70:
93-104,
1992[ISI][Medline].
21.
Pierce, KL,
Tohgo A,
Ahn S,
Field ME,
Luttrell LM,
and
Lefkowitz RJ.
Epidermal growth factor (EGF) receptor-dependent ERK activation by G protein-coupled receptors: a co-culture system for identifying intermediates upstream and downstream of heparin-binding EGF shedding.
J Biol Chem
276:
23155-23160,
2001
22.
Prenzel, N,
Zwick E,
Daub H,
Leserer M,
Abraham R,
Wallasch C,
and
Ullrich A.
EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF.
Nature
402:
884-888,
1999[ISI][Medline].
23.
Seger, R,
and
Krebs EG.
The MAPK signaling cascade.
FASEB J
9:
726-735,
1995
24.
Sugden, PH,
and
Clerk A.
Regulation of the ERK subgroup of MAP kinase cascades through G protein-coupled receptors.
Cell Signal
9:
337-351,
1997[ISI][Medline].
25.
Zoukhri, D,
and
Dartt DA.
Cholinergic activation of phospholipase D in lacrimal gland acini is independent of protein kinase C and calcium.
Am J Physiol Cell Physiol
268:
C713-C720,
1995
26.
Zoukhri, D,
Hodges RR,
Sergheraert C,
Toker A,
and
Dartt DA.
Lacrimal gland PKC isoforms are differentially involved in agonist-induced protein secretion.
Am J Physiol Cell Physiol
272:
C263-C269,
1997