©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Down-regulation of m2 Muscarinic Receptor Gene Expression in Human Embryonic Lung (HEL 299) Cells by Protein Kinase C (*)

(Received for publication, December 19, 1994)

Jonathan Rousell El-Bdaoui Haddad Judith C. W. Mak Peter J. Barnes (§)

From the Department of Thoracic Medicine, National Heart and Lung Institute, London SW3 6LY, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

m2 muscarinic receptor gene expression was investigated following stimulation of protein kinase C (PKC) with the phorbol ester 4beta-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 [^3H]N-methyl-scopolamine binding. Preincubation with the PKC inhibitor GF-109203X inhibited the reduction in M(2) 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(2) receptors. Furthermore, the synthesis of an unidentified factor is required for the down-regulation process.


INTRODUCTION

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 beta-adrenergic receptor kinase or beta-adrenergic receptor kinase-like enzymes in an agonist-dependent manner(10) , protein kinase C (PKC) (^1)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(3) muscarinic receptors in HT-29 cells(20) . However, it appears that mammalian M(1) and M(3) muscarinic receptors are more sensitive to the effects of phosphorylation by PKC than are M(2) 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 beta(2) receptor gene transcription through a cAMP response element(24) , whereas persistent inhibition of adenylate cyclase results in up-regulation of the beta(2) 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(2) muscarinic receptor and m2 receptor gene expression in HEL 299 cells, a primary cell line derived from human embryonic lung, that express predominantly M(2) muscarinic receptors (28) .


EXPERIMENTAL PROCEDURES

Cell Culture

All tissue culture reagents with the exception of Hanks' balanced salt solution and Dulbecco's modified Eagle's medium (Life Technologies, Inc., Paisley, United Kingdom) were obtained from Sigma (Poole, UK). HEL 299 cells were obtained from the American Type Culture Collection (ATCC code CCL 137; Rockville, MD) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mML-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/l amphotericin B in 95% air and 5% CO(2) at 37 °C in a humidifier incubator. All experiments were performed on cells at passage 9, as it has been reported that muscarinic receptor levels decrease with increasing passage number in NIE-115 murine neuroblastoma cells(29) . The medium was replaced every 3-4 days and on reaching confluence cells were subcultured by detaching the monolayer with 0.05% trypsin, 1 mM EDTA. Treatments were carried out such that cells could be harvested simultaneously at preconfluence.

Cell Stimulation

Cells were exposed to one or more of the following; 4beta-PDBu (100 nM), 4alpha-PDBu (100 nM), GF-109203X (1 µM), cycloheximide (10 µg/ml), actinomycin D (5 µg/ml), forskolin (100 µM), pertussis toxin (400 ng/ml), calcium ionophore A23187 (1 µM), zardaverine (10 µM), and carbachol (100 µM). Control cells were incubated with an equivalent dilution of dimethyl sulfoxide (Me(2)SO) where stocks of drugs were prepared in Me(2)SO. All of the above reagents with the exception of GF-109203X (CalBiochem-NovaBiochem, Nottingham, UK) were obtained from Sigma.

Binding Studies

Radioligand binding experiments were performed with a washed membrane preparation at 4 °C. Cells (approximately 5-10 times 10^6 for each binding reaction) were washed twice with ice-cold Tris-HCl buffer (25 mM, pH 7.4), harvested by cell scraping, and homogenized with an Ultra-Turax homogenizer (one 30-s burst). Membranes were isolated by centrifugation at 40,000 times g for 20 min and resuspended in an appropriate volume of Tris buffer. The protein concentration was measured according to the method of Lowry(30) .

[^3H]N-methyl-scopolamine ([^3H]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) .

Northern Analysis

Cells were washed twice with Hanks' balanced salt solution and harvested by detachment of the monolayer with trypsin/EDTA. Poly(A) RNA was isolated directly from the cell pellet using an mRNA micro-extraction kit (Invitrogen, Abingdon, UK) according to the manufacturer's instructions. Northern blots to nylon N membranes (Amersham, Amersham, UK) were prepared subsequent to size fractionation by gel electrophoresis of the denatured mRNA on 1% agarose/formaldehyde gels containing 20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA, pH 7.0. Cloned human muscarinic cDNAs (hm1-hm4) (gifts from Dr. N. J. Buckley, London, UK), and a 1272-base pair PstI fragment specific to rat glyceraldehyde-3-phosphate dehydrogenase mRNA respectively were used as probes for the Northern analyses. The cDNAs specific to the muscarinic receptors corresponded to the third cytoplasmic loop and consisted of a HindIII/EcoRI fragment of hm1 cDNA, an EcoRI/PstI fragment of hm2 cDNA, a PstI/PvuII fragment of hm3 cDNA and a KpnI/HindIII fragment of hm4 cDNA.

Prehybridizations and hybridizations were carried out at 42 °C with the probes labeled to approximately 1.5 times 10^6 cpm/ml in a buffer containing 50% formamide, 50 mM Tris-HCl, pH 7.5, 5 times 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 times 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).

Measurement of Run-on Gene Transcription in Isolated Nuclei

Nuclei were prepared as described by Greenberg et al.(32) . Isolated nuclei were resuspended in Tris-HCl (10 mM, pH 7.4), MgCl(2) (5 mM), glycerol (50%), sorbitol (0.5 M), Ficoll (2.5%), spermidine (0.008%), and dithiothreitol (1 mM) and were stored at -70 °C until use. Nuclei (5 times 10^7) were incubated for 30 min at 27 °C with 300 µCi [P]UTP, ATP (0.625 mM), CTP, and GTP (0.31 mM), Tris-HCl (40 mM), NH(4)Cl (150 mM), MgCl(2) (7.5 mM), and RNasin (120 units). DNA digestion was then carried out with a 15 min incubation at 27 °C with RQ-1 DNase (75 units) in the presence of RNasin (40 units) before protein digestion for 3 h at 37 °C with proteinase K (1 mg/ml) in buffer containing Tris-HCl (pH 7.4, 10 mM), EDTA (15 mM), SDS (3%), and heparin (3 mg/ml). RNA extraction was then carried out with a phenol, phenol/chloroform (1:1), and a chloroform wash and then precipitated three times with 100% EtOH in the presence of 1.33 M ammonium acetate. The radiolabeled RNA was dissolved in 100 µl of TE buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA) and added to 2 ml hybridization solution (50% formamide, 5 times SSC, 0.1% SDS, 1 mM EDTA, 10 mM Tris-Cl pH 7.5, 5 times Denhardt's solution, 50 µg/ml yeast tRNA, 100 µg/ml salmon sperm DNA, 0.02 µg poly(A) and 0.02 µg poly G RNA). Hybridization was carried out at 42 °C for 72 h to 10 µg of the immobilized plasmid pGEM3Z and to plasmids containing inserts of rat glyceraldehyde-3-phosphate dehydrogenase cDNA and hm2 muscarinic receptor cDNA as described previously under Northern analyses. The filters were washed first in buffer A (300 mM NaCl, 10 mM Tris-HCl pH 7.4, 2 mM EDTA, 0.1% SDS, 1 µg/ml RNase A and 10 U/ml RNase T1) at 37 °C for 30 min then in buffer B (10 mM NaCl, 10 mM Tris-HCl pH 7.4, 2 mM EDTA and 0.4% SDS) to a stringency of 55 °C for 30 min before autoradiography.

Cyclic AMP Measurements

Following stimulation cells were washed with Hanks' balanced salt solution and the cAMP-phosphodiesterase inhibitor zardavarine (10 µM) was added to fresh media for 20 min. From each group of treatments basal levels of cAMP were measured, as well as accumulation following exposure to forskolin (10 µM) for 10 min in the presence and absence of carbachol (100 µM). Cells were harvested by addition of 1 ml of boiling water directly to each well. Cells were then boiled for a further 2 min before centrifugation at full speed in a microcentrifuge at 4 °C for 10 min. The supernatant was collected and stored at -20 °C before being assayed by radioimmunoassay for acetylated cAMP (I-acetyl-cAMP). Protein assays were performed using a Bio-Rad protein assay (Hemel Hempstead, UK), according to the manufacturers instructions.


RESULTS

Receptor Binding Studies

Receptor binding studies were performed to determine muscarinic receptor protein levels in HEL 299 cells following PKC stimulation. All receptor binding studies were performed on cells at approximately 70% confluence as a significant loss in the number of receptor binding sites was observed when the cells were allowed to reach confluence (data not shown). Saturation studies performed with the nonselective hydrophilic muscarinic antagonist [^3H]NMS revealed a single class of binding site (B(max), 353 ± 12 fmol/mg protein) with a dissociation constant K(d) of 0.21 ± 0.09 nM. Following PDBu (100 nM) treatment the number of receptor binding sites decreased (Fig. 1A) such that after 12 and 24 h, a 60 and 70% reduction in receptor density was seen, respectively. The equilibrium dissociation constant remained unaltered by the same treatment. The inactive alpha-form of PDBu (100 nM) had no significant effect on M(2) receptor density and preincubation with the specific PKC inhibitor GF-109203X (1 µM) significantly inhibited PDBu-mediated M(2) receptor loss following 24-h PDBu incubation (Fig. 1B). Co-incubations with the calcium Ionophore A23187 (1 µM), and PDBu resulted in no further decrease in the density of [^3H]NMS binding sites after 24 h (data not shown).


Figure 1: Time-dependent effects of PDBu on [^3H]NMS binding in HEL 299 cells. A shows [^3H]NMS binding following PDBu treatment for the times indicated. B shows the effect of the PKC inhibitor GF-109203X (1 µM) and the inactive alpha-form of PDBu (100 nM) on [^3H]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 alpha-form of PDBu (alpha-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.



Cyclic AMP Measurements

Experiments were conducted to determine the functional coupling of the muscarinic receptors in HEL 299 cells and whether phorbol ester treatment resulted in desensitization of the M(2) receptors. In control cells exposure to forskolin (10 µM) for 10 min induced an increase in cAMP accumulation which could be partially inhibited (approximately 30%) by co-incubation with carbachol (100 µM). In cells treated with either pertussis toxin (4 h) or PDBu (24 h) the inhibitory effect of carbachol was lost indicating a functional uncoupling of the receptor (Fig. 2). Rapid desensitzation of the M(2) receptors did not occur on PDBu treatments. Short PDBu incubations (20 min) resulted in inhibition of forskolin-stimulated cAMP accumulation by carbachol to the same degree to that seen in control cells.


Figure 2: Functional coupling of the M(2) 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.



Muscarinic Receptor Gene Expression

Northern blot analyses on isolated messenger RNAs revealed the presence of a 6.1-kilobase transcript corresponding to the m2 muscarinic receptor with no evidence of m1, m3, or m4 receptor mRNA in agreement with findings of Koman et al.(28) . Stimulation with 100 nM PDBu resulted in a time-dependent decrease in m2 receptor mRNA steady-state levels such that a 70% reduction in m2 mRNA was seen after 12 or 24 h incubation (Fig. 3). Incubations with the calcium Ionophore A23187 (1 µM) had no effect on the steady-state levels of m2 receptor mRNA and co-incubations with the calcium Ionophore A23187 and PDBu did not cause any further reduction in the m2 receptor mRNA steady-state levels. Preincubations with the specific PKC inhibitor GF-109203X completely inhibited the down-regulation seen with PDBu (Fig. 4), indicating activation of PKC is essential for the m2 receptor down-regulation.


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 (circle) and absence (bullet) 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 (circle) was compared with actinomycin D treatment alone (bullet). 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 times 10^7) 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.




DISCUSSION

In this study we have shown that PKC stimulation results in down-regulation of M(2) receptors and m2 receptor gene expression through a decrease in the rate of transcription in HEL 299 cells.

Specific [^3H]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 [^3H]NMS binding and the steady-state levels of m2 muscarinic receptor mRNA. A marked decrease in M(2) 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(2) 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(2) receptors (Fig. 1B). Incubations with the inactive form of PDBu (4alpha-PDBu) also confirmed a PKC-mediated effect as 24-h treatments (100 nM) had no effect on [^3H]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(2) 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 [^3H]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(2)) receptors.

To determine whether the decrease in receptor number was accompanied by a desensitization of the receptor following PDBu treatment, the ability of M(2) receptors to inhibit cAMP accumulation was measured. As reported elsewhere (5, 8, 9) M(2) 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(2) receptors(35) , but muscarinic receptors have been shown to be phosphorylated by PKC in an agonist-independent manner. M(1) and M(3) receptors are highly sensitive to stimulation of PKC, and are rapidly internalized and down-regulated, whereas mammalian M(2) 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(2) and M(3) receptors), short term treatment with PDBu resulted in a reduction in the [^3H]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(3) receptors are more sensitive to the effect of PKC than the M(2) receptors in this tissue. In HEL 299 cells loss of [^3H]NMS binding sites and the functional desensitization occurred slowly, suggesting that direct phosphorylation of the M(2) 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(1) and M(3) 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-kappaB 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-kappaB 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(2) receptors and their functional uncoupling from adenylate cyclase. Mammalian M(2) 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.


FOOTNOTES

*
This work was supported by the Medical Research Council (United Kingdom). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 071-351-8174; Fax: 071-351-5675.

(^1)
The abbreviations used are: PKC, protein kinase C; PDBu, 4beta-phorbol 12,13-dibutyrate; [^3H]NMS, [^3H]N-methyl-scopolamine; MOPS, morpholinosulfonic acid.


ACKNOWLEDGEMENTS

We thank Dr. I. M. Adcock and Dr. M. A. Giembycz for helpful discussion throughout this study.


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