Role of microtubule structure in the maintenance of m3-muscarinic acetylcholine receptor rnRNA levels

MOLECULAR

AND

CELLULAR

NEUROSCIENCES

2,X%129

(19%)

Role of Microtubule Structure in the Maintenance of m3-Muscarinic Acetylcholine Receptor mRNA Levels FUMIHIKO FUKAMAUCHI, CHRISTOPHER HOUGH, AND DE-MAW CHUANG~ Section

on Molecular Building

Neurobiology, Biological Psychiatry 10, Room 3N-212, 9000 Rockuille

Branch, National Institute Pike, Bethesda, Marylund

of Mental

Health,

20892

Receivedfor publication January 9, 1991

The regulation of m3-muscarinic acetylcholine receptor (mAChR) mRNA following exposure to microtubuledisrupting agents was studied in cultured cerebellar granule cells. Colchicine (10 fl), an antimicrotubular agent, decreased the levels of m3-mAChR mRNA in a time-dependent fashion. Concentration levels of m3mAChR mRNA were attenuated by 29,49, and 85% of original values at 4, 8, and 24 h, respectively, after exposure to 10 pM colchicine in these cells on the eighth day of culture. This colchicine-induced m3-mAChR mRNA down-regulation correlated with a decrease of mAChR sites measured by binding with N[SH]methylscopolamine and loss of carbachol-inducible phosphoinositide turnover. No changes in morphology or levels of total cellular RNA or &actin mRNA were detected after 8 h of exposure to 10 fl colchicine. The levels of m3-mAChR mRNA decreased in cells exposed to colchicine in a dose-dependent manner. An inactive derivative of colchicine, &lumicolchicine, did not change the steady-state level of m3-mAChR mRNA. The presence of taxol(l0 PM), a microtubule stabilizer, reversed colchicinebut not nocodazole-induced m3-mAChR mRNA down-regulation that occurred at 8 h. Taxol was also able to reverse, at least in part, carbachol-induced m3-mAChR mRNA down-regulation after 8 h of exposure. These results indicate that an intact microtubule structure is critical for the maintenance of m3-mAChR mRNA levels. Q mei Academic PWS, I~C.

INTRODUCTION The cytoskeleton can be classified into three types of fibers, i.e., microtubules, microfilaments, and intermediate filaments, which are involved in such diverse intracellular functions as cell mitosis, secretion, endocytosis, regulation of cell shape, and organelle translocation (l-3). Alterations in the state of microtubules, microfilaments, and their associated proteins can modify the internalization, 1To whom correspondenceshould be addressed.

distribution, and processing of cell surface receptors (46). Microtubule-disrupting agents such as colchicine interfere with microtubule assembly and affect cell mitosis (7,8) and the anchorage of ligand-receptor complexes (4, 5). Moreover, the tubulin-GTP complex associated with synaptic membranes can increase adenylate cyclase activity mediated by G-protein-coupled receptors (9, 10). Treatment of cells with colchicine results in a significant reduction in the level of tubulin mRNA caused by the destabilizing effect of increased levels of free tubulin on /3-tubulin mRNA (11, 12). At least five specific genes encoding muscarinic acetylcholine receptors (mAChRs), termed ml, m2, m3, m4, and m5, have been cloned from rat, porcine, and human tissues (for review, see (13)). Their corresponding receptor subtypes (i.e., Ml, M2, M3, M4, and M5) display differential sensitivities to selective mAChR antagonists such as pirenzepine, AF-DX 116, and 4-diphenylacetoxy-Nmethylpiperidine methiodide and are coupled to distinct signal transduction systems. Thus, according to one mAChR classification, stimulation of receptors encoded by ml, m3, and m5 leads to increases in phosphoinositide turnover and CAMP accumulation, while m2- and m4encoded mAChRs are negatively coupled to adenylate cyclase (13). In addition m3-encoded receptors are linked to arachidonic acid release, activation of Ca2+-dependent K+ channel, and inhibition of M current (13, 14). We have previously demonstrated that in vitro primary cultures of cerebellar granule cells express mRNA for m2and m3-mAChRs (15). Stimulation of these neuronal cultures with a mAChR agonist, carbachol, results in differential down-regulation of m2- and m3-mAChR mRNA (15). At 8 h after carbachol stimulation, the m3-mAChR mRNA down-regulation is associated with a loss of carbachol-induced phosphoinositide turnover as well as mAChR binding sites (15). Using neurohybrid NG108-15 cells, Ray et al. (16) reported that treatment with the antimicrotubular agents colchicine and nocodazole prevents carbachol-induced loss of total mAChRs but not cell surface receptors, suggesting that microtubular structures are involved in the transport of mAChRs from 123

1044-7431/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

124

FUKAMAUCHI,

HOUGH,

the plasma membrane to intracellular degradation loci. The aim of the present study was to determine whether disruption of microtubules by colchicine would alter the level of mRNA and density of m3-mAChR and affect carbachol-induced down-regulation of the m3-mAChR mRNA species. EXPERIMENTAL

PROCEDURES

Material-s Cell culture medium and serum were purchased from GIBCO (Grand Island, NY). Carbachol, colchicine, plumicolchicine, and nocodazole are products of Sigma Chemical Co. (St. Louis, MO). N-[3H]Methylscopolamine (NMS) (87.0 Ci/mmol) and [3H]myo-inositol (16.5 Ci/ mmol) were purchased from New England Nuclear (Boston, MA). Taxol was a gift from the National Cancer Institute, NIH (Bethesda, MD). Primary

CHUANG

and the remaining material was electrophoresed on 1% agarose-formaldehyde gels, blotted, and fixed to nitrocellulose. The level of m3-mAChR mRNA was determined by hybridization with a radiolabeled probe made from 0.7kb StuI-NheI fragment of rat Rm3p8; cDNA clone, generously provided by Dr. T. I. Bonner (Laboratory of Cell Biology, NIHM, Bethesda, MD). Each fragment contains a portion of the i3 region between membrane-spanning regions 5 and 6. The P-actin probe was derived from a cDNA clone of the chicken ,&actin gene (19), kindly supplied by Dr. C. B. Thompson (Howard Hughes Medical Center, Ann Arbor, MI). Hybridized blots were washed at 56°C for 15-20 min in 0.1X SSC (1X SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7) and 1% sodium dodecyl sulfate. Specific m3-mAChR mRNA bands were quantified by using Petagen betascope blot analyzer (Waltham, MA) or A

Cultures of Cerebellar Granule Cells

Cerebellar granule cells were prepared and cultured as described previously (17). Briefly, cerebella were removed from 8-day postnatal Sprague-Dawley rats, chopped into 0.4-mm cubes, washed, and trypsinized in Krebs-Ringer bicarbonate buffer followed by trituration with a Pasteur pipet to dissociate the cells. The cells were then suspended in basal modified Eagle’s medium containing 10% fetal bovine serum, 2 mM glutamine, 50 pg/ml gentamicin, and 25 miV KC1 and plated into 60-mm petri dishes precoated with poly-L-lysine to yield 9-10 X lo6 cells per dish. The cells were maintained at 37°C in a 6% COz humidified atmosphere. Cytosine arabinoside (10 plkf) was added 24 h later to arrest the growth of nonneuronal cells. Unless otherwise specified, cells were treated on the eighth day of culture by addition of drugs dissolved at loo-fold final concentrations in physiological saline solution (PSS). For time course studies, drugs were added sequentially such that all reactions were terminated at the same time. The treatment was terminated by aspiration of the medium from the dish, addition of 2.5 ml of guanidinium isothiocynate solution, and gentle shaking of the dish. Previous studies have established that primary cultures of cerebellar granule cells represent a highly homogeneous population of neurons with a purity greater than 90% after 8 days in culture (17). They differentiate in vitro into a state capable of synthesizing and releasing glutamate in response to stimulus. Northern

AND

Blotting

Total RNA was isolated by the method of Chirgwin et al. (18). The RNA so obtained was rinsed with 75% ethanol and dissolved without further manipulation in formaldehyde gel sample buffer. A one-tenth aliquot of the sample was used for the quantitation of total RNA,

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FIG. 1. Time course of changes of m3-mAChR mRNA following treatment with 10 pM colchicine. Cerebellar granule cells after 8 days in culture were exposed to 10 pM colchicine for indicated periods of time and the reaction was terminated si+multaneously. (A) Autoradiographs of mRNA hybridization to cDNAs of m3-mAC!hR and @-actin mRNA in each sample. (B) Densitometry of autoradiograms. Levels of m3-mAChR mRNA have been normalized to total cellular RNA present at each time point. The data represent the mean 1?I SD of duplicate samples. The experiment has been repeated three times with virtually identical results.

ROLE 1.2

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STRUCTURES

PI turnover mRNA

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FIG. 2. Colchicine-induced decrease in mAChR binding, carbacholinduced phosphoinositide turnover, and m3-mAChR mRNA level in intact granule cells. mAChR sites measured by binding 13H]NMS (2 n&f) to monolayer granule cells and 100 &f carbachol-induced phosphoinositide (PI) turnover was carried out as described previously (20). The data are means -+ SEM of quadruplicate samples and are expressed as relative level to that 0 time. At 0 time the values of [3H]NMS binding and carbachol-induced [3H]inositol monophosphate accumulation were 243 fmol/dish of 3 X 10s cells and 23217 dpm, respectively. The specific activity of [$H]NMS was 63 cpm/fmol.

by laser densitometry of autoradiograms. Total RNA was quantified by laser densitometry of photographic negatives taken of ethidium bromide-stained agarose gels on which all the samples had been electrophoresed in parallel with ribosomal RNA standards. Specific hybridization of the probes was then normalized to total cellular RNA or @-actin mRNA in each sample and to the experimental controls. Measurements of t3H]NMS Binding Phosphoinositide Turnover

IN

m3-mAChR

the loss of carbachol-induced phosphoinositide turnover (Fig. 2). The incorporation of [3H]myo-inositol into inositol lipid was unaffected by treatment with 10 PM colchicine for at least up to 8 h (data not shown). The level of m3-mAChR mRNA after 4 h of exposure to colchicine decreased in a concentration-dependent manner, while the levels of /3-actin mRNA and total cellular RNA remained relatively unchanged up to 100 PM colchicine (Fig. 3). /3-Lumicolchicine, an inactive derivative of colchicine (21), failed to change the steady-state level of m3-mAChR mRNA or carbachol-induced m3-mAChR mRNA downregulation that occurred after exposure for 8 h (Fig. 4). An 8-h treatment of cells with either colchicine (10 PM) or carbachol(lO0 PM) produced approximately the same extent of m3-mAChR mRNA down-regulation (Fig. 5). The simultaneous presence of both drugs induced an effect greater than that produced by either drug alone, but less than that produced when the two were added together. To assess whether the effects of colchicine and carbachol are related to disruption of microtubule structures, we examined the effects of taxol, a microtubule stabilizer (21, 22), on m3-mAChR mRNA down-regulation induced by these two drugs. The presence of 10 PM taxol fully reversed the down-regulation of m3-

A

Colchlcine

RESULTS

Levels of m3-mAChR mRNA in cerebellar granule cells were altered in a time-dependent manner in response to treatment with 10 PM colchicine (Fig. 1). The amount of m3-mAChR mRNA was attenuated to 71+ 7% of an untreated control at 4 h and then further decreased to 51 -t 3 and 15 + 3% at 8 and 24 h, respectively, after the addition of colchicine to the culture media. Levels of total RNA and @-actin mRNA were not significantly changed at 8 h but markedly decreased at 24 h after colchicine treatment. The degree of down-regulation of m3-mAChR mRNA at 4,8, and 24 h of exposure to 10 PM colchicine correlated with the decrease in mAChR sites measured by [3H]NMS binding to intact granule cells at 37°C and

(PM) F nnnn

Tote1 RNA

and

Binding of [3H]NMS (2 ti) to monolayer granule cells was performed in PSS at 37°C as described previously (20). mAChR-mediated phosphoinositide turnover was measured as lithium-induced accumulation of [3H]inositol monophosphate in cells prelabeled with [3H]myo-inositol as described previously (20).

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FIG. 3. Dose-response changes of m3-mAChR mRNA following colchicine exposure (1, 10, 100 PM) for 4 h. Cerebellar granule cells after 8 days in culture were exposed to indicated concentrations of colchicine for 4 h. The levels of fl-actin and m3-mAChR mRNAs were determined by Northern blot hybridization. Levels of m3-mACbR mRNA have been normalized to amounts of total RNA. The data represent the mean + SD of duplicate samples. The experiment has been repeated three times with identical results.

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In this study, we report the effect of colchicine on mAChR mRNA in cerebellar granule cells. Colchicine treatment caused a marked down-regulation of m3mAChR mRNA that was prevented by taxol and mimicked by nocodazole. Colchicine and nocodazole were unable to prevent the ability of carbachol to induce m3mAChR mRNA down-regulation. Colchicine and nocodazole, in fact, enhanced this down-regulation. Taxol, on the other hand, partially reversed carbachol-induced m3mAChR mRNA down-regulation but had little effect alone. Colchicine binds to tubulin heterodimers with high affinity. This complex can then bind to microtubule ends where it inhibits the binding of additional free subunits. Colchicine also destabilizes existing microtubules (21,22). As a consequence, the intracellular concentration of tu-

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mAChR mRNA induced by 8 h of treatment with colchicine and at least partially restored the loss of m3mAChR mRNA induced by carbachol. Taxol by itself did not induce significant changes in m3-mAChR mRNA levels. Nocodazole (10 PM), which inhibits microtubule assembly and induces rapid disassembly of existing microtubules (21, 22) also enhanced carbachol-induced down-regulation of m3-mAChR mRNA at 8 h (data not shown). Taxol did not, however, prevent down-regulation of m3-mAChR mRNA induced by nocodazole alone (Fig. 6). The levels of total RNA and B-actin remained relatively unaffected during the course of colchicine and nocodazole treatment. Table 1 summarizes effects of drugs that affect microtubule structures on m3-mAChR mRNA levels. Figure 7 shows the morphology of cerebellar granule cells after exposure to 10 FM colchicine for 0, 8, and 24 h. Exposure for 8 h did not produce a significant change in cell morphology and neurite formation, while 24 h of exposure resulted in a marked decrease in the number of neurites and detachment of some neurons. The remaining neurons appeared to decrease in size and the cell aggregates seemed to disintegrate.

COL

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FIG. 4. Effects of fl-lumicolchicine (100 pA4) on the levels of m3mAChR mRNA and carbachol-induced m3-mAChR mRNA down-regulation. Cells were pretreated with 100 WM @-lumicolchicine for 30 min and then treated with 100 pit4 carbachol for 8 h. The data are means + SD of duplicate samples. The experiment has been performed three times with nearly identical results.

CCh

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FIG. 6. Effects of taxol on carbacholand colchicine-induced m3mAChR mRNA down-regulation in cerebellar granule cells. Cerabellar granule cells after 8 days in culture were pretreated with 10 pM tax01 (TAX) for 30 min and then, where indicated, treated with 100 pA4 carbachol (CCh) or 10 ti colchicine (COL) for 8 h. (A) Autoradiographs of mRNA hybridization to cDNAs of m3-mAChR and @-actin. (B) Densitometry of autoradiograms. Levels of m3-mAChR mRNA were normalized to values of total cellular RNA in each sample. The data are means + SEM of triplicate samples. The experiment has been repeated three times with identical results.

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FIG. 6. Effects of taxol on nocodazole-induced m3-mAChR mRNA down-regulation in cerebellar granule cells. Cerebellar granule cells after 8 days in culture were pretreated with 10 PM taxol (TAX) for 30 min and then treated with 10 pM nocodazole (NOC) for 8 h. (A) Autoradiograms of mRNA hybridization to cDNAs of m3-mAChR and fl-actin. (B) Densitometry of autoradiograms. The mRNA results have been normalized to total RNA in each sample. The experiment has been repeated three times with virtually identical results.

bulin heterodimers increases. This in turn leads to a specific decrease in both (Y- and fi-tubulin mRNA within 6 h, while actin mRNA species remain unaffected (23). Later events in cerebellar granule cells in culture included the retraction of neurite extensions and detachment from the surface of the culture dish. In view of the role of microtubules in intracellular membrane trafficking and cytoarchitecture (6), the effects of microtubule disruption are likely to be profound. After 8 h of treatment with 10 pM colchicine, however, neurite extensions and the aggregation of granule cells were not significantly affected. At this time there was already a significant decrease in the level of m3-mAChR mRNA, while the level of /3-actin mRNA remained virtually unchanged. This fact argues against the possibility that the effect of microtubule disruption on mAChR mRNA regulation is secondary to changes in morphology and extracellular organization. However, since nocodazole was able to mimic and taxol was able to prevent the effect

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of colchicine on m3-mAChR mRNA levels, this effect must stem from colchicine’s ability to destabilize microtubules. The kinetics of m3-mAChR down-regulation induced by colchicine treatment suggests that mAChR mRNA may be directly regulated by the state of microtubule assembly in some way. This might occur at the level of transcription or by modulation of mRNA stability. The fact that 30 min of pretreatment with taxol could not reverse the effect of nocodazole may stem from the more rapid action and efficacy of nocodazole to cause microtubule disassembly (21, 22). Colchicine was able to enhance carbachol-induced down-regulation of m3-mAChR mRNA; however, the enhanced effect was less than additive. The fact that taxol, a microtubule stabilizer, was able to reverse at least some of carbachol-induced m3-mAChR mRNA down-regulation argues that microtubules may mediate at least some of the effects of both carbachol and colchicine on mAChR mRNA. Numerous effects of colchicine treatment have been reported. These include increasing CAMP levels (24) perhaps via the direct transfer of GTP to G-proteins coupled to adenylate cyclase (9) and the induction of proliferation (1). Neither of these mechanisms, however, can adequately explain the role of microtubule assembly states in regulating m3-mAChR mRNA levels. This mechanism might perhaps be inferred from the function of microtubules to maintain neurite-neurite and cell membranesubstrate contacts. We have observed that, in primary culture of cerebellar granule cells, the level of m3-mAChR mRNA increased in parallel with cell attachment and the formation of interconnecting neurite extensions (15). Colchicine treatment effectively reversed this process, causing retraction of these neurite extensions and cellular detachment. This reversal was accompanied-actually preceeded, as pointed out above-by a loss of m3-mAChR mRNA and mAChR sites. While this relationship remains to be demonstrated, we speculate that cerebellar granule

TABLE

1

Effects of Microtubule-Affecting Agents on the Level of mRNA of m3-mAChR

Drug

used

Colchicine P-Lumicolchicine Nocodazole Taxol Taxol + colchicine Taxol + nocodazole Carbachol Taxol + carbachol

Action on microtubules Disruption Inactive Disruption Stabilization -

Effects mAChR

on m3mRNA levels

Decrease No effect Decrease No effect Complete reversal No reversal Decrease Partial reversal

!G. 7. Photographs of cerebellar granule cell cultures :olchicine for (A) 0 h, (B) 8 h, and (C) 24 b. Magnification

at 8 days in uitro. Cells is X92 for all pictures. 128

dissociated

from

8-day-old

rat cerebella

were

exposed

to 10

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OF

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STRUCTURES

cells are able to regulate the level of mAChR protein expression and transport to functionally relevant membrane areas through the dynamic states of microtubule structures.

12.

13. 14.

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McClain, D. A., and G. M. Edelman (1980). Density-dependent stimulation and inhibition of cell growth by agents that disrupt microtubules. Proc. Natl. Acad. Sci. USA 77: 2148-2752.

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Y., E. Nishida, and H. Sakai (1989). Initiation of DNA synthesis by microtubule disruption in quiescent rat 3Yl cells. Eur. J. Biochem. 183: 275-280.

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Stebbings, H. (1990). How is microtubule-based location regulated? J. Cell Sci. 95: 5-7.

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Edelman, G. M. (1976). Surface modulation cell growth. Science 192: 218-226.

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C. R. (1986). Membrane boundaries involved in the uptake and intracellular processing of cell surface receptors. Trends Biachem. Sci. 11: 473-477.

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Kelly, R. B. (1990). ganization. Cell 61:

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J. L., G. R. Gunther, and G. M. Edelman (1975). by colchicine of the mitogenic stimulation of lymphocytes the S phase. J. Cell Bial. 66: 128-144.

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Baker, M. E. (1976). Colchicine inhibits roblastoma cells that have been arrested

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Rasenick, M. M., and N. Wang (1988). Exchange of guanine nucleotides between tubulin and GTP-binding proteins that regulate adenylate cyclase: cytoskeletal modification of neuronal signal transduction. J. Neurochem. 51: 300-311.

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