- Email: [email protected]
m2- and m3-muscarinic acetylcholine receptor mRNAs have different responses to microtubule-affecting drugs
MOLECULAR
AND CELLULAR
NEUROSCIENCES
2, 315-319
(1991)
m2- and m3-Muscarinic Acetylcholine Receptor mRNAs Have Different Responses to Microtubule-Affecting Drugs FUMIHIKO Section
FUKAMAUCHI,
on Molecular Building
CHRISTOPHER
Neurobiology, Biological Psychiatry 10, Room 3N-212, 9000 Rockville Received
for publication
INTRODUCTION
Primary cultures of cerebellar granule cells prepared from neonatal rats have proven to be an excellent model system for studying the regulation of muscarinic acetylcholine receptors (mAChRs). This nearly homogenous preparation of neurons expresses in culture m2- and m3mAChRs negatively coupled to adenylate cyclase and correspondence
should
AND
DE-MAW CHUANG’
Branch, National Institute Pike, Bethesda, Maryland July
of Mental 20892
Health,
17, 1991
positively linked to phospholipase C, respectively (l-3). Prolonged stimulation of these neurons with the mAChR agonist carbachol results in desensitization of the receptor-mediated effector response and a subsequent loss of total mAChR binding sites (2, 4). Using cloned mAChR cDNA, we have demonstrated that the early phase of carbachol-induced mAChR down-regulation is due, at least in part, to the reduction of m3- and m2-mAChR mRNAs (3). Conversely, persistent occupancy of mAChR in cerebellar granule cells by antagonists causes up-regulation of m3-mAChR mRNA (3). These observations suggest that some factors may be involved in transmitting the signal from the receptors at the plasma membrane to the nucleus to influence the expression of surface receptor mRNA. Several lines of indirect evidence support a role of the cytoskeleton in relaying stimuli from the cell surface to the nucleus to affect gene expression. For example, detachment of 3T3 fibroblasts from their solid support, a condition known to produce cytoskeletal reorganization, induces morphological changes and a concomitant loss of DNA, RNA, and protein synthesis (5,6). These changes are restored by reattachment of suspended cells to their growth surface. There are also reports that organization of cytoarchitecture affects the growth and differentiation of several cell types (7-9) and disruption of the cytoskeleton with cytochalasin D promotes c-fos gene expression (10). Cerebellar granule cells have been shown to differentiate in culture with an early burst in the expression of /3-actin mRNA (3), which coincides with a period of active neurite outgrowth in these cells. These neurites in cerebellar granule cells have been shown to contain abundant actin filaments (11). The appearance of neurites is followed by subsequent development of microtubules, microfilaments, and cytoskeleton-associated proteins such as MAPS and spectrin (11). Additionally, using cultured granule cells, we have recently demonstrated that a microtubule-disrupting agent, colchicine, selectively decreases m3-mAChR mRNA level in a time-dependent manner (12). This m3-mAChR mRNA down-regulation correlates with a loss of mAChR sites and a decrease
We have recently shown that colchicine induces a timeand dose-dependent decrease of m3-muscarinic acetylcholine receptor (mAChR) mRNA in rat cerebellar granule cells (F. Fukamauchi, C. Hough, and D.-M. Chuang, 1991, Mol. Cell. Neurosci. 2: 123-129). We now report the regulation by colchicine of m2-mAChR mRNA level determined by Northern blot hybridization. Colchicine (10 pM) increased m2-mAChR mRNA level in cerebellar granule cells by 14, 33, and 63% after treatment for 2, 4, and 8 h, respectively, but markedly decreased it at 24 h. The colchicine-induced up-regulation of m2-mAChR mRNA was temporally associated with a decrease in intracellular cyclic AMP levels but an increase in c-Fos mRNA. The level of m2-mAChR mRNA also increased by 21,62, and 78% following exposure to 1, 10, and 100 pM of colchicine for 8 h, respectively. The ECso value of colchicine for this increase was approximately 5 PM. cFos mRNA was increased in parallel by 8 h of treatment with colchicine in a concentration-dependent manner. An inactive derivative of colchicine, /I-lumicolchicine, had no effect on the steady-state levels of m2- or m3-mAChR mRNA. The presence of the microtubule stabilizer, taxol (10 @), reversed the colchicine-induced m2-mAChR mRNA up-regulation at 8 h. These results strongly suggest that microtubules are involved in the homeostasis of the level of both m2- and m3-mAChR mRNAs in cerebellar granule cells; however, these two mAChR mRNA species are oppositely regulated by perturbation of microtubule structures. 0 1991 Academic PWKI, 1~.
1 To whom
HOUGH,
be addressed. 315
1044-7431/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
316
FUKAMAUCHI,
HOUGH,
of carbachol-induced phosphoinositide turnover (12). Moreover, colchicine’s effect on m3-mAChR mRNA is completely reversed by taxol, a microtubule stabilizer (12, 13), and overlaps with the m3-mAChR mRNA down-regulation induced by prolonged stimulation with carbachol (12). We have suggested that microtubules play an important role in the maintenance of the steady-state of m3-mAChR mRNA level and further speculated that the nucleus can selectively adjust the regulation of mAChR gene expression in response to perturbation of microtubule structures. In this study, conducted simultaneously with the latter study, we examined the change in m2-mAChR mRNA level under the same conditions in an attempt to define further the role of microtubules in regulating mAChR gene expression. As in the previous study, the use of microtubule-affecting agents such as colchicine or taxol in the present study has elicited interest in the regulation of mAChR mRNAs. The regulation of m2-mAChR mRNA level mediated by microtubule-affecting agents was strikingly different from that of m3-mAChR mRNA level. Possible roles of cyclic AMP (CAMP) and c-Fos expression in microtubule-mediated m2-mAChR mRNA regulation have also been explored. MATERIALS
AND METHODS
AND
CHUANG
by centrifugation through 5.7 M cesium chloride. A onetenth aliquot of total RNA from each sample was electrophoresed in parallel with ribosomal RNA standards on ethidium bromide-stained, 1% agarose gels for quantification of total RNA. For this purpose the RNA was allowed to migrate approximately 5 mm from the sample well and then was quantified by using laser densitometry of photographic negatives of the entire RNA band. The remaining RNA was denatured, fractionated by 1% agarose-formaldehyde gels, and transferred to a nitrocellulose membrane by capillary action. The blot was hybridized with an m2-mAChR probe made from a 0.5-kb AvaI fragment of human Hm2p9 (a gift from Dr. T. I. Bonner, Laboratory of Cell Biology, NIMH, Bethesda, MD), murine c-Fos cDNA probe, or a chicken /3-actin cDNA probe (gifts from Dr. C. B. Thompson, Howard Hughes Medical School, Ann Arbor, MI) at 42’C overnight. Each probe had been labeled with [32P]dCTP (3000 Ci/mmol) by the random primer labeling method (16). Washing was performed three times by using 2X SSC and 0.1% SDS for 5 min at room temperature, followed by high stringency washes at 48°C for 15 min in 0.1X SSC and 0.1% SDS in the case of m2-mAChR and 56°C for c-Fos and P-actin. Specific radiolabeled bands were determined by direct counting of the autoradiographs with a Petagen betascope or laser densitometer. Levels of m2-mAChR mRNA were normalized to total cellular RNA in all cases.
Materials Colchicine and P-lumicolchicine were purchased from Sigma Chemical Co. (St. Louis, MO). Taxol was kindly provided by the National Cancer Institute, NIH (Bethesda, MD). Basal modified Eagle’s medium (BMEM), fetal calf serum, and glutamine were from GIBCO Laboratories (Grand Island, NY). Cell Culture and Northern
Blot Analysis
Primary cultures were prepared from &day-old Sprague-Dawley rats as described previously (14). Cells were seeded in 60-mm Costar culture dishes precoated with poly-L-lysine at a density of 9-10 X lo6 cells per dish and maintained in BMEM, 10% fetal calf serum, 2 mM glutamine, 50 pg/ml gentamicin, and 25 mM KC1 at 37°C in air containing 6% CO,. After 24 h, @-cytosine arabinoside (10 pM) was added to inhibit the replication of nonneuronal cells. Drugs were added to 8-day-old culture from 100X stocks made by dissolving the drugs in physiological saline solution (PSS), dimethyl sulfoxide (DMSO), or 70% ethanol. In control experiments, none of these agents had an effect on m2-mAChR mRNA at the concentrations used in this study. The reaction was terminated by aspiration of the media and the immediate addition of 2.5 ml of guanidinium thiocyanate solution to each dish. RNA preparation was carried out essentially as described by Chirgwin et al. (15). Total RNA was isolated
[3HjOuabain Binding Measurement
Assay and Intracellular
CAMP
[3H]ouabain binding to intact cells was carried out using cells grown in a Costar 24-well cell culture cluster following the procedure described by Markwell et al. (17). CAMP quantification was performed in cells grown in 60-mm dishes using a [3H]cAMP binding protein assay kit from Amersham (18, 19). RESULTS AND DISCUSSION Figure 1A shows that treatment of cerebellar granule cells with 10 pM colchicine for at least 8 h produced no significant change in the amount of total RNA and pactin mRNA illustrated by Northern blot hybridization. However, normalization of m2-mAChR mRNA to total RNA revealed a time-dependent up-regulation of relative m2-mAChR level with increases of 14 + 3,33 + 9, and 63 f 8% at 2, 4, and 8 h, respectively (Fig. 1B). These increases were tightly associated with up-regulation of 2.2kb C-FOS mRNA. For comparison, colchicine-induced down-regulation of relative m3-mAChR mRNA level obtained from the same blot was also shown in Fig. 1B. At 24 h, levels of total RNA and mRNAs of #?-actin, C-Fos, m2-mAChR, and m3-mAC!hR were also markedly reduced. This coincided with a dramatic decrease in cell number, size, and neurite outgrowth (12) and is probably
EFFECTS
OF
COLCHICINE
A Hours of Treatment Total RNA ml-mAChR
mRNA
c-Fos mRNA B-actln mRNA
0-r 0
.
I 2
Time
.
I 4
after
.
I
.
6
Treatment
I
II-
0
24
ON
m2-mAChR
317
mRNA
CAMP content was decreased in a time-dependent manner with a 47% loss at 8 h (Fig. 2). The level of CAMP was further decreased by about 70% at 24 h when the toxic effect was evident. These results indicate that the loss of CAMP induced by short-term (<8 h) colchicine treatment is not the result of neurotoxicity and further suggest that up-regulation of m2-mAChR mRNA by colchicine could be mediated by inversed regulation through CAMP. This possibility is strengthened by our preliminary findings that an increase in intracellular CAMP by treatment with 8BrcAMP or forskolin results in attenuation of m2mAChR mRNA content. The increase in m2-mAChR mRNA induced by colchicine at 8 h was dose-dependent for up to 100 pM colchicine with a concomitant increase of c-Fos mRNA but little or no change in total RNA or @actin mRNA levels. In contrast, a marked down-regulation of m3-mAChR mRNA occurred (Fig. 3). The effect of colchicine could be reversed by 10 pM taxol, the microtubule bundle stabilizing agent, and the inactive derivative of colchicine, @-lumicolchicine, had no effect on m2-mAChR mRNA (Fig. 4). Thus, as for m3-mAChR mRNA, the effect of colchicine on m2-mAChR mRNA must be a consequence of colchicine’s effects on microtubule assembly (12). It has been reported that the intraventricular injection of colchicine led to a substantial increase of proneurotensin mRNA in the dorsomedial part of the caudate putamen and periventricular area of the hypothalamus (20).
(hrs.)
FIG. 1. Time course of changes of m2-mAChR and c-Fos mRNA following treatment with 10 pM of colchicine. Cerebellar granule cells after 8 days in culture were treated with 10 FM colchicine for the indicated periods of times and the reaction was terminated simultaneously. (A) Autoradiographs of mRNA hybridization to cDNAs of m2-mAChR, c-Fos, and @-actin in each sample. (B) Densitometry of autoradiograms. Levels of m2-mAChR and c-Fos mRNA have been normalized to total cellular RNA present at each time point and expressed relative to the control at 0 time. The data represent the mean and range of duplicate samples. Similar results have been obtained in three independent experiments. For comparison, colchicine-induced down-regulation of m3mAChR mRNA obtained from the same blot was included. These data have been published recently (12).
08
0.6
0.4
0.2
00
the result of the colchicine’s toxicity on cerebellar neurons. The toxicity of colchicine was further examined by measuring the binding of [3H]ouabain to Na+, K+-ATPase in cells. This method was recently shown to be an effective tool for quantifying neuronal survival in a mixed culture (17). As shown in Fig. 2, binding of [3H]ouabain to granule cells was unaffected by 10 &f colchicine up to 8 h of treatment, while at 24 h, the binding was drastically decreased, confirming colchicine’s toxicity with long-term treatment. To search for a possible second messenger mediating colchicine’s effect, in a parallel experiment intracellular CAMP levels were assessed at various time periods after colchicine treatment. The results show that the
0
4
2
Time
after
6
Treatment
a
24
(hrs.)
FIG. 2. Time course of colchicine-induced changes in [sH]ouabain binding to intact cells and intracellular CAMP levels. Cerebellar granule cells were incubated with colchicine (10 a) for the indicated time periods at 37’C. Reactions were terminated by aspirating media. Binding of [3H]ouabain (25 r&f) to intact granule cells was measured in the presence of ATP (2.5 mM) and glucose (2.5 mi%4) (17). CAMP quantification was performed using a CAMP assay kit from Amersham (18, 19). The data are means + SEM of three independent experiments performed in triplicate samples and are expressed as levels relative to that at 0 time. At 0 time the values of [3H]ouabain binding and intracellular CAMP were 23186 + 1646 dpm/dish and 61.2 + 4.3 pmol/dish, respectively.
318
FUKAMAUCHI,
HOUGH,
(PM,
0
1
10
100
Total RNA
m&mAChR
mRNA
C-FOS mRNA B-actln mFtNA
W
CHUANG
mAChR-mediated phosphoinositide turnover (3). This would suggest either that m3-mAChR mRNA is much more abundant than m2-mAChR mRNA or that m3mAChR mRNA is more efficiently translated into the receptor protein than m2-mAChR mRNA, such that total mAChR number fluctuates more closely with changes in m3-mAChR mRNA. Regardless of the event involved, our preliminary receptor binding data using subtype-selective antagonists as the displacer revealed that m3-mAChR did greatly outnumber m2-mAChR in cerebellar granule cells. The opposite influence of microtubule disruption by colchicine strengthens a prominent role of this cytoskeleton in the complex regulation of mAChR gene expression and provides a tool to further dissect out components involved in this differential regulation. Experiments are also in progress to explore whether similar opposing effects of
A Colchlcine
AND
m2
A :TRL R-lumi
TAX
COL
TAX CAL
Total RNA 0
1
Colchiclne
100
10
(PM)
FIG. 3. Dose-response changes of m2-mAChR and c-Fos mRNA following colchicine exposure (1, 10,100 p&f) for 8 h. Cerebellar granule cells after 8 days in culture were treated with the indicated concentrations of colchicine for 8 h. The levels of m2-mAChR, c-Fos, and @actin mRNAs were determined by Northern blot hybridization. Levels of m2mAChR and c-Fos mRNA have been normalized to amounts of total RNA and expressed as values relative to untreated control. The data represent the means and ranges of duplicate samples. The experiment has been performed three times with nearly identical results. For comparison, dose-dependent decrease of m3-mAChR mRNA was also shown. These data are from our recent publication (12).
The observations could be due to the blockade of intracellular transport of neurotensin mRNA. This explanation would not be satisfactory for cerebellar granule cells in culture, since mRNA species within the same cells are differentially affected by colchicine. The association of changes in c-Fos mRNA and CAMP levels with the upand down-regulation of mAChR mRNAs suggests the involvement of these two gene regulators in mAChR gene expression; however, their precise role remains for further study. It is of interest to note that total mAChR number was markedly decreased at 8 and 24 h after 10 FM colchicine treatment (12), despite a significant increase in the level of m2-mAChR mRNA at 8 h (Fig. 1). The loss of total mAChR sites was also temporally correlated with m3-
5.2Kb
m2-mRNP
R-actin mRNA
rr”
CTRL
O-lumi
TAX
Ccc
COL+TAX
FIG. 4. Effects of @lumicolchicine or tax01 on the levels of m2mAChR mRNA or colchicine-induced up-regulation in cerebellar granule cells. Cerebellar granule cells after 8 days in culture were exposed to 10 pit4 tax01 (TAX) for 30 min and then, where indicated, treated with 10 $kf of colchicine (COL) for 8 h. (A) Autoradiographs of mRNA hybridization to cDNAs of m2-mAChR and @-actin. (B) Densitometry of autoradiograms. Levels of m2-mAChR mRNA were normalized to values of total cellular RNA in each sample and expressed relative to untreated control. The data are the means and ranges of duplicate samples. Comparable results have been obtained in three independent experiments.
EFFECTS
OF
COLCHICINE
microtubule-disrupting drugs exist for other classes of neurotransmitter receptors.
ON
10.
11. Xu, J., and D.-M. Chuang (1986). Muscarinic receptor inhibit adenylate cyclase of membranes from rat cerebellar cells in primary culture. Fed. Proc. 45: 661.
2.
Xu, J., and D.-M. Chuang (1987). Muscarinic acetylcholine receptor-mediated phosphoinositide turnover in cultured cerebellar granul cells: desensitization by receptor agonists. J. Pharmacol. Exp. Ther. 242: 238-244.
3.
agonists granule
Fukamauchi, F., C. Hough, and D.-M. Chuang (1991). Expression and agonist-induced down-regulation of mRNAs of m2- and m3muscarinic acetylcholine receptors in cultured cerebellar granule cells. J. Neurochem. 56: 716-719.
4.
Dillon-Carter, O., and D.-M. Chuang (1989). Homologous desensitization of muscarinic cholinergic, histaminergic, adrenergic, and serotonergic receptors coupled to phospholipase C in cerebellar granule cells. J. Neurochem. 52: 598-603.
5.
Benecke, B.-J., A. Ben-Ze’ev, and S. Penman (1978). The control of mRNA production, translation and turnover in suspended and reattached anchorage-dependent fibroblasts. Cell 14: 931-939. Farmer, S., K. Wan, A. Ben-Ze’ev, and S. Penman (1983). Regulation of actin mRNA levels and translation responds to changes in cell configuration. Mol. Cell. Biol. 3: 182-189.
6.
Rothstein, T. (1986). of anti-immunogloblin 136: 813-816.
Stimulation antibody
of B cells by sequential addition and cytochalasin. J. Immunol.
Rovera, G., D. Santoli, and C. Damsky (1979). Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with a phorbol diester. Proc. Not.!. Acad. Sci. USA 76: 2779-2783. Penman, S., D. Capco, E. Fey, P. Chattejee, T. Reiter, S. Ermish, and K. Wan (1983). The three-dimensional structural networks of
mRNA
cytoplasm and nucleus. In Modern Cell Biology Eds.), Vol. 2, pp. 385-415. A. R. Liss, New York.
REFERENCES 1.
m2-mAChR
12.
13.
14.
15.
16.
17.
18. 19.
20.
319 (J. R. McIntosh,
Zambetti, G., A. Ramsey-Ewing, R. Bortell, G. Stein, and J. Stein (1991). Disruption of the cytoskeleton with cytochalasin D induces c-fos gene expression. Exp. Cell Res. 192: 93-101. Burgoyne, R. D., and M. A. Cambray-Deakin (1988). The cellular neurobiology of neuronal development: The cerebellar granule cell Brain Res. Reu. 13:77-101. Fukamauchi, F., C. Hough, and D.-M. Chuang (1991). Role of microtubule structure in the maintenance of m3-muscarinic acetylcholine receptor mRNA levels. Mol. Cell. Neurosci. 2: 123-129. Schiff, P. B., and S. B. Horwitz (1980). Tax01 stabilizes microtubules in mouse fibroblast cells. Proc. Natl. Acad. Sci. USA 77: 15611565. Gallo, V., M. T. Ciotti, A. Coletti, F. Aloisi, and G. Levi (1982). Selective release of glutamate from cerebellar granule cells differentiating in culture. Proc. Natl. Acad. Sci. USA 79: 7919-7923. Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299. Feinberg, A. P., and B. Vogelstein (1983). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. Markwell, M. A. K., H. Z. Sheng, D. E. Brenneman, and S. M. Paul (1991). A rapid method to quantify neurons in mixed cultures based on the specific binding of [3H]ouabain to neuronal Na+, K+ATPase. Brain Res. 538: l-8. Gilman, A. G. (1970). A protein binding assay for adenosine 3’:5’cyclic monophosphate. Proc. Natl. Acad. Sci. USA 67: 305-312. Brown, B. L., J. D. M. Albano, R. P. Ekins, and A. M. Sgheni (1971). A simple and sensitive saturation assay method for the measurement of adenosine 3’:5’-cyclic monophosphate. Biochem. J. 121: 561-562. Kiyama, H., and P. C. Emson (1991). Colchicine-induced expression of proneurotensin mRNA in rat striatum and hypothalamus. Mol. Brain Res. 9: 353-358.
AND CELLULAR
NEUROSCIENCES
2, 315-319
(1991)
m2- and m3-Muscarinic Acetylcholine Receptor mRNAs Have Different Responses to Microtubule-Affecting Drugs FUMIHIKO Section
FUKAMAUCHI,
on Molecular Building
CHRISTOPHER
Neurobiology, Biological Psychiatry 10, Room 3N-212, 9000 Rockville Received
for publication
INTRODUCTION
Primary cultures of cerebellar granule cells prepared from neonatal rats have proven to be an excellent model system for studying the regulation of muscarinic acetylcholine receptors (mAChRs). This nearly homogenous preparation of neurons expresses in culture m2- and m3mAChRs negatively coupled to adenylate cyclase and correspondence
should
AND
DE-MAW CHUANG’
Branch, National Institute Pike, Bethesda, Maryland July
of Mental 20892
Health,
17, 1991
positively linked to phospholipase C, respectively (l-3). Prolonged stimulation of these neurons with the mAChR agonist carbachol results in desensitization of the receptor-mediated effector response and a subsequent loss of total mAChR binding sites (2, 4). Using cloned mAChR cDNA, we have demonstrated that the early phase of carbachol-induced mAChR down-regulation is due, at least in part, to the reduction of m3- and m2-mAChR mRNAs (3). Conversely, persistent occupancy of mAChR in cerebellar granule cells by antagonists causes up-regulation of m3-mAChR mRNA (3). These observations suggest that some factors may be involved in transmitting the signal from the receptors at the plasma membrane to the nucleus to influence the expression of surface receptor mRNA. Several lines of indirect evidence support a role of the cytoskeleton in relaying stimuli from the cell surface to the nucleus to affect gene expression. For example, detachment of 3T3 fibroblasts from their solid support, a condition known to produce cytoskeletal reorganization, induces morphological changes and a concomitant loss of DNA, RNA, and protein synthesis (5,6). These changes are restored by reattachment of suspended cells to their growth surface. There are also reports that organization of cytoarchitecture affects the growth and differentiation of several cell types (7-9) and disruption of the cytoskeleton with cytochalasin D promotes c-fos gene expression (10). Cerebellar granule cells have been shown to differentiate in culture with an early burst in the expression of /3-actin mRNA (3), which coincides with a period of active neurite outgrowth in these cells. These neurites in cerebellar granule cells have been shown to contain abundant actin filaments (11). The appearance of neurites is followed by subsequent development of microtubules, microfilaments, and cytoskeleton-associated proteins such as MAPS and spectrin (11). Additionally, using cultured granule cells, we have recently demonstrated that a microtubule-disrupting agent, colchicine, selectively decreases m3-mAChR mRNA level in a time-dependent manner (12). This m3-mAChR mRNA down-regulation correlates with a loss of mAChR sites and a decrease
We have recently shown that colchicine induces a timeand dose-dependent decrease of m3-muscarinic acetylcholine receptor (mAChR) mRNA in rat cerebellar granule cells (F. Fukamauchi, C. Hough, and D.-M. Chuang, 1991, Mol. Cell. Neurosci. 2: 123-129). We now report the regulation by colchicine of m2-mAChR mRNA level determined by Northern blot hybridization. Colchicine (10 pM) increased m2-mAChR mRNA level in cerebellar granule cells by 14, 33, and 63% after treatment for 2, 4, and 8 h, respectively, but markedly decreased it at 24 h. The colchicine-induced up-regulation of m2-mAChR mRNA was temporally associated with a decrease in intracellular cyclic AMP levels but an increase in c-Fos mRNA. The level of m2-mAChR mRNA also increased by 21,62, and 78% following exposure to 1, 10, and 100 pM of colchicine for 8 h, respectively. The ECso value of colchicine for this increase was approximately 5 PM. cFos mRNA was increased in parallel by 8 h of treatment with colchicine in a concentration-dependent manner. An inactive derivative of colchicine, /I-lumicolchicine, had no effect on the steady-state levels of m2- or m3-mAChR mRNA. The presence of the microtubule stabilizer, taxol (10 @), reversed the colchicine-induced m2-mAChR mRNA up-regulation at 8 h. These results strongly suggest that microtubules are involved in the homeostasis of the level of both m2- and m3-mAChR mRNAs in cerebellar granule cells; however, these two mAChR mRNA species are oppositely regulated by perturbation of microtubule structures. 0 1991 Academic PWKI, 1~.
1 To whom
HOUGH,
be addressed. 315
1044-7431/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
316
FUKAMAUCHI,
HOUGH,
of carbachol-induced phosphoinositide turnover (12). Moreover, colchicine’s effect on m3-mAChR mRNA is completely reversed by taxol, a microtubule stabilizer (12, 13), and overlaps with the m3-mAChR mRNA down-regulation induced by prolonged stimulation with carbachol (12). We have suggested that microtubules play an important role in the maintenance of the steady-state of m3-mAChR mRNA level and further speculated that the nucleus can selectively adjust the regulation of mAChR gene expression in response to perturbation of microtubule structures. In this study, conducted simultaneously with the latter study, we examined the change in m2-mAChR mRNA level under the same conditions in an attempt to define further the role of microtubules in regulating mAChR gene expression. As in the previous study, the use of microtubule-affecting agents such as colchicine or taxol in the present study has elicited interest in the regulation of mAChR mRNAs. The regulation of m2-mAChR mRNA level mediated by microtubule-affecting agents was strikingly different from that of m3-mAChR mRNA level. Possible roles of cyclic AMP (CAMP) and c-Fos expression in microtubule-mediated m2-mAChR mRNA regulation have also been explored. MATERIALS
AND METHODS
AND
CHUANG
by centrifugation through 5.7 M cesium chloride. A onetenth aliquot of total RNA from each sample was electrophoresed in parallel with ribosomal RNA standards on ethidium bromide-stained, 1% agarose gels for quantification of total RNA. For this purpose the RNA was allowed to migrate approximately 5 mm from the sample well and then was quantified by using laser densitometry of photographic negatives of the entire RNA band. The remaining RNA was denatured, fractionated by 1% agarose-formaldehyde gels, and transferred to a nitrocellulose membrane by capillary action. The blot was hybridized with an m2-mAChR probe made from a 0.5-kb AvaI fragment of human Hm2p9 (a gift from Dr. T. I. Bonner, Laboratory of Cell Biology, NIMH, Bethesda, MD), murine c-Fos cDNA probe, or a chicken /3-actin cDNA probe (gifts from Dr. C. B. Thompson, Howard Hughes Medical School, Ann Arbor, MI) at 42’C overnight. Each probe had been labeled with [32P]dCTP (3000 Ci/mmol) by the random primer labeling method (16). Washing was performed three times by using 2X SSC and 0.1% SDS for 5 min at room temperature, followed by high stringency washes at 48°C for 15 min in 0.1X SSC and 0.1% SDS in the case of m2-mAChR and 56°C for c-Fos and P-actin. Specific radiolabeled bands were determined by direct counting of the autoradiographs with a Petagen betascope or laser densitometer. Levels of m2-mAChR mRNA were normalized to total cellular RNA in all cases.
Materials Colchicine and P-lumicolchicine were purchased from Sigma Chemical Co. (St. Louis, MO). Taxol was kindly provided by the National Cancer Institute, NIH (Bethesda, MD). Basal modified Eagle’s medium (BMEM), fetal calf serum, and glutamine were from GIBCO Laboratories (Grand Island, NY). Cell Culture and Northern
Blot Analysis
Primary cultures were prepared from &day-old Sprague-Dawley rats as described previously (14). Cells were seeded in 60-mm Costar culture dishes precoated with poly-L-lysine at a density of 9-10 X lo6 cells per dish and maintained in BMEM, 10% fetal calf serum, 2 mM glutamine, 50 pg/ml gentamicin, and 25 mM KC1 at 37°C in air containing 6% CO,. After 24 h, @-cytosine arabinoside (10 pM) was added to inhibit the replication of nonneuronal cells. Drugs were added to 8-day-old culture from 100X stocks made by dissolving the drugs in physiological saline solution (PSS), dimethyl sulfoxide (DMSO), or 70% ethanol. In control experiments, none of these agents had an effect on m2-mAChR mRNA at the concentrations used in this study. The reaction was terminated by aspiration of the media and the immediate addition of 2.5 ml of guanidinium thiocyanate solution to each dish. RNA preparation was carried out essentially as described by Chirgwin et al. (15). Total RNA was isolated
[3HjOuabain Binding Measurement
Assay and Intracellular
CAMP
[3H]ouabain binding to intact cells was carried out using cells grown in a Costar 24-well cell culture cluster following the procedure described by Markwell et al. (17). CAMP quantification was performed in cells grown in 60-mm dishes using a [3H]cAMP binding protein assay kit from Amersham (18, 19). RESULTS AND DISCUSSION Figure 1A shows that treatment of cerebellar granule cells with 10 pM colchicine for at least 8 h produced no significant change in the amount of total RNA and pactin mRNA illustrated by Northern blot hybridization. However, normalization of m2-mAChR mRNA to total RNA revealed a time-dependent up-regulation of relative m2-mAChR level with increases of 14 + 3,33 + 9, and 63 f 8% at 2, 4, and 8 h, respectively (Fig. 1B). These increases were tightly associated with up-regulation of 2.2kb C-FOS mRNA. For comparison, colchicine-induced down-regulation of relative m3-mAChR mRNA level obtained from the same blot was also shown in Fig. 1B. At 24 h, levels of total RNA and mRNAs of #?-actin, C-Fos, m2-mAChR, and m3-mAC!hR were also markedly reduced. This coincided with a dramatic decrease in cell number, size, and neurite outgrowth (12) and is probably
EFFECTS
OF
COLCHICINE
A Hours of Treatment Total RNA ml-mAChR
mRNA
c-Fos mRNA B-actln mRNA
0-r 0
.
I 2
Time
.
I 4
after
.
I
.
6
Treatment
I
II-
0
24
ON
m2-mAChR
317
mRNA
CAMP content was decreased in a time-dependent manner with a 47% loss at 8 h (Fig. 2). The level of CAMP was further decreased by about 70% at 24 h when the toxic effect was evident. These results indicate that the loss of CAMP induced by short-term (<8 h) colchicine treatment is not the result of neurotoxicity and further suggest that up-regulation of m2-mAChR mRNA by colchicine could be mediated by inversed regulation through CAMP. This possibility is strengthened by our preliminary findings that an increase in intracellular CAMP by treatment with 8BrcAMP or forskolin results in attenuation of m2mAChR mRNA content. The increase in m2-mAChR mRNA induced by colchicine at 8 h was dose-dependent for up to 100 pM colchicine with a concomitant increase of c-Fos mRNA but little or no change in total RNA or @actin mRNA levels. In contrast, a marked down-regulation of m3-mAChR mRNA occurred (Fig. 3). The effect of colchicine could be reversed by 10 pM taxol, the microtubule bundle stabilizing agent, and the inactive derivative of colchicine, @-lumicolchicine, had no effect on m2-mAChR mRNA (Fig. 4). Thus, as for m3-mAChR mRNA, the effect of colchicine on m2-mAChR mRNA must be a consequence of colchicine’s effects on microtubule assembly (12). It has been reported that the intraventricular injection of colchicine led to a substantial increase of proneurotensin mRNA in the dorsomedial part of the caudate putamen and periventricular area of the hypothalamus (20).
(hrs.)
FIG. 1. Time course of changes of m2-mAChR and c-Fos mRNA following treatment with 10 pM of colchicine. Cerebellar granule cells after 8 days in culture were treated with 10 FM colchicine for the indicated periods of times and the reaction was terminated simultaneously. (A) Autoradiographs of mRNA hybridization to cDNAs of m2-mAChR, c-Fos, and @-actin in each sample. (B) Densitometry of autoradiograms. Levels of m2-mAChR and c-Fos mRNA have been normalized to total cellular RNA present at each time point and expressed relative to the control at 0 time. The data represent the mean and range of duplicate samples. Similar results have been obtained in three independent experiments. For comparison, colchicine-induced down-regulation of m3mAChR mRNA obtained from the same blot was included. These data have been published recently (12).
08
0.6
0.4
0.2
00
the result of the colchicine’s toxicity on cerebellar neurons. The toxicity of colchicine was further examined by measuring the binding of [3H]ouabain to Na+, K+-ATPase in cells. This method was recently shown to be an effective tool for quantifying neuronal survival in a mixed culture (17). As shown in Fig. 2, binding of [3H]ouabain to granule cells was unaffected by 10 &f colchicine up to 8 h of treatment, while at 24 h, the binding was drastically decreased, confirming colchicine’s toxicity with long-term treatment. To search for a possible second messenger mediating colchicine’s effect, in a parallel experiment intracellular CAMP levels were assessed at various time periods after colchicine treatment. The results show that the
0
4
2
Time
after
6
Treatment
a
24
(hrs.)
FIG. 2. Time course of colchicine-induced changes in [sH]ouabain binding to intact cells and intracellular CAMP levels. Cerebellar granule cells were incubated with colchicine (10 a) for the indicated time periods at 37’C. Reactions were terminated by aspirating media. Binding of [3H]ouabain (25 r&f) to intact granule cells was measured in the presence of ATP (2.5 mM) and glucose (2.5 mi%4) (17). CAMP quantification was performed using a CAMP assay kit from Amersham (18, 19). The data are means + SEM of three independent experiments performed in triplicate samples and are expressed as levels relative to that at 0 time. At 0 time the values of [3H]ouabain binding and intracellular CAMP were 23186 + 1646 dpm/dish and 61.2 + 4.3 pmol/dish, respectively.
318
FUKAMAUCHI,
HOUGH,
(PM,
0
1
10
100
Total RNA
m&mAChR
mRNA
C-FOS mRNA B-actln mFtNA
W
CHUANG
mAChR-mediated phosphoinositide turnover (3). This would suggest either that m3-mAChR mRNA is much more abundant than m2-mAChR mRNA or that m3mAChR mRNA is more efficiently translated into the receptor protein than m2-mAChR mRNA, such that total mAChR number fluctuates more closely with changes in m3-mAChR mRNA. Regardless of the event involved, our preliminary receptor binding data using subtype-selective antagonists as the displacer revealed that m3-mAChR did greatly outnumber m2-mAChR in cerebellar granule cells. The opposite influence of microtubule disruption by colchicine strengthens a prominent role of this cytoskeleton in the complex regulation of mAChR gene expression and provides a tool to further dissect out components involved in this differential regulation. Experiments are also in progress to explore whether similar opposing effects of
A Colchlcine
AND
m2
A :TRL R-lumi
TAX
COL
TAX CAL
Total RNA 0
1
Colchiclne
100
10
(PM)
FIG. 3. Dose-response changes of m2-mAChR and c-Fos mRNA following colchicine exposure (1, 10,100 p&f) for 8 h. Cerebellar granule cells after 8 days in culture were treated with the indicated concentrations of colchicine for 8 h. The levels of m2-mAChR, c-Fos, and @actin mRNAs were determined by Northern blot hybridization. Levels of m2mAChR and c-Fos mRNA have been normalized to amounts of total RNA and expressed as values relative to untreated control. The data represent the means and ranges of duplicate samples. The experiment has been performed three times with nearly identical results. For comparison, dose-dependent decrease of m3-mAChR mRNA was also shown. These data are from our recent publication (12).
The observations could be due to the blockade of intracellular transport of neurotensin mRNA. This explanation would not be satisfactory for cerebellar granule cells in culture, since mRNA species within the same cells are differentially affected by colchicine. The association of changes in c-Fos mRNA and CAMP levels with the upand down-regulation of mAChR mRNAs suggests the involvement of these two gene regulators in mAChR gene expression; however, their precise role remains for further study. It is of interest to note that total mAChR number was markedly decreased at 8 and 24 h after 10 FM colchicine treatment (12), despite a significant increase in the level of m2-mAChR mRNA at 8 h (Fig. 1). The loss of total mAChR sites was also temporally correlated with m3-
5.2Kb
m2-mRNP
R-actin mRNA
rr”
CTRL
O-lumi
TAX
Ccc
COL+TAX
FIG. 4. Effects of @lumicolchicine or tax01 on the levels of m2mAChR mRNA or colchicine-induced up-regulation in cerebellar granule cells. Cerebellar granule cells after 8 days in culture were exposed to 10 pit4 tax01 (TAX) for 30 min and then, where indicated, treated with 10 $kf of colchicine (COL) for 8 h. (A) Autoradiographs of mRNA hybridization to cDNAs of m2-mAChR and @-actin. (B) Densitometry of autoradiograms. Levels of m2-mAChR mRNA were normalized to values of total cellular RNA in each sample and expressed relative to untreated control. The data are the means and ranges of duplicate samples. Comparable results have been obtained in three independent experiments.
EFFECTS
OF
COLCHICINE
microtubule-disrupting drugs exist for other classes of neurotransmitter receptors.
ON
10.
11. Xu, J., and D.-M. Chuang (1986). Muscarinic receptor inhibit adenylate cyclase of membranes from rat cerebellar cells in primary culture. Fed. Proc. 45: 661.
2.
Xu, J., and D.-M. Chuang (1987). Muscarinic acetylcholine receptor-mediated phosphoinositide turnover in cultured cerebellar granul cells: desensitization by receptor agonists. J. Pharmacol. Exp. Ther. 242: 238-244.
3.
agonists granule
Fukamauchi, F., C. Hough, and D.-M. Chuang (1991). Expression and agonist-induced down-regulation of mRNAs of m2- and m3muscarinic acetylcholine receptors in cultured cerebellar granule cells. J. Neurochem. 56: 716-719.
4.
Dillon-Carter, O., and D.-M. Chuang (1989). Homologous desensitization of muscarinic cholinergic, histaminergic, adrenergic, and serotonergic receptors coupled to phospholipase C in cerebellar granule cells. J. Neurochem. 52: 598-603.
5.
Benecke, B.-J., A. Ben-Ze’ev, and S. Penman (1978). The control of mRNA production, translation and turnover in suspended and reattached anchorage-dependent fibroblasts. Cell 14: 931-939. Farmer, S., K. Wan, A. Ben-Ze’ev, and S. Penman (1983). Regulation of actin mRNA levels and translation responds to changes in cell configuration. Mol. Cell. Biol. 3: 182-189.
6.
Rothstein, T. (1986). of anti-immunogloblin 136: 813-816.
Stimulation antibody
of B cells by sequential addition and cytochalasin. J. Immunol.
Rovera, G., D. Santoli, and C. Damsky (1979). Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with a phorbol diester. Proc. Not.!. Acad. Sci. USA 76: 2779-2783. Penman, S., D. Capco, E. Fey, P. Chattejee, T. Reiter, S. Ermish, and K. Wan (1983). The three-dimensional structural networks of
mRNA
cytoplasm and nucleus. In Modern Cell Biology Eds.), Vol. 2, pp. 385-415. A. R. Liss, New York.
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