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J. Biol. Chem., Vol. 278, Issue 1, 672-678, January 3, 2003

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₁-Adrenergic Receptor Subtypes Differentially Control the Cell Cycle of Transfected CHO Cells through a cAMP-dependent Mechanism Involving p27^Kip1*

Katsushi Shibata, Susumu Katsuma, Takaaki Koshimizu, Hitomi Shinoura, Akira Hirasawa, Akito Tanoue, and Gozoh Tsujimoto

From the Department of Molecular and Cell Pharmacology, National Center for Child Health and Development Research Institute, 3-35-31 Taishido, Setagaya-Ku, Tokyo 154-8567, Japan

Received for publication, February 11, 2002, and in revised form, October 15, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Three distinct subtypes of ₁-adrenergic receptors (₁A-, ₁B-, and ₁D-AR) play a prominent role in cell growth. However, little is known about subtype-specific effects on cell proliferation. The activation of ₁A- or ₁B-AR inhibits serum-promoted cell proliferation, whereas ₁D-AR activation does not show such an inhibitory effect. Notably, cell-cycle progression was blocked at G₁/S transition after activation of ₁A/₁B-AR but not of ₁D-AR. In agreement with the differential cell proliferation effect, cAMP production was increased after activation of ₁A/₁B-AR but not ₁D-AR, whereas all ₁-AR subtypes are associated with inositol 1,4,5-trisphosphate production and mitogen-activated protein kinase activation in a similar fashion. Furthermore, the serum-induced reduction in the levels of the cyclin-dependent kinase inhibitor, p27^Kip1, was blocked after activation of ₁A/₁B-AR but not ₁D-AR. These results show that ₁-AR subtypes differentially activate the cAMP/p27^Kip1 pathway and thereby have differential inhibitory effects on cell proliferation. Subtype-dependent effects should be taken into consideration when assessing the physiological response of native cells where ₁-AR subtypes are generally co-expressed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cellular responses to a variety of external stimuli alter the proliferation or differentiation status via receptor-mediated intracellular signaling cascades. Plasma membrane receptors coupled to heterotrimeric G proteins (G protein-coupled receptor) a protein family with ~1,000 members, mediate a large variety of important physiological actions induced by hormones, neurotransmitters, and sensory signals. ₁-Adrenergic receptors (₁-ARs)¹ are members of the superfamily of G protein-coupled receptor and mediate the effects of the sympathetic nervous system, especially those effects related to the regulation of cellular hypertrophy and proliferation (1-3). There is substantial evidence indicating that stimulation of ₁-ARs by catecholamines generally enhances growth-related gene expression and cell growth in a variety of cells including cardiac myocytes, vascular smooth muscle cells, hepatocytes, and also adipocytes (4-6).

The ₁-ARs initiate their physiological effects by activating phospholipase C at the cell membrane, resulting in production of inositol 1,4,5-trisphosphate. Inositol 1,4,5-trisphosphate, in turn, mobilizes intracellular calcium and diacylglycerol, which activates protein kinase C (7, 8). More recently, the ₁-ARs were found to activate a variety of other effectors such as the mitogen-activated protein kinase (MAPK) pathway, cAMP metabolism, phospholipase D, and A₂ in various cells (9-14). Both molecular and pharmacological studies have shown that ₁-ARs consist of three subtypes: ₁A-, ₁B-, and ₁D-AR (15-19). The three different ₁-AR subtypes are expressed in different tissues and various cell types. Therefore, studies on the physiological effects mediated by ₁-ARs in individual tissues are complicated by the co-existence of multiple ₁-AR subtypes (20). Although the pharmacological properties of each subtype are well documented (21-23), little is known regarding the physiological role of each receptor subtype at a cellular level and in the regulation of cell proliferation. In this study, we explored the role of each ₁-AR subtype in cell proliferation using a heterologous expression system. Our results show that these highly homologous receptor subtypes have distinct signal transduction coupling properties and have differential effects on cell proliferation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Phentolamine hydrochloride was from Ciba Pharmaceutical Corp. (Summit, NJ). Phenylephrine was from Sigma. Ham's F-12 medium, Lipofectin, and G418 (Geneticin disulfate) were from Invitrogen. Fura-2/acetoxymethyl ester (Fura-2/AM) was from Dojindo (Kumamoto, Japan). Triton X-100 (polyoxyethylene (10) octhylphenyl ether) was from Wako Pure Chemical Industries (Osaka, Japan). MAPK substrate peptide and Whatman P81 phosphocellulose discs were from Invitrogen. RpcAMP (Rp-isomer cyclic AMP) and dibutyrl cyclic AMP were from Sigma. Anti-p27^Kip1 polyclonal antibody (sc-776) was from Santa Cruz Biotechnologies (Santa Cruz, CA). All other chemicals were of reagent grade. The CHO-K1 cell line was obtained from American Type Culture Collection (Rockville, MD).

Transfections and Culture Conditions-- Genes encoding the human ₁A-, ₁B-, or 1D-AR were ligated into the EcoRI site of the eukaryotic expression vector, pSVK3, containing the neomycin-resistance gene of pMAM-neo (pSVK3neo) as described previously (23). Wild-type CHO-K1 cells were grown in Ham's F-12 medium containing L-glutamine supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in 5% CO₂ in an air-ventilated humidified incubator. Cells were passaged using trypsin. For stable expression of human ₁A-, ₁B-, or ₁D-AR subtype, CHO-K1 cells were seeded at a density of 2 × 10⁴ cells in 35-mm tissue culture dishes. One day after seeding the cells, the medium was removed, and 1 ml of serum-free F12 medium containing 13.8 µg of Lipofectin (24) and 9.2 µg of the recombinant expression plasmid was added to the cells. At 24 h after DNA-lipid treatment, 1 ml of F12 medium containing 20% fetal bovine serum was added. The cells were passaged at a low density ~72 h later. Single colonies resistant to the antibiotic G418 (600 µg/ml) were isolated and maintained in F12 medium with 10% fetal bovine serum and 200 µg/ml G418. Three clonal CHO cell lines for each ₁-AR subtype (₁A-, ₁B-, or ₁D-AR), which stably express the ₁-AR at similar level, were established and used for subsequent studies. Specifically, one line of CHO stable transfectants was designated as CHO1A, CHO1B, and CHO1D.

The ligand-binding characteristics of the receptors expressed in CHO cells were determined in a preliminary series of radioligand-binding studies performed as described previously (23) using [¹²⁵I]HEAT (specific activity 2,200 Ci/mmol, PerkinElmer Life Science) as the radioligand for ₁-AR. Membrane preparations from CHO cells stably expressing the cloned human ₁-ARs showed saturable binding of [¹²⁵I]HEAT; B_max values were 1.3 ± 0.2, 5.5 ± 0.1, and 1.1 ± 0.1 pmol/mg protein with K_d values of 110 ± 21, 60 ± 1, and 300 ± 26 pM for the CHO1A, CHO1B, and CHO1D (n = 3 each), respectively. Competition isotherms showed the expected pharmacology for each receptor subtype (23).

[³H]Thymidine Incorporation-- We employed the method of Kato et al. (25). Confluent cells grown in 96-well plates were rendered quiescent by serum deprivation for 24 h. The cells were then incubated with 10% serum with or without 10 µM phenylephrine for 18 h and for an additional 6 h with [6-³H]thymidine (1 µCi/ml, PerkinElmer Life Sciences). The cells were rinsed twice with ice-cold phosphate-buffered saline, precipitated with cold 5% trichloroacetic acid, and then washed with cold ethanol and solubilized with 0.5 N NaOH. DNA radioactivity was measured by scintillation counting.

Cell Cycle Analysis by Flow Cytometry-- Cells at 50% confluence in 35-mm culture dishes were rendered quiescent by serum deprivation for 24 h. The cells were then incubated with 10% serum with or without 10 µM phenylephrine for 48 h. Flow cytometric analysis of each CHO cell stained with propidium iodide was used to estimate the cell cycle phase distributions using the CycleTEST^TM Plus DNA Regent Kit (BD Biosciences). Subsequent flow-cytometric analysis was performed with a FACScan flow cytometer (BD Biosciences). Data were analyzed using the CellFIT software (BD Biosciences).

Measurement of Inositol 1,4,5-Trisphosphate-- Cells at 50% confluence in 35-mm culture dishes were rendered quiescent by serum deprivation for 24 h and treated with phenylephrine (10 µM) for 10 s at 37 °C. The medium was subsequently removed, and cells were scraped into 1 ml of 20% (w/v) perchloric acid on ice. After centrifugation, the supernatant was adjusted to pH 7.0 with HEPES-KOH solution, and the sediment was eliminated by centrifugation. Amounts of inositol 1,4,5-trisphosphate in a sample were measured by a radioreceptor assay with the D-myo-inositol 1,4,5-triphosphate [³H] assay kit (TRK 1000, Amersham Biosciences).

MAPK Assay-- Cells were rendered quiescent by serum deprivation for 24 h before stimulation with 10% serum in the presence or absence of phenylephrine for 30 min at 37 °C. Cell monolayers were washed in phosphate-buffered saline, pH 7.4, and lysed, and the overall activity of ERK1/2 MAPK in the lysate was determined using the Biotrak^TM MAPK assay (Amersham Biosciences) in accordance with the manufacturer's instructions. Aliquots of 15 µl of cell lysates freshly obtained or stored at 80 °C corresponding to 10 µg of protein were incubated at 30 °C with 15 µl of a reaction mixture containing a synthetic specific peptide substrate and [-³²P]ATP (1 µCi; 1.2 mM, PerkinElmer Life Sciences). After 30 min, the reaction was stopped and samples were spotted on 3-cm discs of binding paper. After several washes in 0.75% phosphoric acid and water, the radioactivity on each disc was then determined by scintillation counting.

Measurements of cAMP Production-- cAMP production in intact transfected CHO cells was determined as described previously (26). Each transfected CHO cell population was seeded in 6-well plates at a density of 5 × 10⁵ cells/well and cultured for 12-16 h. The cells were washed twice with phosphate-buffered saline, incubated for 30 min in HEPES buffer, and then the medium was replaced with the fresh HEPES buffer containing 1 mM isobutylmethylxanthine for 10 min. The reaction was started by adding phenylephrine (10 µM) or forskolin (10 µM). After incubation for 10 min, the medium was aspirated and the reaction was stopped with 6% (w/v) trichloroacetic acid. cAMP levels were determined by radioimmunoassay (Yamasa cAMP Assay Kit, Yamasa Shoyu Co., Chiba, Japan). Phentolamine was added 10 min prior to agonist stimulation in HEPES buffer containing 1 mM isobutylmethylxanthine and 1 µM propanolol.

Adenylyl Cyclase Assay-- Each transfected CHO cell population was cultured in 15-cm dishes, suspended in lysis buffer (62.5 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, 5 µg/ml pepstatin A, and 1 µg/ml leupeptin), and homogenized using the Cell Disruption Bomb (Parr Instrument Co., Moline, IL) at 400 p.s.i. for 30 min. The homogenate was centrifuged at 35,000 × g for 20 min at 4 °C, and the pellet was resuspended in 10 mM Tris-HCl, pH 7.4, containing 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, 5 µg/ml pepstatin A, and 1 µg/ml leupeptin and then used as membrane preparations. The standard assay mixture contained 20 µg of membrane proteins in 100 µl of 50 mM HEPES, pH 7.4, 1 mM EDTA, 5 mM MgCl₂, 1 mM isobutylmethylxanthine, 0.5 mM ATP, 1 mM dithiothreitol, 1 µM guanosine 5'-3-O-(thio)triphosphate or guanosine 5'-O-(3-thiotriphosphate), and 1 µM propanolol.

Reactions were started by the addition of the agonists, carried out for 15 min at 37 °C, and terminated by boiling at 90 s. The cAMP levels were determined using a cAMP radioimmunoassay kit (Yamasa Shoyu Co.).

Immnoblotting with Anti-p27^Kip1-- Following the experimental treatments, cells were washed with phosphate-buffered saline and lysed with 1% Nonidet P-40 in a buffer consisting of 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 10% glycerol, 0.4 mM sodium orthovanadate, 10 mM EDTA, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 2.5 µg/ml each of leupeptin and aprotinin). After 30 min on ice, the cell extracts were clarified by centrifugation for 10 min at 14,500 × g at 4 °C. The p27^Kip1 quantitation was adjusted for protein load, and equal amount of the lysates (10 µg of protein) were mixed with 1× Laemmli sample buffer, boiled for 5 min, and resolved by SDS-polyacrylamide gel electrophoresis on a 12% gel. The proteins were electrophoretically transferred to polyvinylidine fluoride membranes as recommended by the manufacturer. The blots were first incubated with blocking buffer (l% bovine serum albumin in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) and probed with rabbit polyclonal anti-p27^Kip1 antibody (sc-776). The immune complexes were detected using the ECL reagents from Amersham Biosciences and quantitated by densitometry using NIH Image software. The protein concentration was measured using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL).

Statistics-- Values are expressed as the mean ± S.E. Two-way analysis of variance with 95 or 99% confidence limits followed by a Student's t test on individual sets of data was performed with the analytical software StatView 4.0 (SAS Institute Inc., Cary, NC).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activation of ₁A-AR or ₁B-AR but Not ₁D-AR Inhibits Serum-stimulated Cell Proliferation-- The cell proliferation of CHO cells stably expressing each ₁-AR subtype (CHO1A, CHO1B, and CHO1D) was measured by [³H]thymidine incorporation after serum deprivation. Cells were then exposed to serum in either the presence or the absence of ₁-AR stimulation. The addition of 10% serum to serum-deprived cells markedly stimulated DNA synthesis in all CHO cell lines expressing any of the ₁-AR subtypes. The activation of ₁-AR by phenylephrine (10 µM) inhibited the serum-promoted [³H]thymidine incorporation in CHO1A and CHO1B cells, whereas such an inhibitory effect was not observed when treating CHO1D cells with phenylephrine (Fig. 1). [³H]Thymidine incorporation experiments were also performed for additional two clonal CHO stable transfectants for each ₁-AR (₁A-, ₁B-, or ₁D-AR), and we observed similar subtype-specific anti-proliferative effects (data not shown). Thus, the results clearly showed ₁-AR subtype-dependent differential effects on the cell proliferation.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Effect of 1-AR subtype activation on serum-stimulated cell proliferation. [3H]Thymidine incorporation was measured in each CHO cell line stably expressing 1A-, 1B-, or 1D-AR cDNA following growth in serum-free medium (Basal) or in serum-enriched medium alone (Serum) or serum with phenylephrine (Serum/PE) or serum with phenylephrine/phentolamine (Serum/PE/Phent). The results are expressed as the mean ± S.E. of four different experiments. **, p < 0.01 versus serum alone.

Activation of ₁A- or ₁B-AR but not ₁D-AR Inhibits Serum-promoted Cell Cycle Progression-- To explore the mechanism underlying the ₁-AR subtype-dependent differential effects on DNA synthesis and morphological alterations, the effects of ₁-AR activation on the cell cycle progression were examined by flow cytometric analysis in each of the CHO cell lines. Three distinct cell populations corresponding to the G₁, S, and G₂/M phases of the cell cycle were readily observed in serum-deprived cells. In addition, there were no significant differences among the cell cycle profiles of CHO1A, CHO1B, and CHO1D cells (Fig. 2). Treatment with serum resulted in a 2- to 3-fold increase in the cell population in S phase with a concomitant reduction in the cell population in G₁ in all CHO cell groups, indicating entry of a significant number of cells into S phase. The addition of 10 µM phenylephrine to serum, however, markedly and reproducibly blocked the cell cycle progression in CHO1A and CHO1B. In contrast, the addition of 10 µM phenylephrine to CHO1D cells failed to block the serum-induced proliferation (Fig. 2). These results indicate that ₁-AR activation has an inhibitory effect on serum-promoted cell cycle progression in CHO1A and CHO1B cells, whereas stimulation of CHO1D may have been mildly growth promoting. The phenylephrine-stimulated cell cycle blockade observed in CHO1A and CHO1B was reversed by treatment with the ₁-AR antagonist, phentolamine, at 10 µM (Fig. 2).

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Effect of 1-AR subtype activation on cell cycle progression as analyzed by flow cytometry. Cells rendered quiescent by serum deprivation for 24 h were treated with 10% serum in the presence or absence of 10 µM phenylephrine for 48 h. Furthermore, the effect of direct stimulation of Gs protein by cholera toxin on cell proliferation was examined in each 1-AR-expressing CHO cell (bottom). Cells were then collected by trypsinization, fixed, and stained with propidium iodide. Subsequent flow-cytometric analysis was performed with a FACScan flow cytometer. Data were analyzed using the CellFIT software. Histograms show relative DNA content (x axis) and relative cell number (y axis). Flow-cytometric analysis was performed on six independent pools of CHO cells in each treatment group, and the results of the cell cycle study are presented as percent of cells in the indicated cell cycle stage. PE, phenylephrine; Phent, phentolamine; CTX, cholera toxin.

₁-AR Subtypes Similarly Stimulate Inositol 1,4,5-Trisphosphate Production and Serum-stimulated MAPK Activation but Differentially Induce cAMP Production-- We postulated that participation of different intracellular signaling transduction pathways might explain the differential regulation of cell proliferation by ₁-AR subtype activation. To evaluate this possibility, we first examined the production of inositol 1,4,5-trisphosphate after ₁-AR activation. ₁-AR-activated production of inositol 1,4,5-trisphosphate was observed in all ₁-AR-expressing CHO cells (Fig. 3A) but not in untransfected CHO-K1 cells (data not shown). When MAPK activity (ERK isoforms) was measured, ₁-AR activation had no discernible effect on serum-stimulated MAPK activity in all CHO cells stably expressing each ₁-AR subtype (Fig. 3B). These results indicated that these early events caused after receptor stimulation were not different upon activation of different ₁-AR subtypes.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Effect of 1-AR subtype activation on intracellular signal transduction pathways. A, effect of 1-AR subtype activation on inositol 1,4,5-triphosphate production. B, effect of 1-AR subtype activation on the serum-promoted MAPK activation. C, effect of 1-AR subtype activation on intracellular cAMP production. D, effect of 1-AR subtype activation on adenylyl cyclase activity in CHO cells stably expressing each 1-AR subtype. In all experiments, phentolamine (10 µM) treatment was started 30 min prior to the phenylephrine stimulation. Values are the mean ± S.E. of at least three different experiments, each performed in duplicate. **, p < 0.01 versus basal. IP3, inositol 1,4,5-triphosphate; PE, phenylephrine; Phent, phentolamine; FSK, forskolin.

We next decided to examine the ability of ₁-AR activation to induce production of cAMP in CHO1A, CHO1B, and CHO1D cells. As shown in Fig. 3C, ₁-AR stimulation resulted in marked cAMP production in CHO1A and CHO1B but not in CHO1D. This was also found to be true for additional two clonal CHO stable transfectants for each ₁-AR (₁A-, ₁B-, or ₁D-AR) (data not shown). On the other hand, an adenylyl cyclase activator, forskolin (5 µM), elicited an ~10- to 25-fold increase in cAMP production above basal levels in all ₁-AR-expressing CHO cells (Fig. 3C) as well as in untransfected CHO-K1 cells (data not shown). Furthermore, we examined the effects of ₁-AR activation on the adenylyl cyclase activity of membranes from each transfected CHO cell line. ₁-AR activation increased the adenylyl cyclase activity in the CHO1A and CHO1B membranes but not in the CHO1D (Fig. 3D). The GTP analog, GTPS (10 µM), elicited an ~2- to 4-fold increase in adenylyl cyclase activity above basal levels in all CHO1A, CHO1B, and CHO1D cells. ₁-AR-mediated cAMP production and adenylyl cyclase activation in CHO1A and CHO1B were abolished by the ₁-AR antagonist, phentolamine (10 µM). The ₁-AR-induced cAMP response was not observed in wild-type CHO-K1 cells (data not shown). Hence, the above data show that stimulation of ₁A- and ₁B-AR but not 1D-AR promotes cAMP synthesis in part by activating adenylate cyclase.

cAMP Suppresses Cell Proliferation in Transfected CHO Cells-- We next examined the correlation between cAMP signaling pathway and ₁-AR-mediated anti-proliferative effects as observed in CHO1A and CHO1B. cAMP-inducing agents, cholera toxin, forskolin, and dibutyrl cyclic AMP, all inhibited serum-promoted [³H]thymidine incorporation to 30~40% of control levels in all three transfected CHO cells (Fig. 4), suggesting that cAMP signaling is an important anti-mitogenic signal in CHO cells. On the other hand, treatment with RpcAMP, an antagonist of cAMP, significantly blocked ₁-AR-mediated inhibitory effects on cell proliferation in CHO1A and CHO1B (Fig. 4). A series of experiments using cAMP modulators were also performed for additional two clonal CHO stable transfectants for each ₁-AR (₁A-, ₁B-, or ₁D-AR), and we observed basically similar subtype-dependent behavior (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Effect of various agents that modulate cAMP pathway on serum-stimulated cell proliferation. [3H]Thymidine incorporation was measured in each CHO cell line stably expressing 1A-, 1B-, or 1D-AR cDNA. The results are expressed as the mean ± S.E. of four different experiments. ++, p < 0.01 versus serum free. **p < 0.01 versus serum alone. PE, phenylephrine; Phent, phentolamine; CTX, cholera toxin; FSK, forskolin.

Enhanced cAMP Production Inhibits the Serum-promoted Cell Cycle-- We next examined whether ₁-AR-mediated cAMP production may have an inhibitory effect on the cell cycle progression of CHO1A and CHO1B cells. The effect of direct stimulation of Gs protein by cholera toxin on cell proliferation was examined in each ₁-AR-expressing CHO cell. As shown in Fig. 2, bottom, the addition of 10 µM cholera toxin resulted in a decreased cell population in S phase in all CHO cell lines. This change in cell cycle profile was similar to that observed in the CHO1A and CHO1B cells. Thus, the data indicate that the differential effect of ₁-AR subtype stimulation on cell proliferation may be attributed to the differential activation of the cAMP-mediated signaling pathway by each ₁-AR subtype.

cAMP-mediated Cell Cycle Blockade Caused by ₁A-AR or ₁B-AR but Not by ₁D-AR Is Associated with an Induction of the Cyclin-dependent Kinase Inhibitor p27^Kip1-- p27^Kip1 is a cyclin-dependent kinase inhibitor that negatively regulates cell cycle progression and enforces cell cycle arrest in a cAMP-dependent manner (25). Hence, we further examined whether changes in p27^Kip1 expression levels could account for the differential effect of ₁-AR subtype stimulation on cell cycle progression. As shown in Fig. 5, immunoblot with a specific anti-p27^Kip1 antibody revealed that serum treatment caused a significant reduction in the protein expression level of p27^Kip1 with similar kinetics in all ₁-AR-expressing CHO cell lines. On the other hand, the activation of ₁-AR by phenylephrine blocked this serum-induced reduction of p27^Kip1 expression only in the CHO1A and CHO1B cells but not in the CHO1D cells (Fig. 5). These phenylephrine-induced changes in the expression of p27^Kip1 in CHO1A and CHO1B were blocked by pretreatment with phentolamine (data not shown). Corresponding with the effects on serum-induced cell proliferation, we observed that treatment with cholera toxin and forskolin inhibited the serum-induced reduction in p27^Kip1 expression in all three CHO cell lines as observed when ₁A/₁B-AR are activated (Fig. 6). Also, these phenylephrine-induced changes in the expression of p27^Kip1 in CHO1A and CHO1B were blocked by RpcAMP (Fig. 6).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Effect of 1-AR subtype activation on serum-induced decrease in the level of the cyclin-dependent kinase inhibitor p27Kip1. Serum-starved cells were stimulated with 10% serum in the presence or absence of phenylephrine or cholera toxin. Cells were then harvested at the indicated times, and total cell lysates were electrophoresed and immunoblotted with anti-p27Kip1 antibody. Both the autoradiograph (A) and quantitative data obtained by densitometry using NIH Image software (B) are shown. PE, phenylephrine.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Effect of various agents that modulate cAMP pathway on the level of the cyclin-dependent kinase inhibitor p27Kip1. Serum-starved cells were stimulated with 10% serum in the presence or absence of phenylephrine. Cells were then harvested 48 h after serum addition, and total cell lysates were electrophoresed and immunoblotted with anti-p27Kip1 antibody. Both the autoradiograph (A) and quantitative data obtained by densitometry using NIH Image software (B) are shown. In all experiments, phentolamine (10 µM) treatment was started 30 min prior to the phenylephrine stimulation. The results are expressed as the mean ± S.E. of three different experiments. *, p < 0.05; **, p < 0.01 versus serum alone (lane 2). Lane 1, basal; lane 2, serum; lane 3, serum with phenylephrine; lane 4, serum with phenylephrine/phentolamine; lane 5, serum with phenylephrine/RpcAMP; lane 6, serum with cholera toxin; and lane 7, serum with forskolin.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we explored the roles of each ₁-AR subtype in cell proliferation using a heterologous expression system. The activation of ₁A- or ₁B-AR but not ₁D-AR inhibited serum-promoted cell proliferation as evidenced by changes in the cell cycle profiles. In view of the differential expression profiles obtained between ₁A/₁B-AR- and ₁D-AR-activated cells, we examined the molecular mechanism for the ₁-AR subtype-dependent differential effect on cell proliferation. We found that all of the ₁-AR subtypes share induction of inositol 1,4,5-trisphosphate production and activation of MAPK pathways (ERK isoforms), but only ₁A- and ₁B-ARs are coupled to the cAMP signaling pathway. Furthermore, we observed that ₁A- or ₁B-AR but not ₁D-AR can block the serum-induced depletion of the cell cycle-dependent kinase inhibitor p27^Kip1, which is known to mediate cAMP-induced G₁ phase arrest. Taken together, our present data show that the ₁A- and ₁B-ARs but not ₁D-AR are coupled to cAMP synthesis and block serum-promoted depletion of p27^Kip1, and thereby inhibit serum-promoted cell proliferation.

₁-AR stimulation generally promotes cell growth in various cell types. However, recent studies have reported an anti-proliferative effect of ₁-AR activation in certain cell types (27-29). In the process of hepatic regeneration after hepatectomy, ₁-AR stimulation is thought to act as anti-proliferative signal in hepatic cells (28, 29). Also, it was reported that the activation of ₁B-AR in HepG2 human hepatoma cells stably transfected with a rat ₁B-AR cDNA inhibited cell growth via a p42^MAPK and p21^WAF1/CIP1-dependent mechanism (28). This study confirms an inhibitory effect of ₁-AR activation on the serum-promoted cell proliferation in a heterologous expression system and shows that the inhibitory effect is subtype-dependent and mediated through cAMP production. In agreement with our findings, Keffel et al. (30) recently has reported that ₁-AR subtypes are differentially coupled to growth promotion and growth inhibition in CHO cells (30). However, Keffel et al. (30) concluded that the opposite effects of ₁-AR subtypes on cellular growth are because of quantitative differences in the ERK isoforms in the MAPK signal transduction pathway. Although we confirmed the subtype-dependent differential extent of MAPK activation that is previously reported by Keffel et al. (30) in the serum-free conditions (data not shown), we also found that serum activates the MAPK pathway more dramatically than ₁-AR activation. In fact, as shown in Fig. 3B of this study, ₁-AR activation had a small additive stimulatory effect on the serum-promoted MAPK activation in all subtypes. Therefore, we consider that the anti-proliferative effect observed upon activation of ₁A/₁B-AR but not of ₁D-AR may not be solely explained by the quantitative difference in MAPK activation.

₁-ARs have been reported to have a stimulating effect on adenylyl cyclase activity in certain native tissues. In fact, the activation of ₁-AR increases cAMP production in tissue sections of rat brain or rat liver (9, 10). However, the molecular mechanism and physiological significance of the ₁-AR-induced cAMP production have remained uncertain. This report proposes for the first time that ₁-ARs activate the cAMP cascade in a subtype-dependent manner and that this differential activation is closely associated with the anti-proliferative effect of the ₁-AR subtype. We previously examined the mechanism of ₁-AR-mediated cAMP synthesis using ₁B-AR as a model and found that ₁B-AR-mediated cAMP synthesis is because of a direct coupling of ₁B-AR to the Gs protein (26). However, in other studies, the mechanism of ₁-AR-mediated induction of cAMP synthesis was shown to be secondary to its phosphatidylinositol hydrolysis/protein kinase C activation (12, 31, 32). Hence, ₁-AR-stimulated cAMP production is now considered to involve multiple mechanisms including direct activation of Gs/adenylyl cyclase and a secondary effect through activation of phosphatidylinositol hydrolysis. However, our data showing that all subtypes are efficiently coupled to phosphatidylinositol hydrolysis but that only ₁A- and ₁B-AR promote cAMP production provide support for a mechanism by which ₁-AR-mediated cAMP synthesis is the result of direct coupling to a Gs protein.

This study shows that activation of ₁-ARs inhibits the serum-promoted cellular growth in a cAMP-dependent manner. Several studies (34, 35) have reported that cAMP inhibits growth factor-induced mitogenesis by antagonizing the ERK/MAPK pathway at the Raf-1 level. In this study, however, we did not obtain any evidence in support of such a mechanism. Cell cycle blockade is recognized as another potential mechanism for cAMP/protein kinase A-mediated inhibition of cell proliferation in certain cells (36-38). In mammalian cells, the cell division cycle is controlled by the formation and activation of protein kinase complexes consisting of cyclins and cyclin-dependent kinases (39-41). The activity of cyclin-cyclin-dependent kinase complexes controls the cell cycle checkpoints in a coordinated way. By inhibiting the kinase activity of the cyclin-dependent kinases, cyclin-dependent kinase inhibitor proteins interfere with phosphorylation events critical for cell cycle transition (33, 41). Recently, it was reported that cAMP blocks colony-stimulating factor-induced mitogenesis in macrophages by inhibiting the cyclin D1-cyclin-dependent kinase 4 activity via increasing the expression of p27^Kip1 (25). Our results show that serum stimulation is capable of activating the G₁/S transition with a concomitant decrease in the expression level of p27^Kip1 and that these events are inhibited by ₁-AR subtype-dependent activation of cAMP. Therefore, it is possible that serum-promoted cell growth may be mediated by a decrease in the steady-state level of p27^Kip1 and that the subtype-dependent anti-proliferative effect could be caused by a cAMP-dependent inhibition of serum-promoted down-regulation of p27^Kip1. This study provides evidence for the first time for regulation of cell proliferation through a cAMP/p27^Kip1-related mechanism rather than through an ERK/MAPK-related mechanism.

In conclusion, we hereby report that a gene family of highly related ₁-ARs exert subtype-dependent differential effects on cell proliferation, although ₁-ARs are generally viewed to promote cell proliferation. Moreover, we found that the subtype-dependent differential effects are because of the different signal coupling associated with each subtype. Thus, activation of ₁A- and ₁B-ARs but not ₁D-AR, exerts anti-proliferative effects via the cAMP/p27^Kip1 pathway. The present study was performed by using an ₁-AR overexpression system where receptor couplings are sometimes promiscuous. Hence, direct extrapolation of the present findings to a natural system requires caution. Furthermore, in vivo cells naturally co-express varying levels of multiple or all subtypes. The physiological output of adrenergic stimulation in vivo system may consist of complex cellular responses, which involve subtype-dependent differential effects as observed in this study.

	ACKNOWLEDGEMENT

We thank Y. Kitagawa for help with cell culture.

	FOOTNOTES

^* This work was supported in part by research grants from the Scientific Fund of the Ministry of Education, Science, and Culture of Japan; the Japan Health Science Foundation and Ministry of Human Health and Welfare; the Organization for Pharmaceutical Safety and Research; and a grant for Liberal Harmonious Research Promotion System from the Ministry of Education, Culture, Sports, Science and Technology of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-3-3419-1252; Fax: 81-3-3419-1252; E-mail: gtsujimoto@nch.go.jp.

Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M201375200

	ABBREVIATIONS

The abbreviations used are: ₁-AR, ₁-adrenergic receptor; MAPK, mitogen-activated protein kinase; CHO1A, CHO1B, and CHO1D, CHO cells stably expressing each ₁-AR subtype; HEAT, 2-[2-(4-hydroxy-3-[¹²⁵I], iodophenyl)ethylaminomethyl]--tetralone; RpcAMP, cyclic AMP Rp-isomer; ERK, extracellular signal-regulated kinase.

	REFERENCES

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