(Received for publication, December 19, 1994)
From the
m2 muscarinic receptor gene expression was investigated
following stimulation of protein kinase C (PKC) with the phorbol ester
4-phorbol dibutyrate (PDBu) in HEL 299 cells. PDBu (100
nM) caused a time-dependent decrease in the steady-state
levels of m2 receptor mRNA and in specific
[
H]N-methyl-scopolamine binding.
Preincubation with the PKC inhibitor GF-109203X inhibited the reduction
in M
receptor and mRNA levels induced by PDBu, confirming
the involvement of PKC. Chronic PDBu treatment also caused
desensitization of the receptor as forskolin-stimulated cAMP
accumulation, inhibited by carbachol in control cells, was lost upon
treatment with PDBu for 24 h. Co-incubation with PDBu and the protein
synthesis inhibitor cycloheximide, inhibited PDBu-mediated reduction of
m2 receptor mRNA, indicating new protein synthesis is required for
down-regulation. Half-life studies using the transcriptional inhibitor
actinomycin D suggested that the stability of the m2 receptor mRNA was
not altered by PDBu treatment (t
= 2 h).
Nuclear run-on assays showed a 50% reduction in the rate of m2 receptor
gene transcription after treatment with PDBu for 12 h. In conclusion we
have provided evidence for heterologous regulation of m2 receptor gene
expression through changes in gene transcription resulting in
uncoupling of M
receptors. Furthermore, the synthesis of an
unidentified factor is required for the down-regulation process.
Five muscarinic acetylcholine receptor subtypes (m1-m5) are known to exist within rat and human genomes, and coding regions of these genes have been cloned, sequenced, and expressed(1, 2, 3, 4) . Through the stable expression of these cloned muscarinic receptors a large body of data regarding the structure, function, and pharmacology of muscarinic receptor subtypes has accumulated(5, 6) . Each of the five cloned muscarinic receptor subtypes is preferentially coupled to a distinct signal transduction pathway; m1, m3, and m5 muscarinic receptors are preferentially coupled to the stimulation of phosphatidylinositol hydrolysis(4, 6, 7) , whereas m2 and m4 muscarinic receptors are coupled to the inhibition of adenylate cyclase(5, 8) , along with weak stimulation of phosphatidylinositol breakdown(5, 9) . However, much less is known about the factors that regulate the expression of muscarinic receptors, in part because the noncoding promoter and enhancer regions that directly control transcription of the individual muscarinic receptor genes have not yet been sequenced.
Chronic
agonist stimulation of muscarinic receptors induces desensitization and
down-regulation of the receptor. Several studies have demonstrated the
crucial role of phosphorylation by specific cellular kinases in the
receptor desensitization process. Muscarinic receptors are known to be
phosphorylated by -adrenergic receptor kinase or
-adrenergic
receptor kinase-like enzymes in an agonist-dependent
manner(10) , protein kinase C (PKC) (
)in an
agonist-independent manner (11, 12) , protein kinase
A(13) , and by a kinase activity that seems to be different
from any known protein kinase(14) . Recently it has become
clear that prolonged exposure to agonist also has effects on the gene
expression of muscarinic
receptors(15, 16, 17, 18) , causing
both changes in the stability of the mRNA and in the rate of
transcription, often leading to their subsequent down-regulation.
Intracellular messengers are also involved in the regulation of a
number of G protein-coupled receptors, including muscarinic receptors.
PKC activation induces a rapid sequestration of muscarinic receptors in
neuroblastoma cells (19) and down-regulation of M muscarinic receptors in HT-29 cells(20) . However, it
appears that mammalian M
and M
muscarinic
receptors are more sensitive to the effects of phosphorylation by PKC
than are M
muscarinic
receptors(21, 22, 23) . The gene expression
of G protein-coupled receptors may also be influenced by changes in the
levels of second messengers. Adrenergic receptors provide a good
example where the crucial role of cAMP in their regulation has been
elucidated. Elevation of cAMP levels results in stimulation of
receptor gene transcription through a cAMP response
element(24) , whereas persistent inhibition of adenylate
cyclase results in up-regulation of the
receptors(25) . However, although several studies have
investigated the action of second messengers on muscarinic receptors at
the protein level, few have focused on gene expression. In dissociated
chick heart cells (cm2 and cm4 receptors), phorbol 12-myristate
13-acetate treatment causes a modest (15-20%), but significant,
decrease in the steady-state levels of cm2 and cm4 muscarinic receptor
mRNA(26) . Recently it has also been shown that endothelin-1
increases both the levels of mRNA and protein for m2 and m3 receptors
in cerebellar granule cells(27) .
Here we investigate the
effect of PKC stimulation on the regulation of the M muscarinic receptor and m2 receptor gene expression in HEL 299
cells, a primary cell line derived from human embryonic lung, that
express predominantly M
muscarinic receptors (28) .
[H]N-methyl-scopolamine
([
H]NMS; 79.5 Ci/mmol; New England Nuclear,
Stevenage, UK) saturation curves were elucidated using a concentration
range varying from 0.04 to 8 nM. Nonspecific binding was
measured in the presence of 1 µM atropine (Sigma).
Incubations were performed at 30 °C for 2 h and terminated by rapid
vacuum filtration over 0.2% polyethyleneimine pre-treated Whatman GF/C
glass fiber filters using a Brandel cell harvester. The filters were
washed three times with 4 ml of ice-cold Tris buffer and placed in
vials with 4 ml of scintillation mixture (Filtron X, National
Diagnostics, Manville, NJ) and counted on a Packard liquid
scintillation counter (Packard 2200 CA model). Binding data were
analyzed with the computerized nonlinear regression program
LIGAND(31) .
Prehybridizations and hybridizations were carried out at 42 °C
with the probes labeled to approximately 1.5 10
cpm/ml in a buffer containing 50% formamide, 50 mM Tris-HCl, pH 7.5, 5
Denhardt's solution, 0.1% sodium
dodecyl sulfate (SDS), 5 mM EDTA, and 250 µg/ml denatured
salmon sperm DNA. Following hybridization the blots were washed to a
stringency of 0.1
SSC, 0.1% SDS at 65 °C before exposure to
Kodak XAR-5 film. After suitable exposure times, autoradiographs were
analyzed by laser densitometry (PDI, Huntington Station, NY).
Figure 1:
Time-dependent effects of PDBu on
[H]NMS binding in HEL 299 cells. A shows
[
H]NMS binding following PDBu treatment for the
times indicated. B shows the effect of the PKC inhibitor
GF-109203X (1 µM) and the inactive
-form of PDBu (100
nM) on [
H]NMS binding. Cells were
treated with vehicle (control), PDBu for 24 h (PDBu),
pre-treated with GF-109203X before 24-h treatment with the active PDBu (PDBu + GF 109203X) and with the inactive
-form of
PDBu (
-PDBu). Data are the mean (±S.E.) of five
independent experiments. Significance of difference between control and
treated cells at** = p < 0.01;*** = p < 0.001 determined by Student's t test.
Figure 2:
Functional coupling of the M receptor in HEL 299 cells. Accumulation of cAMP was measured in
the presence of forskolin (100 µM) in untreated cells,
pretreatment with 100 nM PDBu (20 min or 24 h) or 400 ng/ml
pertussis toxin (PTx, 4 h). Inhibition of cAMP accumulation
was measured in the presence of carbachol (100 µM). In
control cells and cells treated with PDBu for 20 min a significant
inhibition of cAMP accumulation (30%) was seen that was lost in cells
treated with PDBu (24 h) or pertussis toxin (4 h). Data represent the
mean (±S.E.) of cAMP (n = 6) accumulation.
Significance compared with control at * = p <
0.05.
Figure 3: Time-dependent effects of PDBu on steady-state levels of m2 mRNA in HEL 299 cells. Northern blot analyses were performed with cDNA labeled probes for m2 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) after isolation of messenger RNA. A shows a representative Northern blot following PDBu (100 nM) treatment for the times indicated. B represents m2 receptor mRNA levels relative to glyceraldehyde-3-phosphate dehydrogenase in the presence of 100 nM PDBu for the indicated times after assessment by laser densitometry. All data points are the mean (±S.E.) of at least four independent experiments.
Figure 4: Effect of the PKC inhibitor GF-109203X on PDBu-mediated m2 mRNA down-regulation. Northern blots were performed on isolated mRNA from HEL 299 cells after preincubation with the specific PKC inhibitor GF-109203X. Data represents the mean (±S.E.) of six independent experiments. Significance compared with control at** = p < 0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Experiments were also performed to determine the mechanism of the m2 receptor down-regulation. Incubations with the protein synthesis inhibitor cycloheximide (10 µg/ml) in the presence and absence of PDBu had no effect on m2 receptor gene expression (Fig. 5), even after 12 h when a large decrease in m2 receptor mRNA had been seen with PDBu alone. Thus, synthesis of at least one protein factor is required after PKC stimulation to alter m2 receptor mRNA levels. Half-life studies with the RNA polymerase inhibitor actinomycin D (5 µg/ml) were carried out to measure the rate of m2 mRNA degradation in treated and untreated cells. The degradation rate of m2 receptor mRNA in the presence of actinomycin D was not significantly reduced following 4-h PDBu treatment (half lives of 2 and 2.5 h, respectively; Fig. 6). Therefore, changes in m2 receptor mRNA stability do not appear to be responsible for the loss of m2 receptor mRNA, indicating a reduction in the rate of m2 receptor gene transcription. This was confirmed by nuclear run-on transcription experiments where production of new m2 receptor mRNA was measured from isolated cell nuclei of control and PDBu-treated cells. The rate of transcription of new m2 receptor mRNA, compared with glyceraldehyde-3-phosphate dehydrogenase transcription (which remained unchanged), was reduced by 50% following 12 h PDBu treatment (Fig. 7).
Figure 5:
Effect of inhibiting protein synthesis on
PDBu-mediated m2 receptor down-regulation. Northern blots were
performed on mRNA isolated from HEL 299 cells after cycloheximide (10
µg/ml) treatments in the presence () and absence (
) of
PDBu (100 nM). Results are presented relative to the levels of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and are the
mean (±S.E.) of three independent
experiments.
Figure 6:
Degradation rate of m2 receptor mRNA.
Cells were treated with vehicle or PDBu for 4 h before incubation with
actinomycin D (5 µg/ml) for the times indicated. Messenger RNA was
then isolated from cells and Northern blots performed. The degradation
rate of m2 receptor mRNA following PDBu incubation () was compared
with actinomycin D treatment alone (
). Data are the average
(±S.E.) of six separate experiments.
Figure 7:
Nuclear run-on transcription. P-Labeled mRNA was transcribed in vitro from
isolated cell nuclei (5
10
) from PDBu-treated (12
h) and untreated cells and was hybridized to plasmid cDNAs immobilized
on nylon membranes. The plasmids used were pGEM3Z (negative control)
and plasmids containing m2 receptor and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA inserts. Data are the average of
two separate experiments and represent the ratio of the optical density
of m2 to glyceraldehyde-3-phosphate dehydrogenase in control and
PDBu-treated cells.
In this study we have shown that PKC stimulation results in
down-regulation of M receptors and m2 receptor gene
expression through a decrease in the rate of transcription in HEL 299
cells.
Specific [H]NMS binding to muscarinic
receptors in HEL 299 cell membranes was saturable and best described by
the interaction of the radioligand with a single population of high
affinity binding sites. Northern blot analysis on isolated messenger
RNA revealed expression of the m2 muscarinic receptor transcript only,
in agreement with previous data obtained from the same cell
line(28) . Direct stimulation of PKC with PDBu (100
nM) resulted in a time-dependent decrease in
[
H]NMS binding and the steady-state levels of m2
muscarinic receptor mRNA. A marked decrease in M
receptor
protein was seen after 12 and 24 h (Fig. 1A) when
receptor density had fallen by 60 and 70%, respectively. This was
mirrored by a fall in the steady-state levels of m2 receptor mRNA which
preceded the fall in receptors (Fig. 3). After 4 h there was a
50% fall in the steady-state levels of m2 receptor mRNA at a time when
there was no significant decrease in receptor density, suggesting the
loss in M
receptor binding sites is a result of the
reduction in mRNA steady-state levels.
The loss of m2 receptor
messenger RNA and protein in HEL 299 cells after exposure to PDBu
appeared to be a PKC-mediated effect since pretreatments with the PKC
inhibitor GF-109203X (33) completely inhibited the PDBu-induced
reduction in m2 receptor mRNA (Fig. 4) and significantly
inhibited the reduction in M receptors (Fig. 1B). Incubations with the inactive form of PDBu
(4
-PDBu) also confirmed a PKC-mediated effect as 24-h treatments
(100 nM) had no effect on [
H]NMS binding
or m2 receptor mRNA levels (data not shown). Potential PKC
desensitization following long term treatment with PDBu was not
observed as the calcium ionophore A23187, which is thought to
potentiate the effect of PKC stimulation, did not produce any further
down-regulation of M
muscarinic receptor protein or mRNA
when used in combination with PDBu (data not shown). This may indicate
a relative insensitivity to calcium of the particular PKC isoform(s)
present in these cells(34) . Elevation of calcium by the
ionophore A23187 had no effect on [
H]NMS binding
capacity or the levels of m2 receptor mRNA, a result which argues
against the involvement of the
Ca
-calmodulin-dependent protein kinase in the
down-regulation of m2 (or M
) receptors.
To determine
whether the decrease in receptor number was accompanied by a
desensitization of the receptor following PDBu treatment, the ability
of M receptors to inhibit cAMP accumulation was measured.
As reported elsewhere (5, 8, 9) M
receptors in HEL 299 cells were coupled to the inhibition of
adenylate cyclase through a pertussis toxin-sensitive G protein.
Forskolin-stimulated cAMP levels in untreated HEL 299 cells were
reduced significantly (30%) in the presence of the muscarinic agonist
carbachol. Both pertussis toxin and PDBu pretreatments (4 and 24 h,
respectively) reversed the effects seen with carbachol on cAMP levels
in control cells, indicating functional uncoupling of the receptor. A
shorter incubation period (20 min) had no effect on the coupling state
of the receptors.
There is some uncertainty concerning the
involvement of PKC in the phosphorylation and desensitization of
M receptors(35) , but muscarinic receptors have
been shown to be phosphorylated by PKC in an agonist-independent
manner. M
and M
receptors are highly sensitive
to stimulation of PKC, and are rapidly internalized and down-regulated,
whereas mammalian M
muscarinic receptors appear to be a
poor substrate for PKC(21, 22, 23) . For
example, in a mouse adrenocarcinoma cell line transfected with m1 and
m2 receptor cDNAs, m1, but not m2, receptors appear sensitive to
internalization induced by the phorbol ester phorbol 12-myristate
13-acetate(21) . In dissociated dog colon smooth muscle cells
(M
and M
receptors), short term treatment with
PDBu resulted in a reduction in the [
H]NMS
binding sites with a loss of the high affinity pirenzepine binding
sites from the cell surface(22) . The loss of receptor was
rapid occurring within 30 min of PKC activation and suggests that
M
receptors are more sensitive to the effect of PKC than
the M
receptors in this tissue. In HEL 299 cells loss of
[
H]NMS binding sites and the functional
desensitization occurred slowly, suggesting that direct phosphorylation
of the M
receptor by PKC is unlikely. However,
desensitization does occur following chronic exposure to PDBu. This
indicates that phosphorylation of the receptor, its G protein, or
adenylate cyclase occurs, possibly through the effects of another
induced kinase or through changes in gene expression as seen in these
cells(36) . In HEL 299 cells loss in receptor number is also
slow and rather than reflecting internalization through phosphorylation
of the receptor, as seen with M
and M
receptors
in other studies, it seems to reflect the fall in the steady-state
levels of m2 receptor mRNA. The delay between protein loss and falling
mRNA levels may be indicative of the inherent stability of the receptor
protein and the rate of receptor turnover within the cell.
While many studies have dealt with the effect of PKC on muscarinic receptor protein few have focused on alterations in gene expression. Habecker and Nathanson (26) showed a modest (15-20%) but significant reduction in steady-state levels of chick m2 and m4 muscarinic receptor mRNAs after treatment with a phorbol ester. Treatment of neuroblastoma SH-SY5Y cells, expressing m1 and m2 receptor subtypes, with the phorbol ester tetradecanoylphorbol acetate by Koman et al.(37) , resulted in changes in the steady-state levels of both m1 and m2 receptor mRNAs. The pattern of regulation, however, was different to that reported here. A small decrease in m2 receptor mRNA after 24 h was preceded by a large increase in m2 receptor mRNA at 8 h, whereas m1 receptor mRNA decreased to 24 h before a large increase at 96 h. The mechanisms of down-regulation were not examined. Conversely, Fukamauchi et al.(27) recently found that PDBu treatment had no effect on m2 or m3 mRNA levels in primary cultures of cerebellar granule cells but inhibited an up-regulation caused by endothelin-1 on both muscarinic receptor mRNAs. Differences such as these may reflect different patterns of expression of transcription factors, PKC isoforms (34) and other intracellular messengers present in different cells.
The mechanism of the down-regulation seen in HEL 299 cells was investigated further using cycloheximide, a potent protein synthesis inhibitor. Cycloheximide alone had no effect on m2 receptor mRNA levels up to 12 h but blocked completely the down-regulation induced by PDBu, indicating that the synthesis of at least one protein factor is required (Fig. 5). This factor itself, or a subsequently induced protein, might alter transcription of the m2 receptor gene directly or change the degradation rate of the m2 receptor mRNA. Half-life studies were performed to investigate whether there was any change in the stability of the m2 receptor mRNA following PDBu treatment (Fig. 6). Incubations with the RNA polymerase inhibitor actinomycin D (5 µg/ml) were performed in the absence of PDBu or following incubation for 4 h with PDBu. The half-life study revealed no change in the stability of the m2 receptor mRNA following PDBu treatment. This result suggests that a change in the rate of m2 gene transcription is responsible for the reduction in the steady-state levels of m2 receptor mRNA. Nuclear run-on transcription assays showed that transcription of m2 receptor gene was reduced by 50% following 12 h PDBu treatment (Fig. 7). Induction, possibly through new protein synthesis of a transcription factor is therefore required to cause the down-regulation seen.
The nature of the protein(s) induced by PKC activation in
these cells are unknown but PKC is known to phosphorylate and induce
DNA binding activity of a number of proteins, including transcription
factors such as NF-B and AP-1 which may in turn alter the
expression of other genes(38, 39, 40) . In
HEL 299 cells, preliminary data indicate both AP-1 and NF-
B
binding can be induced by PDBu (data not shown), but without any
knowledge of the promoter sequence of the m2 receptor gene, it is
difficult to speculate on whether these factors are involved in the
down-regulation process.
In summary, we have shown modulation of m2
receptor gene expression through changes in transcription by PKC that
is also reflected in down-regulation of the M receptors and
their functional uncoupling from adenylate cyclase. Mammalian M
receptors are not coupled to PKC via the PLC pathway, nor do they
appear to be sensitive to direct phosphorylation by PKC. However, an
interaction between these second messenger systems clearly exists in
this and other cell types. Such interactions which have been described
for adrenergic receptors and recently for muscarinic receptors are
certain to become a common element in the control of receptor gene
expression.