Retinoic acid-induced differentiation of a human neuroblastoma cell line alters muscaric receptor expression

305

Del,elopmental Brain Research, 72 (1993) 305-308 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00

BRESD 60487

Retinoic acid-induced differentiation of a human neuroblastoma cell line alters muscaric receptor expression Melissa K. Baumgartner, Jian Wei and Robert S. Aronstam Department of Pharmacolog.v and Toxicology, Medical College of Georgia Augusta, GA 30912-2300 (USA) (Accepted 9 December 1992)

Key words: Muscarinic receptor; Retinoic acid; Differentiation; Sk-N-SH neuroblastoma

Muscarinic receptor density increased by approximately 36% after differentiation induced by retinoic acid (Bin. ~, control = 126_+ 13 fmol/mg protein; B . ~ , retinoic acid-treated = 170+_ 17 fmol/mg protein; P < 0.05), corresponding to a 170% increase in receptor content per cell. The affinity of [3H]NMS for the receptors was somewhat lower in the retinoic acid-treated cells (K d, control =0.14+_0.04 nM; Kd, retinoic acid-treated = 0.25 +-0.04 riM; P < 0.05). Reverse transcriptase/polymerase chain reaction analysis using subtype-specific primers revealed that undifferentiated Sk-N-SH cells transcribed mRNA for all 5 receptor subtypes; this pattern was not affected by retinoic acid treatment. [3H]NMS displacement curves with subtype selective receptor ligands (pirenzepine, ml; AFDX-116, m2; 4-DAMP, m3) indicated the predominant expression of m3 and ml receptor subtypes, and differentiation did not affect the pharmacological profile of the expressed receptor populations. The present results indicate that differentiation induces selective changes in the expression and activity of muscarinic receptors in a neuronal cell line.

Neuroblastoma cell lines have been used as models to study the differences between undifferentiated (or immature) neurons and differentiated (or mature) neurons ''2. Neuroblastoma cells have been isolated from tumors of sympathetic ganglia and adrenal medulla 1~. These tumor cells do not express their full differentiated functions, but can be induced to differentiate by retinoic acid into various forms of a mature neuron ~°''5'~'. Thus, differentiated neuroblastoma cells can be compared to their normal counterparts, developing autonomic neurons. Differentiation with retinoic acid induces changes in neurotransmitter receptor populations and second messenger systems ~2'tv'19'2°. Pharmacological and biochemical studies indicate that the human neuroblastoma cell line Sk-N-SH expresses the m3 and ml subtypes of muscarinic receptor 5"~'1~. The purpose of the present study was to determine the effects of retinoic acid-induced differentiation on muscarinic receptor populations in Sk-N-SH cells. It is demonstrated that retinoic acid-induced differentiation of Sk-N-SH neuroblastoma cells (11 increases the size of the muscarinic receptor population (Bma x) while decreasing

[3H]NMS binding affinity, and (2) does not alter muscarinic receptor pharmacology or expression of muscarinic receptor subtypes. Sk-N-SH cells grown in Dulbecco's modified Eagle's medium supplemented with nonessential amino acids, 10% fetal bovine serum (Biocell, Rancho Dominguez, CA), 1 mM sodium pyruvate, streptomycin (100 ~ g / m l ) , and penicillin (100 I U / m l ) were induced to differentiate with 1 /.tM retinoic acid for 1 week. Differentiation was characterized by an increase in neurite formation and inhibited cell growth (not shown). The amount of protein in each neuroblastoma cell increases after differentiation ~3. In the present study, the total amount of protein per cell was assessed by counting the number of cells after 7 days of growth in a 75 cm 2 flask with a hemocytometer. Protein content was determined using the method of Lowry et al.'( The total protein content of the differentiated cells (1241 _+ 239 pg/cell) was approximately double that of the undifferentiated cells (676-741 pg/cell) (Table 1). For the muscarinic receptor binding assays, cells were harvested in a buffer containing 50 mM Tris-HC1, 2 mM MgC12, and 1 mM dithiothreito[ (DTT), pH 7.4.

Corresponden¢~." M.K. Baumgartner, Department of Pharmacology and Toxicology. Medical College of Georgia, Augusta. Georgia 30912-2300. USA. Fax: (1) (706) 721 2347.

306 TABLE I

The influence of retinoic acid-induced differentiation on protein content, / ~ H / N M S bindhzg parameters" (Bin, ~ and K,t ). and rec~7~tor pharma~'ologv m muscarinic receptors in Sk-N-SH cells Group

Control 0.1% Ethanol 1 p,M Retinoic acid

pg protein / cell

Bm~× (fmol / m g membrane protein)

741 ± 121 (n = 6) 676 ± 107 ( n = 5) 1241 +239 * (n = 4)

125 + 13 (n = 10) 123 .+ t 1 (n = 8) 170-+17 * (n = 11)

B .... (receptors / cell) 37,267 36,058 100,256 *

K,/ (nM)

0.14 ± 0.04 (n = 10) 0.15 ± 0.02 (n = 8) 0.25±0.04 * (n = 11)

IC5o (tz M) 4-DA MP

I('~ o (la M) Pirenzepine

0.03 -+ 0.01 (n = 4) 0.04 _+0.01 (n = 4) 0.02_+0.01 (n = 4)

0.50 _+0.12 (n = 4) 0.80 ± 0.09 (n = 4) 0.63±0.09 (n = 4)

1( '~ (~ M) ,4 FI)X- I I (~

6.50 -+ 0.811 (n = 2) ND 8.00±0.5(t (n = 2)

ND = not determined * Parameter is significantly different ( P < 0.05) from control and 0.1% ethanol group values as assessed by analysis of variance and Fisher PLSD tests. Means _+S.E.M. for the indicated number of independent experiments. Membrane-associated protein represented 67. 72 and 79% of total cellular protein in control, 0.1% ethanol-treated, and 1 izM retinoic acid-treated cells, respectively. Binding data were analyzed by nonlinear regression fit to a model incorporating a single population of non-interacting binding sites, as follows: B = (Bma x × C ) / ( C + Ko), where B is binding ( m o l / u n i t membrane protein), Bmax is the density of binding sites, C is the concentration of [3H]NMS, and K d is the equilibrium dissociation constant.

The pellet was homogenized in fresh buffer using a Teflon-glass tissue grinder, and the homogenate was spun at 17,000 rpm for 20 min at 4°C. The pellet was resuspended in the original buffer and used without further treatment. [ 3H]N-methylscopolamine ( [ N - m e t h y l - 3 H ] s c o p o l a mine methyl chloride; [3H]NMS; 81.5 Ci/mmol, NENDuPont, Boston, MA) binding to muscarinic receptors was measured in a medium containing 0.032 to 3.2 nM [3H]NMS, 50 mM Tris-HCl (pH 7.4), 2 mM MgCI 2 and 1 mM DTT. After incubation for 1 h at room temperature, the reaction was terminated by filtration through Whatman G F / B glass fiber filters. The tubes and filters were washed twice with 5 ml of cold (~ 4°C) 50 mM Tris-HC1, pH 7.4, and the radioactivity content of the filters was determined by liquid scintillation counting. Nonspecific binding (5-35% of total binding, depending on [3H]NMS concentration) was determined in the presence of 100 ~M (-)scopolamine methyl bromide. In some assays, the ability of subtype-specific muscarinic receptor antagonists (pirenzepine; ml; (Sigma), AFDX-116; m2; (Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT), 4-DAMP; m3; (Research Biochemicals, Inc., Natick, MA)) to inhibit the specific binding of 1.0 nM [3H]NMS was determined, Specific [3H]N-methylscopolamine ([3H]NMS) binding to membranes isolated from undifferentiated Sk-NSH cells was saturable with an apparent K d of 0.14 nM (control) or 0.15 nM (0.1% ethanol, the vehicle for retinoic acid) and a Bma x of 125 + 13 fmol/mg protein (control) or 123 _+ 11 fmol/mg protein (0.1% ethanol) (Fig, 1; Table I). Differentiation with retinoic acid induced a significant (P < 0.05) increase in Omax (170 + 17 fmol/mg membrane protein) and in the apparent

K d (0.25 nM) (Fig. 1; Table I). The reasons for this selective decrease in antagonist affinity are not obvious. Retinoic acid-induced differentiation is associated with an increase in vasoactive intestinal peptide (VIP) and mu opioid receptor populations in SH-SY5Y cells, a subclone of the Sk-N-SH neuroblastoma cells t4Ag. Recently, it has been shown in the LA-N-1 human neuroblastoma cell line that differentiation with retinoic acid increases muscarinic receptor expression 17. Our findings are consistent with these studies in that differentiation of Sk-N-SH cells was found to engender 200

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Fig. 1. The effects of differentiation induced by retinoic acid on [3H]NMS binding to muscarinic receptors in cultured Sk-N-SH neuroblastoma cells. Cells were grown for 7 days in the absence (control) of retinoic acid or in the presence of 1 p.M retinoic acid. One group of control cells was grown in the presence of 0.1% ethanol, the vehicle for retinoic acid. Ethanol, at the concentration present in the vehicle (0.1%), did not affect cell morphology or cell growth. The retinoic acid-treated group was significantly different from the control and ethanol-treated groups ( P < 0.05). All binding experiments were performed in duplicate, and data are presented as means.+ S.E.M. for 8-11 experiments. Lines are drawn according to nonlinear regression analysis which revealed the binding parameters listed in Table I.

3O7 a 36% increase in the muscarinic receptor density, corresponding to a 170% increase in the receptor content of each cell. Displacement of 1 nM [3H]NMS binding by subtype-selective antagonists (pirenzepine, m l; AFDX-116, m2; 4-DAMP, m3) was consistent with the prominent expression of m3 a n d / o r m l subtypes (Table I). In terms of receptor affinity, the following relationship was obtained: 4-DAMP (ICs0 = 0.03 p.M) > pirenzepine (ICs0 = 0.50 # M ) > AFDX-116 (IC 5 = 6.50 /xM). The selectivity of the expressed receptor populations for the subtype-selective antagonists was not altered after retinoic acid-induced differentiation (Table I). Genes encoding 5 distinct muscarinic receptor subtypes (m l - m 5 ) have been identified 4'~ L. These subtypes differ in their primary structure, ligand binding properties, interactions with G proteins, control of effeetor mechanisms, and expression in different tissues and brain regions. These subtypes can account for much of the pharmacological and biochemical heterogeneity of muscarinic receptors noted in earlier studies 3'<7. Pharmacological studies have suggested that Sk-N-SH cells express mainly m3 and ml muscarinic receptor subtypes 5a. In light of the increased muscarinic receptor population after differentiation, the question arose as to whether the increase involved a change in the pattern of subtype expression. To determine the species of muscarinic receptor m R N A transcribed by the cultured cells, mRNA was isolated from the undifferentiated and differentiated cells using a FastTrack kit (Invitrogem San Diego, CA). The isolated m R N A was used to direct the synthesis of eDNA by reverse transcriptase, and amplified by a qualitative polymerase chain reaction ( G e n e A M P RNA PCR Kit; Perkin-Elmer, Norwalk, CT) using subtype specific primers. The amplified products were analyzed by gel electrophorcsis (1.8% agarose) and visualized by staining with ethidium bromide. Somewhat unexpectedly, all five receptor subtypes were expressed in both control and differentiated cells (Fig. 2). Thus, differentiation of Sk-N-SH cells with retinoic acid results in an increase in muscarinic receptor density, whether considered on a wet weight, membrane protein, or unit cell basis. Cell protein in the particulate membrane fraction increased from 67 to 79% of total cellular protein after differentiation, probably reflecting the increase in ncurite outgrowth. Receptor pharmacology and pattern of receptor subtype expression are not affected by differentiation. [3bl]NMS displacement curves with specific antagonists to m l (pirenzepine), m2 (AFDX-116), and m3 (4-DAMP) receptors did not reveal any change in receptor phar-

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I000 700 5O0 400 3OO 200 Fig. 2. Expression of muscarinic receptor mRNA in Sk-N-SH cells. Reverse transeriptase/PCR was used to amplify, signals from iso lated mRNA. The sizes (bp) of the standard nucleic acid fragments in the first lane are indicated on the left. For each set of subtypespecific primers, mRNA was run in lhc presence ~ + } and absence ( ) of reverse transcriptase. The blank lanes in the absence of reverse transcriptase attest to the absence of contamination from cellular DNA or amplified product. The mRNA was isolated from control (A) or retinoic acid-differentiated (I3) cells. Primers w e r e synthesized using a Model 392 nucleotidc synthesizer (Applied Biosystcms; Foster City, CA). Primer sequences, corresponding base sites (coding initiation site I), size of lhe PCR producl, and sequence number (Genbank) are indicated below. ml: 5 ' - G A A G A A G A G G A A ( i A G G A C G A A 3' upper, bases $2(~847 5 ' - C A G G A G A G G G G A ( ' T A T ( ' A ( ~ ( ' A - 3 ' h~wcr, bases 137S 1399 PCR product: 573 bp; sequence numbcr X15263 m2: 5'-GGGTCCTCTCTTT('ATCCTCT 3', upper, bases 443-464 5'-TCCTGGGTTATTTCATCATCT-3', lower, bases S01 t~l 2 PCR product: 469 bp: sequence number Xl52~4 m3: 5 ' - A G C ( ' A A A C G A A C A A C A A A ( ; A G - 3 ' , uppel, bases 531

552 5'-TTGAAGGACAGAGGTAGA(VIG-3', h}wer, bases 13561377 PCR product: 846 bp; sequencc number XI32{m m4: 5 ' - C G C T A T G A G A C G G T G G A A A T G 3', upper, bases, 76 98 5'-CGTCTTGGCTTTCTTCTCCTT-3', lower, bases 7{}3 724 PCR product: 648 bp: sequencc number X152{'~5 m5: 5 ' - G G A A A C A G A G A A G C G A A C C A A - 3 ' . upper, bases {}54 675 5 ' - A G C A C A A ( ' C A A T A G ( ' C C A A G T - 3 ' , lower, bases 1433 1454 PCR product: 80(I bp; scquence number MNII333

macology upon differentiation, suggesting that m3 and ml remained the predominant receptor specics. It is interesting to note, however, that mRNA for all five

308 subtypes was detected by reverse transcriptase/PCR in both undifferentiated and differentiated cells. Thus, while SK-N-SH cells express mainly m3 and m l muscarinic receptors, m2, m4, and m5 muscarinic receptor subtypes may also be expressed in at least small quantities. The increase in Bmax induced by differentiation probably reflects a coordinated increase in the expression of both m3 and ml. Acknowledgement. The authors are grateful for the expert technical assistance of Latha Narayanan and the gift of Sk-N-SH cells from Dr. Stephen K. Fisher. This work was supported by GM-46408 and the Medical Research Service of the Department of Veterans Affairs. 1 Pahlman, S., Mamaeva, S., Meyerson, G., Mattsson, M.E.K., Bjelfman, C., Ortoft, E. and Hammerling, U., Human neuroblastoma cells in culture: a model for neuronal cell differentiation and function, Acta Physiol. Scand. Suppl., 592 (1991) 24-37. 2 Abemayor, E. and Sidell, N., Human neuroblastoma cell lines as models for the in vitro study of neoplastic and neuronal cell differentiation, Environ. Health Perspect., 80 (1989) 3-15. 3 Birdsall, N.J.M. Hulme, E.C. and Burgern, A.S.V., The character of the muscarinic receptors in different regions of the rat brain, Proc. R. Soc. B, 207 (19801 1-12. 4 Bonnet, T.I., Buckley, N.J., Young, A.C. and Brann, M.R., Identification of a family of muscarinic acetyleboline receptor genes, Science, 237 (1987) 527-532. 5 Fisher, S.K. and Heacock, A.M., A putative m3 muscarinic cholinergic receptor of high molecular weight couples to phosphoinositide hydrolysis in human SK-N-SH neuroblastoma cells, J. Neurochem.., 50 (1988) 984-987. 6 Hulme, E.C., Birdsall, NJ.M. and Buckley, N.J., Muscarinic receptor subtypes, Annu. Ret'. Pharmacol. Toxicol., 30 (1990) 633-673. 7 lkeda, S.R., Aronstam, R.S. and EIdefrawi, M.E., Nature of regional and chemically induced differences in the binding properties of muscarinic acetylcholine receptors from rat brain, Neuropharmacology, 19 (1980) 575-585. 8 Lambert, D.G., Ghataorre, A.S. and Nahorski, S.R., Muscarinic receptor binding characteristics of a human neuroblastoma SK-

N-SH and its clones SH-SY5Y and SH-EPt, t:ul..L Pharmacol., 165 (19891 7/-77. tl Lowry, O.tt., Rosebrough, N.J., Farr, A.L. and Randell, R.J., Protein measurement with the Folin phenol reagent .,~ Biol. Chem., 193 (195l) 265-275. 10 Lotan, R., Lotan, D. and Sacks, P.G., Inhibition of tumor cell growth by retinoids, Methods Enzymol., 190 (19901 100-1111. II Peralta, E.G., Ashkenazi, A., Winslow, J.W., Smith, D.H., Ramachandran, J. and Capon, D.J., Distinct primary structures, ligand-binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors, EMBO Z, 6 (1987) 3923-3920. 12 Ponzoni, M. and Lanciotti, M., Retinoic acid rapidly decreases phosphatidylinositol turnover during neuroblastoma cell differentiation, J. Neurochem., 54 (1990) 540-546. 13 Prasad, K,N,, Differentiation of neuroblastoma cells in culture, Biol. Rez,., 50 (19751 129-265. 14 Preis, P.N., Saya, H., Nadasdi, L., Hochhaus, G., Levin, V. and Sadee, W., Neuronal cell differentiation of human neuroblastoma cells by retinoic acid plus herbimycin A, Cancer Res., 48 (1988) 6530-6534. 15 Sidell, N., Altman, A., Haussler, M.R. and Seeger, R.C., Effects of retinoic acid (RA) on the growth and phenotypic expression of several human neuroblastoma cell lines, Exp. Cell Res., 148 (19831 21-30. 16 Sidell, N., Sarafian, T., Kelly, M., Tsuchida, T. and Haussler, M., Retinoic acid-induced differentiation of human neuroblastoma: A cell variant system showing two distinct responses, ExpL Cell Biol., 54 (1986) 287-300. 17 Sorrentino, G., Singh, I.N., Hubscb, A., Kanfer, J.N., Mykita, S. and Massarelli, R., Muscarinic binding sites in a catecholaminergic human neuroblastoma cell line, Neurochem. Res., 17 (t992) 215-222. 18 Wall, S.J., Yasuda, R.P., Li, M. and Wolfe, B.B~, Development of an antiserum against m3 muscarinic receptors: distribution of m3 receptors in rat tissues and clonal cell lines, Mol. Pharmacol., 40 (19911 783-789. 19 Waschek, J.A., Muller, J.-M., Duan, D.-S. and Sadee, W., Retinoic acid enhances VIP receptor expression and responsiveness in human neuroblastoma cell, SH-SY5Y, FEBS Letc, 250 (19891 611-614. 20 Yu, V.C., Hochhaus, G., Chang, F., Richards, M.L., Sadee, W., Differentiation of human neuroblastoma cells: marked potentiation of prostaglandin E-stimulated accumulation Of cyclic AMP by retinoic acid, J. Neurochen., 51 (19881 1892-1899.