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Endocrinology Vol. 145, No. 11 5157-5167
Copyright © 2004 by The Endocrine Society

Differential Regulation of the Cell Cycle by {alpha}1-Adrenergic Receptor Subtypes

Pedro J. Gonzalez-Cabrera, Ting Shi, June Yun, Dan F. McCune, Boyd R. Rorabaugh and Dianne M. Perez

Department of Physiology and Pharmacology (J.Y.), Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272; and Department of Molecular Cardiology (P.J.G.-C., T.S., D.F.M., B.R.R., D.M.P.), The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195

Address all correspondence and requests for reprints to: Dianne M. Perez, The Department of Molecular Cardiology NB50, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195. E-mail: perezd{at}ccf.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
{alpha}1-Adrenergic receptors have been implicated in growth-promoting pathways. A microarray study of individual {alpha}1-adrenergic receptor subtypes ({alpha}1A, {alpha}1B, and {alpha}1D) expressed in Rat-1 fibroblasts revealed that epinephrine altered the transcription of several cell cycle regulatory genes in a direction consistent with the {alpha}1A- and {alpha}1D-adrenergic receptors mediating G1-S cell cycle arrest and the {alpha}1B-mediating cell-cycle progression. A time course indicated that in {alpha}1A cells, epinephrine stimulated a G1-S arrest, which began after 8 h of stimulation and maximized at 16 h, at which point was completely blocked with cycloheximide. The {alpha}1B-adrenergic receptor profile also showed unchecked cell cycle progression, even under low serum conditions and induced foci formation. The G1-S arrest induced by {alpha}1A- and {alpha}1D-adrenergic receptors was associated with decreased cyclin-dependent kinase-6 and cyclin E-associated kinase activities and increased expression of the cyclin-dependent kinase inhibitor p27Kip1, all of which were blocked by prazosin. There were no differences in kinase activities and/or expression of p27Kip1 in epinephrine {alpha}1B-AR fibroblasts, although the microarray did indicate differences in p27Kip1 RNA levels. Cell counts proved the antimitotic effect of epinephrine in {alpha}1A and {alpha}1D cells and indicated that {alpha}1B-adrenergic receptor subtype expression was sufficient to cause proliferation of Rat-1 fibroblasts independent of agonist stimulation. Analysis in transfected PC12 cells also confirmed the {alpha}1A- and {alpha}1B-adrenergic receptor effect. The {alpha}1B-subtype native to DDT1-MF2 cells, a smooth muscle cell line, caused progression of the cell cycle. These results indicate that the {alpha}1A- and {alpha}1D-adrenergic receptors mediate G1-S cell-cycle arrest, whereas {alpha}1B-adrenergic receptor expression causes a cell cycle progression and may induce transformation in sensitive cell lines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CATECHOLAMINES EPINEPHRINE and norepinephrine mediate the functions of the sympathetic nervous system by activating G protein-coupled adrenergic receptors (ARs). The {alpha}1 family of ARs is comprised of three cloned and pharmacologically defined subtypes (termed {alpha}1A-, {alpha}1B-, and {alpha}1D-AR). These are coexpressed within most sympathetic tissues and preferentially activate the Gq/11 family of G proteins, thus raising the question whether discrete {alpha}1-AR subtype functions exist. Dissecting subtype-specific function in vivo has proven difficult with conventional subtype-selective antagonists due to poor selectivity; although recently published work (1, 2, 3) with knockout models has added new insights into this issue, particularly in the vasculature in which {alpha}1-ARs mediate vasoconstriction. An additional function of {alpha}1-AR subtypes in the vasculature has been proposed based on in vitro studies, which indicate that {alpha}1-AR stimulation mediates cell growth, proliferation, and migration of vascular smooth muscle cells (4, 5, 6). These data suggest that {alpha}1-ARs may have implications in vascular remodeling. Although the growth/proliferation effects have been largely studied in vascular smooth muscle cells (7, 8, 9), there is new evidence that indicates that {alpha}1-ARs are expressed in surrounding fibroblasts (10) and may participate in remodeling events.

Little is known about the molecular mechanism by which distinct {alpha}1-AR subtypes regulate cellular proliferation. In an independent microarray approach intended to identify novel pathways stimulated by {alpha}1-AR subtypes, we discovered that in Rat-1 fibroblasts expressing individual {alpha}1-AR subtypes, epinephrine activation differentially altered the transcription of several cell cycle regulatory genes, including: cyclin E, cyclin D1, cyclin-dependent kinase (CDK)-2{alpha}, proliferating cell nuclear antigen (PCNA), cyclin B, CDK-1, p27Kip1, p21Cip1, and DNA polymerases I-IV vs. nontransfected cells. These gene products are key regulators of cell cycle progression through the gap1 (G1) phase of the cell cycle, transition into the S (DNA synthesis) phase, and progression through G2 phase before mitosis (M). Progression through G1 takes place by formation and activation of G1 cyclin/CDK complexes, predominantly cyclin D-CDK-4/6 and cyclin E-CDK-2 (recently reviewed in Ref.11). In addition to binding of regulatory subunits (cyclins), the activity of CDK enzymes is regulated by phosphorylation-dephosphorylation events and interaction with CDK inhibitors (CDKIs), including p27Kip1 and p21Cip1. The latter are negative regulators of CDK activity that bind to cyclin/CDK complexes leading to cell cycle arrest, a function that has been shown to operate in growth regulation, wound repair, and vascular remodeling in vitro (12, 13, 14).

In this study, we investigated the effect of {alpha}1-AR subtypes on proliferation of Rat-1 cells. We show that expression of the {alpha}1B-AR subtype in Rat-1 fibroblasts is sufficient to induce unchecked cell cycle progression and stimulate cellular proliferation by inducing unchecked CDK-6- and cyclin E-associated kinase activities, whereas {alpha}1A- and {alpha}1D-AR subtypes have antiproliferative effects and mediate G1-S cell cycle arrest by inhibiting CDK-6 and cyclin E-associated kinase activities. These effects are likely through {alpha}1A- and {alpha}1D-AR stimulated up-regulation of the cell cycle kinase inhibitor p27Kip1 or in the case of {alpha}1B-ARs, dysregulation of G1-S cell cycle checkpoints. We also confirmed these effects in transfected PC12 cells and native DDT1-MF2 cells that endogenously express predominantly {alpha}1B-ARs. These results suggest that {alpha}1-AR subtypes may have differential effects in the control of the cell cycle and may regulate its proliferative effects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
(–)-Epinephrine bitartrate, (S)-(–)-propranolol hydrochloride, L-phenylephrine hydrochloride, rauwolscine hydrochloride, prazosin hydrochloride, and water-soluble dexamethasone were from Sigma-Aldrich (St. Louis, MO). DMEM, penicillin, streptomycin, trypsin-EDTA, Hanks’ balanced salt solution, and Ca2+-Mg2+-free PBS were obtained from The Cleveland Clinic Foundation (CCF) house facility (Cleveland, OH). Fetal bovine serum (FBS) and horse serum were obtained from BioWhittaker (Walkersville, MD). G418 was purchased from Calbiochem (La Jolla, CA). M-Per lysis reagent and West-Pico enhanced chemiluminescent reagent were from Pierce (Rockford, IL). [32P]-ATP and Na2-ATP were from Amersham Biosciences (Chicago, IL) and Roche Diagnostics (Indianapolis, IN), respectively. Protein A-agarose and anti-cyclin E (M20) antibody were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-CDK-6 and anti-p27Kip1 antibodies were from Cell Signaling Technologies (Beverly, MA). The anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Novus Biologicals (Littleton, CO). Histone H1 was kindly provided by Dr. Alexandru Almasan (CCF). Horseradish peroxidase-conjugated antirabbit IgG was from Jackson Laboratories (Bar Harbor, ME). Isopropyl-ß-D-thiogalactopyranoside was from USB Pharmaceuticals (Cleveland, OH).

Cell culture
Rat-1 fibroblasts stably transfected with human {alpha}1-AR cDNAs corresponding to the {alpha}1A-, {alpha}1B-, or {alpha}1D-AR subtypes were a gift of GlaxoSmithKline (Uxbridge, UK). Both Rat-1 and DDT1-MF2 cells were maintained in DMEM as described (15, 16). Nontransfected PC12 cells and those transfected with either {alpha}1A- or {alpha}1B-AR subtypes were a gift from Dr. Kenneth P. Minneman (Emory University, Atlanta, GA) and were maintained as described (17). Both the fibroblasts and the PC12 cell are made from clonal isolates and have been previously characterized in their signal transduction properties (15, 17). Both the fibroblasts and PC12 cells had similar receptor densities between the {alpha}1-AR subtypes and displayed no evidence of constitutive activity.

Preparation of biotin-labeled cRNA and hybridization to oligonucleotide arrays
Five pooled flasks (150 mm2 each) of either nontransfected or {alpha}1-AR subtype-transfected Rat-1 fibroblasts were used for individual hybridization to two oligonucleotide microarrays (Rat Genome U34A arrays; Affimetrix, Santa Clara, CA). Two separate hybridizations from two separate RNA preparations were performed per cell group. For drug treatments, confluent monolayers were preincubated for 30 min with propranolol (1 µM) and rauwolscine (0.1 µM) to block endogenous ß-AR and putative {alpha}2-ARs, respectively. Preincubation was followed by a 60-min treatment with 10 µM epinephrine. The subsequent steps for poly (A)+ RNA isolation, cDNA synthesis, and purification and synthesis of biotin-labeled cRNA were performed as reported (15). The Gene Expression Core Service at the CCF carried out the fragmentation of biotin-labeled cRNAs, the hybridization of these fragments to the oligonucleotide arrays, and the quality control test chips. The hybridization signal was amplified by the antibody amplification protocol as described in the Affimetrix GeneChip expression analysis manual.

For data analysis, all combinations of pair comparisons were made between the control (nontransfected) Rat-1 fibroblasts and those transfected with each {alpha}1-AR subtype [i.e. {alpha}1A-AR (chip 1) vs. non transfected (chip 1); {alpha}1A-AR (chip 2) vs. nontransfected (chip 1), etc.]. Therefore, for each subtype, a four-way comparison was performed. This results in four numerical values for each cell line. For each comparison, changes in the expression of a particular gene had to exhibit an average of 2.0-fold or greater and be present in each of the four-way analyses to be included in the table. This decision was based on previous reports that Taqman verification of the microarray is valid in the 1.7- to 1.8-fold range (18). Table 1Go reflects the mean values of the four-way comparisons ± SEM.


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  		TABLE 1. {alpha}1-AR subtype changes in cell-cycle regulatory gene expression after epinephrine (10 µM, 1 h) exposure

 
Cell cycle analysis
To determine the percentage of cells in different phases of the cell cycle, cells were seeded in 60-cm dishes at approximately 50% confluence and allowed to grow for 3 h in DMEM containing regular growth serum (10% FBS). The monolayers were then washed twice with sterile Hank’s balanced salt solution to remove traces of serum, and incubated for 48 h in media containing 0.2% FBS (low serum) to remove the cells from the cell cycle. In the initial experiments, a time course was followed to determine changes in the cell cycle phases due to 10% FBS (as a mitogen); 10% FBS and 10 µM epinephrine; or 10% FBS, epinephrine, and 25 µg/ml of cycloheximide, a translational inhibitor. Thereafter, after incubation in low serum, cells were incubated for 18 h in DMEM containing 10% FBS (as a mitogen), 10% FBS with 10 µM epinephrine, or 10% FBS with 10 µM epinephrine plus 1 µM of the {alpha}1-AR antagonist prazosin. After incubation, the monolayers were washed twice with Ca2+ and Mg2+-free PBS, and the adherent cells were collected by trypsinization. Cells were then washed once with PBS to remove traces of trypsin and immediately fixed in 70% ethanol for 30 min at 4 C. The analysis of the cell cycle was performed by the CCF Flow Core Facility with the cellular DNA flow cytometric analysis kit (Roche Diagnostics), which uses propidium iodide for DNA staining. Multiparameter cell cycle analysis of 10,000 cells was performed on a FACScan (Becton Dickinson, San Jose, CA) using Modfit LT software (Verify Software House Inc., Becton Dickinson).

Analysis of the cell cycle in PC12 cells was determined by using identical incubation times and agonist/antagonist concentrations as shown above, after inducing receptor expression with incubation with isopropyl-ß-D-thiogalactopyranoside (1 mM) for 48 h before experiments, as previously described (17).

Immunoprecipitation and histone H1 kinase assay
The kinase assay was modified (from Ref.19). The specificity of the antibodies was verified through Western blotting (data not shown) and has been used previously in CDK kinase assays (20). The effect of {alpha}1-AR subtype stimulation on G1 cell cycle kinases (CDK-6 and CDK-2) was determined by immunoprecipitation of Rat-1 cell lysates with anti-CDK-6 and anti-cyclin E (to pull down CDK-2) antibodies from low-serum synchronized cells, and those that were additionally treated with DMEM containing 10% FBS (as a mitogen), epinephrine, or epinephrine plus the {alpha}1-AR blocker prazosin. Cells were lysed in buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 25 mM ß-glycerophosphate, 1% Triton X-100, 1 mM EDTA, 1 mM Na3VO4, 1 mM dithiothreitol, and 1 mM NaF plus protease inhibitors (cocktail set III, Calbiochem) at 4 C. Lysates were centrifuged (10,000 x g) for 10 min at 4 C to remove insoluble components. Endogenous CDK-6 or cyclin E was immunoprecipitated overnight at 4 C from equal amounts (500 µg) of total protein lysates using an anti-CDK-6 (Cell Signaling) antibody at 1:100 dilution or 0.5 µg anti-cyclin E (M20) antibody (Santa Cruz Biotechnology), respectively. The immunoprecipitates were then incubated with protein A-Agarose at 4 C for 2 h, and the Agarose beads were washed three times with lysis buffer and once with kinase buffer consisting of 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 25 mM ß-glycerophosphate, 0.1 mM Na3VO4, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM NaF, and 50 µM ATP. Kinase activities were assayed using 1 µg histone H1 as the substrate in a reaction mixture containing kinase buffer and 5 µCi of [{gamma}-32P]ATP (3000 Ci/mmol) per assay in a volume of 30 µl. The reaction was allowed to proceed for 30 min at 30 C and then stopped by addition of 4 x Laemmli loading buffer. Labeled substrate was separated from free nucleotide by 4–15% SDS-PAGE, and the incorporated radioactivity was quantified using the public-domain National Institutes of Health Image program (Bethesda, MD) after fixation of the gel (10% methanol, 10% acetic acid) and autoradiography.

Western blotting
Western blot analysis was performed to determine expression levels of the CDKI p27Kip1 using lysates from cultures that were synchronized with low serum for 48 h and those that were low-serum synchronized and that received DMEM containing 10% FBS or DMEM containing 10% FBS with epinephrine (10 µM) in the presence and absence of prazosin (1 µM). Lysates were obtained by scraping using M-Per lysis reagent. Lysates (30 µg per lane) were separated by SDS-PAGE, transferred to nitrocellulose, and incubated overnight with anti-p27Kip1 according to manufacturer’s instructions. Expression of p27Kip1 was detected by chemiluminescence using West-Pico enhanced chemiluminescent reagent after incubation with secondary anti-horseradish peroxidase antibody (1:2000). Nitrocellulose membranes were stripped and reblotted with an anti-GAPDH (1:5000) antibody to confirm equal protein loading.

Cell counts
{alpha}1-AR subtype-transfected Rat-1 fibroblasts were seeded at similar densities and allowed to grow in regular growth media or media containing 10 µM epinephrine plus/minus 1 µM prazosin. For each treatment, viable cells were counted at time 0 and every 24 h thereafter using triplicate counts with a hemacytometer.

Foci formation
Rat-1 fibroblasts were seeded at a density of 1 x 104 cells in T 25 flasks and allowed to grow for 3 wk in DMEM/10% FBS and geneticin and then photographed. Media were changed as needed. Epinephrine at 10 µM final was then added to each flask and incubated for 4 d and the cells photographed.

Statistical analysis
Data are expressed as mean ± SEM unless otherwise stated. Significance was determined through paired Student’s t test using GraphPad Software (San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Epinephrine differentially modifies the transcription of genes involved in cell cycle control in Rat-1 fibroblasts individually expressing {alpha}1-AR subtypes
To examine signaling differences activated by {alpha}1-AR subtypes, we performed a microarray study of epinephrine-stimulated Rat-1 fibroblasts separately expressing each {alpha}1-AR subtype and compared the transcriptional profiles induced by epinephrine in subtype-transfected cells relative to those in epinephrine-stimulated, nontransfected Rat-1 fibroblasts. We recently reported most of the transcriptional changes evoked by epinephrine in a separate study (15). We previously found that all three {alpha}1-AR subtypes regulated the transcription of many genes in a similar manner, including c-fos and c-jun. In Table 1Go, we provide additional data from the microarray that involves epinephrine-stimulated transcriptional differences in cell cycle regulatory genes. The microarray analysis revealed that epinephrine stimulating {alpha}1A- and {alpha}1D-AR-expressing cells significantly inhibited the transcription of genes whose products are required for progression through G1 and transition into S phase, such as cyclin E and CDK-2{alpha}, and those required for DNA replication during S phase, such as DNA polymerase {alpha}-subunits I-IV. The transcription of PCNA, a marker of proliferating cells (21), was also significantly inhibited in epinephrine-stimulated {alpha}1A- and {alpha}1D-AR cultures relative to nontransfected cells. G2-M phase checkpoints including cdc2 (CDK-1) and its cyclin partner, cyclin B1, were additionally inhibited in {alpha}1A- and {alpha}1D-AR cells vs. nontransfected control cells.

Activation of {alpha}1B-AR fibroblasts with epinephrine resulted in a vastly different transcriptional profile vs. that of {alpha}1A- and/or {alpha}1D-AR cells. In {alpha}1B-AR cells treated with epinephrine, none of the above-mentioned regulators of G1-S or G2-M checkpoints showed significant differences in transcription vs. nontransfected cells. However, epinephrine stimulation of {alpha}1B-ARs resulted in the significant up-regulation of CCND1/cyclin D1, a cyclin involved in G1-S cell cycle transition and whose associated kinase activity has been shown to drive terminally differentiated cells into the cell cycle on overexpression (19).

Activation of all three {alpha}1-AR subtypes with epinephrine resulted in the significant up-regulation of the CDKI p27Kip1 vs. nontransfected cells, although by higher levels (2-fold) in {alpha}1A- and {alpha}1D-AR than {alpha}1B-AR cells. Another CDKI, p21Cip1, shown to have a protective role against apoptotic cell death (22, 23, 24) in addition to having growth arrest functions, was up-regulated only by activated {alpha}1B-AR cells. Overall, the results from Table 1Go raised the possibility that in Rat-1 fibroblasts, epinephrine stimulation induced G1-S cell cycle arrest through the activation of {alpha}1A- and/or {alpha}1D-AR subtypes while augmenting cell cycle progression in {alpha}1B-AR cells.

Activation of {alpha}1A- and {alpha}1D-ARs causes G1-S cell cycle arrest, and {alpha}1B-ARs induce unchecked cell cycle progression in Rat-1 fibroblasts
To examine the effect of {alpha}1-AR activation on Rat-1 fibroblast cell cycle profiles, we performed fluorescence-activated cell sorter (FACS) analysis using synchronized cell cultures that were exposed to mitogenic stimulation alone or mitogenic stimulation with epinephrine and/or the {alpha}1-AR antagonist prazosin. Synchronization of cells into G0 (quiescence) phase was achieved using a low-serum incubation condition (0.2% FBS, 48 h), thus providing a common cycling start point for every cell within the culture. As a positive control, we included mitogenic stimulation (10% FBS) to ensure reentrance of G0-synchronized cultures into the cell cycle. In initial experiments, we determined a time course of the cell cycle S phase changes for the {alpha}1A-AR and {alpha}1B-AR cell line. As shown in Fig. 1AGo, cell cycle arrest for the {alpha}1A-AR began as evidenced by a decrease in the percentage of cells in the S phase beginning at 8 h of epinephrine incubation and ending at 16 h, compared with FBS alone. However, the {alpha}1B-AR displayed no changes in the cell cycle through the same time course but did start with a higher percentage of the cells in S phase as compared with the {alpha}1A-AR cell line. The addition of cycloheximide to the epinephrine-stimulated {alpha}1A-AR cells blocked the effects on the cell cycle for both epinephrine and serum only at the 16 h time point (Fig. 1BGo). The effects of serum on the {alpha}1B-AR cell line were also blocked by cycloheximide. Because these results indicated a transcriptionally dependent effect on the cell cycle, all subsequent experiments used an 18 h time point for epinephrine incubation.



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  		FIG. 1. Time-course studies indicate a transcriptional effect on cell cycle regulation. A, Rat-1 fibroblasts were first serum starved (0.2% FBS) for 48 h, and then 10% FBS was added back to the {alpha}1A-AR cells ({blacktriangleup}) or {alpha}1B-AR cells ({diamondsuit}) only or in the presence of 10 µM epinephrine ({blacksquare}, {alpha}1A-AR; {blacktriangledown}, {alpha}1B-AR). B, The time-course experiment was repeated with cycloheximide, a translational inhibitor for the {alpha}1A-AR cells ({blacksquare}) or {alpha}1B-AR cells ({blacktriangledown}). Data are from three independent experiments. *, P < 0.05.

 
Figure 2Go and Table 2Go show that, as expected, low-serum incubation-arrested cells in quiescence, as evidenced by the substantial percentage (80–90%) of nontransfected {alpha}1A-AR and/or {alpha}1D-AR fibroblasts in the G0-G1 population (P < 0.05). The {alpha}1B-AR cells did not remove readily from the cell cycle after 48 h of low-serum incubation because only approximately 64% of the cells were quiescent, and a relatively large (27%) population were actively synthesizing DNA. Increasing the low-serum incubation time from 48 to 60 h did not further increase the percentage of cells in G0-G1 (for any of the Rat-1 cultures), indicating that synchronization was maximal at 48 h (data not shown). Addition of 10% FBS for 18 h to low-serum synchronized cultures stimulated cell cycle progression into S- and G2-M-phases in nontransfected cells (by 2.7- and 4.7-fold), {alpha}1A-AR cells (by 2.3- and 4.1-fold), and {alpha}1D-AR (by 3.3- and 2.8-fold), respectively. In contrast, stimulation of serum-starved {alpha}1B-AR cells with mitogens (10% FBS) for 18 h resulted in modest but nonsignificant increases in S phase (by 1.2-fold) and G2-M phase (by 1.8-fold) relative to low-serum-treated cells.



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  		FIG. 2. Epinephrine mediates G1-S cell cycle arrest by activating {alpha}1A- and {alpha}1D-AR subtypes, whereas {alpha}1B-ARs cause unchecked cell-cycle progression. After low serum (0.2% FBS) for 48 h, cells were incubated for 18 h in growth serum (10% FBS) or growth serum and 10 µM epinephrine (10% FBS + Epi) in the presence and absence of 1 µM prazosin (10% FBS + Epi + Pra) and subjected to a FACScan flow cytometer. For each treatment, the mean (n = 4) percentage of cells in G0-G1, S- and G2-M phases of the cell cycle are shown (top, middle, and bottom percentages, respectively). Refer to Table 1Go for mean ± SEM. The arrows indicate that epinephrine mediates G1-S cell cycle arrest in {alpha}1A- and {alpha}1D-AR cells, as evident from the reduction in S-phase cells and concomitant increases in G0-G1 cells vs. treatment with 10% FBS.

 

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  		TABLE 2. Percentages of Rat-1 fibroblasts in the different phases of the cell cycle after various cell culture treatments

 
Consistent with the microarray data of a G1-S cell cycle arrest, stimulation of {alpha}1A- and {alpha}1D-AR subtypes with epinephrine inhibited mitogen-induced G1-S transition, leading to only 4 and 14% of the cells in the S phase cell populations, respectively, and substantial increases in respective G0-G1 populations (P < 0.05 vs. 10% FBS treatment controls). The effect of epinephrine in {alpha}1A- and {alpha}1D-AR cells was completely abolished with the {alpha}1-AR antagonist prazosin. On the other hand, incubation of {alpha}1B-AR cells with epinephrine or epinephrine with prazosin did not modify their FACS profile relative to 10% FBS treatment, suggesting that the cells had become insensitive to changes in the cell cycle distribution. Finally, as expected, neither epinephrine nor epinephrine plus prazosin treatments had any effect on the FACS profile of nontransfected Rat-1 fibroblasts, known to lack endogenous {alpha}1-ARs.

FACS analysis of {alpha}1A- and {alpha}1B-AR subtypes expressed in PC12 cells
The cell cycle profile of epinephrine activated {alpha}1A- and {alpha}1B-AR subtypes individually expressed in PC12 cells is shown in Table 3Go. The PC12 cells used in this study, besides being phenotypically different (neuronal) relative to Rat-1 fibroblasts, express a lower density of {alpha}1A- (268 fmol/mg) and {alpha}1B-AR (241 fmol/mg) subtypes (17) vs. Rat-1 fibroblasts (1.3 ± 0.1 and 1.4 ± 0.4 pmol/mg protein, respectively). We did not use the {alpha}1D-AR transfected PC12 cell line because this expressed too little of the receptor to detected differences (data not shown). Table 3Go shows that in nontransfected and {alpha}1A-AR transfected PC12 cells that were incubated with low serum (0.2% FBS) for 48 h, additional stimulation with mitogens (5% FBS, 10% horse serum) for 18 h triggered cell cycle progression from G0-G1 to S-phase. We observed increases in S-phase from 4.7 to 17.8% (nontransfected) and from 16.3 to 21.3% ({alpha}1A-AR transfected), at the expense of G0-G1, which was reduced to 61 and 56%, respectively. Similar to Rat-1 fibroblasts, epinephrine stimulated G1-S cell cycle arrest in mitogen-induced {alpha}1A-AR PC12 cells, characterized by a reduction in S-phase to 7% and an increase to 76% in G0-G1 population (P < 0.05). The G1-S arrest induced by epinephrine was completely blocked by incubation with prazosin. As shown in Table 3Go, {alpha}1B-AR expression in PC12 cells was again, resulting in the same cell cycle distribution as serum starvation. The absence of an S-phase in the {alpha}1B-AR expressing PC12 cells indicates that the time spent dividing is very short and hard to capture with FACS analysis. This is confirmed by a high proliferation rate (data not shown), similar to the growth curves seen in the {alpha}1B-AR fibroblast (see Fig. 6Go).


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  		TABLE 3. Percentages of PC12 cells in the different phases of the cell cycle after various cell culture treatments

 


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  		FIG. 6. The G1-S cell-cycle arrest ({alpha}1A- and {alpha}1D-AR Rat-1 fibroblasts) induced by epinephrine and unchecked cell-cycle progression ({alpha}1B-AR fibroblasts) is consistent with its effect on cellular proliferation. Cells expressing individual {alpha}1-AR subtypes were allowed to grow in regular DMEM containing 10% FBS ({blacksquare}), 10 µM epinephrine ({blacktriangleup}), and 10 µM epinephrine with 1 µM prazosin ({triangledown}). Parental cells without the receptor had a growth curve that was not significantly different from unstimulated {alpha}1A- or {alpha}1D-AR curves (data not shown). For each treatment, cell counts were performed in triplicate every 24 h with a hemacytometer.

 
Effect of {alpha}1-AR subtype activation on G1 CDK activity in Rat-1 fibroblasts
Cell cycle progression is controlled by the sequential activation of CDKs. To investigate whether the observed G1-S cell cycle arrest by {alpha}1A- and {alpha}1D-AR subtypes or unchecked progression by {alpha}1B-ARs was accompanied by changes in the activities of G1 cell cycle regulatory mechanisms, we examined the activity of CDK-6 (a cyclin D1 binding partner) and cyclin-E-associated kinase activity (to pull down CDK-2). Cells were first incubated for 48 h with 0.2% FBS for synchronization and then further stimulated for 18 h with either 10% FBS or 10% FBS containing epinephrine alone or with the {alpha}1-AR antagonist prazosin. CDKs were isolated from cell lysates by immunoprecipitation with anti-CDK-6 or anti-cyclin E antibodies and the immunoprecipitates assayed for their kinase activities in vitro with histone H1 as the substrate.

Figure 3Go shows that the CDK-6 activities obtained from 0.2% FBS-synchronized cells were relatively low, whereas those obtained from cells that were further incubated with 10% FBS were, as expected, much higher. The figure also shows that in {alpha}1A- and {alpha}1D-AR cells, epinephrine (10% FBS + Epi) led to significant decreases in CDK-6 activity, compared with 10% FBS controls (P < 0.05) and that the {alpha}1-AR antagonist prazosin (10% FBS + Epi + Pra) blocked the inhibitory effects of epinephrine. Consistent with the unchecked cell cycle progression obtained by FACS analysis, epinephrine-stimulated {alpha}1B-ARs (10% FBS + Epi) had similar levels of CDK-6 activity to those obtained by mitogenic stimulation alone (10% FBS), which were not significantly different from those obtained in prazosin (10% FBS + Epi + Pra)-treated cells. Parental cells without the receptor did not show any epinephrine-stimulated differences from the 10% FBS treatment (data not shown).



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  		FIG. 3. A, The G1-S cell-cycle arrest ({alpha}1A- and {alpha}1D-AR Rat-1 fibroblasts) induced by epinephrine and unchecked cell-cycle progression ({alpha}1B-AR fibroblasts) are associated with modulation in the activity of CDK-6. After low serum (0.2% FBS) for 48 h, an additional 18 h incubation with regular growth serum (10% FBS) or regular growth serum with 10 µM epinephrine (10% FBS + Epi) was performed in the presence and absence of 1 µM prazosin (10% FBS + Epi + Pra). The activity of CDK-6 was measured by in vitro kinase assays. Parental cells without the receptor did not show any epinephrine-stimulated differences from the 10% FBS treatment (data not shown). B, Bars indicate the mean ± SEM of three independent experiments. For each experiment, the absolute band intensities were first calculated (in arbitrary units) using NIH Image and then normalized as fractional fold values relative to those of 10% FBS (taken to be 1.0). *, P < 0.05 vs. 10% FBS treatment.

 
Because CDK-2 is normally associated with cyclin E, which regulates the G1-S transition, we assayed for the kinase activity associated with cyclin E. As shown in Fig. 4Go, in cells expressing {alpha}1A- and {alpha}1D-AR subtypes, there was a significant decrease in epinephrine-stimulated cyclin E-associated activity (10% FBS + Epi), as compared with respective 10% FBS treatments (P < 0.05). Both cyclin E-associated kinase activities in epinephrine-stimulated {alpha}1A- and {alpha}1D-AR cells were also augmented by incubation with the {alpha}1-AR antagonist prazosin. In contrast, in cells expressing {alpha}1B-ARs, no differences in cyclin E-associated kinase activities were observed between epinephrine alone and epinephrine plus prazosin treatments. However, we consistently observed relatively high levels of cyclin E-associated kinase activity in serum-starved {alpha}1B-AR fibroblasts, relative to those in {alpha}1A- and/or {alpha}1D-AR cells. Parental cells without the receptor did not show any epinephrine-stimulated differences from the 10% FBS treatment (data not shown).



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  		FIG. 4. A, The G1-S cell-cycle arrest ({alpha}1A- and {alpha}1D-AR Rat-1 fibroblasts) induced by epinephrine and unchecked cell-cycle progression ({alpha}1B-AR fibroblasts) are associated with modulation in cyclin E-associated activity. After low serum (0.2% FBS) for 48 h, cells were incubated for 18 h in DMEM containing either regular growth serum (10% FBS) or regular growth serum with 10 µM epinephrine (10% FBS + Epi) in the presence and absence of 1 µM prazosin (10% FBS + Epi + Pra). Cyclin E-associated activity was measured by in vitro kinase assays. Parental cells without the receptor did not show any epinephrine-stimulated differences from the 10% FBS treatment and had no high basal levels (data not shown). B, Bars indicate the mean ± SEM of three independent experiments. For each experiment, the absolute band intensities were first calculated (in arbitrary units) using NIH Image, and then normalized as fractional fold values relative to those of 10% FBS (taken to be 1.0). *, P < 0.05 vs. 10% FBS treatment.

 
{alpha}1-ARs regulate the expression of p27Kip1 in Rat-1 fibroblasts
To investigate whether CDK-6 and cyclin E-associated kinase inhibition observed in {alpha}1A- and {alpha}1D-AR cells were associated with alteration in expression of the CDKI p27Kip1, we examined its levels in low-serum (0.2% FBS for 48 h) synchronized cells that received either 10% FBS or 10% FBS containing epinephrine ± prazosin. As shown in Fig. 5Go by Western blot analysis with an anti-p27Kip1 antibody, incubation of {alpha}1A- or {alpha}1D-AR cells with 0.2% FBS for 48 h led to a substantial elevation in p27Kip1 expression, which is a typically observed response of serum starved cells. In contrast, and as expected of cells that reenter the cell cycle from G0, there was significant degradation of p27Kip1 after 18 h of mitogenic stimulation (10% FBS). In agreement with the microarray data and consistent with the FACS and CDK data, epinephrine stimulated the accumulation of p27Kip1 in {alpha}1A- and {alpha}1D-AR cells, an effect that was completely abolished by incubation with prazosin. However, in {alpha}1B-AR cells, there were no differences in expression levels of p27Kip1 among any treated groups. Parental cells without the receptor did not show any epinephrine-stimulated differences from the 10% FBS treatment (data not shown).



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  		FIG. 5. Epinephrine-mediated G1-S cell cycle arrest in {alpha}1A- and {alpha}1D-AR cells is associated with up-regulation of p27Kip1 protein, whereas unchecked cell cycle progression in {alpha}1B-AR cells is associated with p27Kip1 dysregulation. After low-serum DMEM (02% FBS) for 48 h, cells were incubated for 18 h with DMEM containing either regular growth serum (10% FBS) or regular growth serum with 10 µM epinephrine (10% FBS + Epi) in the presence and absence of 1 µM prazosin (10% FBS + Epi + Pra). Parental cells without the receptor did not show any epinephrine-stimulated differences from the 10% FBS treatment (data not shown). The expression of p27Kip1 was determined from cell lysates by Western blotting. Membranes were stripped and reblotted with anti-GAPDH to compare protein loading. Representative experiments are shown (n = 2).

 
Cell count experiments
Figure 6Go shows that in cultures of {alpha}1-AR-expressing cells seeded at similar densities, epinephrine inhibited the proliferation of {alpha}1A- and {alpha}1D-AR cells that were growing asynchronously in the logarithmic phase (P < 0.05). The antiproliferative effect of epinephrine was blocked with prazosin. On the other hand, {alpha}1B-AR cells proliferated at a faster basal rate, with a doubling time in unstimulated cells which was approximately 24 h vs. the approximately 36–48 h for unstimulated {alpha}1A- and {alpha}1D-AR cultures (P < 0.05). In addition, epinephrine led to a slight increase in proliferation of {alpha}1B-AR cells, blocked by prazosin back to unstimulated conditions. Parental cells without the receptor had a growth curve not significantly different from unstimulated {alpha}1A- or {alpha}1D-AR cells (data not shown).

Foci formation
Because the results indicated that the {alpha}1B-AR maybe causing an unchecked cell cycle progression similar to protooncogenes, we determined the ability of the {alpha}1B-AR to form foci in the fibroblasts. As shown in Fig. 7Go, after 3 wk in culture, the parental and {alpha}1A-AR cell lines formed monolayers and had contact inhibition. However, the {alpha}1B-AR cell line developed foci characterized by a loss of contact inhibition, increased packing, and disordered array. When a maximal concentration of epinephrine was added for 4 d, the parental cell line maintained its monolayer, whereas the {alpha}1A-AR cell line underwent apoptosis, which we have confirmed by DNA fragmentation and caspase-3 activation (our manuscript in preparation), whereas the {alpha}1B-AR cell line continued to increase cell number causing colonies to form.



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  		FIG. 7. Agonist-independent transformation of the {alpha}1B-AR. Rat-1 fibroblasts stably transfected with vector alone (parental control, A) or the human {alpha}1A-AR cDNA (C) or human {alpha}1B-AR cDNA (E) were incubated for 3 wk with DMEM and 10% FBS and geneticin. Magnification, x10 (A, C, E). The cells were then incubated with 10 µM epinephrine for 4 d (B, D, F). Magnification, x4 (B, D, F).

 
The role of {alpha}1B-AR stimulation on the cell cycle of DDT1-MF2 cells
To determine whether the effects of {alpha}1-AR stimulation on the cell cycle could be extended into an endogenous cell line, we employed the tumor smooth muscle cell line, DDT1-MF2, isolated from hamster vas deferens and having been previously characterized to contain predominately {alpha}1B-ARs (25). Because we could not serum starve these cells, we used asynchronous cultures. In a dose response to epinephrine, the G0-G1 population decreased with increasing epinephrine concentration, whereas at the same time, there were simultaneous increases in S- and G2-M phases (Fig. 8Go). At 10–5 M epinephrine, the increases in S-phase and G2-M phases were maximal. Prazosin completely blocked the effect of epinephrine at 10–5 M (data not shown).



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  		FIG. 8. The effect of {alpha}1B-AR stimulation on serum-induced cell cycle progression in the endogenous DDT1-MF2 cell line. After incubation, cells received 5% FBS or increasing concentrations of epinephrine, and the relative percentages of G0-G1({blacksquare}), S ({Delta}), and G2-M ({blacktriangledown}) cell populations were determined by FACS.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is evidence in the literature that {alpha}1-AR stimulation induces proliferation and increases the rate of DNA synthesis in a variety of cell types: vascular smooth muscle cells (9), hepatocytes (26), and even bone cells (27). With the lack of sufficiently selective antagonists for the {alpha}1-AR subtypes, no one knows the specific {alpha}1-AR subtype that controls these responses, and in most cases, a particular cell type seems to express a combination of {alpha}1-AR subtypes. However, no one had previously thought that the {alpha}1-AR subtypes controlled proliferation by directly controlling the cell cycle, but often invoked transcriptional regulators such as c-Fos or c-Jun. {alpha}1-AR mediated control of the cell cycle is unlikely through these common transcription factors because these genes were similarly regulated by all three {alpha}1-AR subtypes (15). We found that microarray analysis of epinephrine-stimulated Rat-1 fibroblasts expressing each of the individual {alpha}1-AR subtypes could not only regulate various genes that control the cell cycle but also that this effect was also {alpha}1-AR subtype dependent (Table 1Go).

As opposed to protein-mediated signal transduction commonly activated by these receptors, the changes in the cell cycle, as suggested by the microarray, was mediated through a late-onset transcription that had maximal effects on the cell cycle at 16 h of stimulation (Fig. 1AGo). This was confirmed through the use of cycloheximide, a translational inhibitor, which only showed effects at 16 h of stimulation, indicating the production of a new protein(s) that mediated the changes on the cell cycle (Fig. 1BGo). The {alpha}1A- and {alpha}1D-AR subtypes appeared to control the cell cycle by not only decreasing the transcription of the cell cycle progressive cyclins, cyclins E and B, but also by down-regulating their associated kinases (CDK-2 and CDK-1, respectively). This was confirmed by a functional assay of the kinase activity of CDK-2 and CDK-6 (Figs. 3Go and 4Go), which decreased on epinephrine stimulation, consistent with a G1-S cell cycle arrest and reversed with prazosin blockade. FACS analysis also confirmed that the arrest occurred during the G1-S transition (Fig. 2Go). Because G1-S cell cycle arrest also can be associated with increases in the CDKIs, and p27Kip1 was increased in the microarray analysis, we confirmed that epinephrine also increased p27Kip1 protein levels in {alpha}1A- and {alpha}1D-AR expressing cells. All in all, the results support the conclusion that stimulation of the {alpha}1A- and {alpha}1D-AR in Rat 1 fibroblasts can cause G1-S cell cycle arrest.

The {alpha}1B-AR demonstrated a different profile in the regulation of the cell cycle. The microarray analysis showed that many of the genes that were down-regulated by stimulation of the {alpha}1A and {alpha}1D-AR fibroblasts were unchanged for the {alpha}1B-AR. However, a completely independent set of genes was up-regulated by stimulation of the {alpha}1B-AR subtype, which are associated with unchecked cell cycle progression: CCND1/cyclin D1. Indeed, in both the Rat-1 and PC12 cells, the {alpha}1B-AR caused an unchecked cell cycle progression. This effect was not due to desensitization, a common regulatory mechanism for G protein-coupled receptors, because desensitization was not seen in the time-course studies (Fig. 1AGo). The chromosomal locus CCND1 is located at 11q13 and encodes the cell cycle-regulating protein, cyclin D1. Cyclin D1 is involved in the G1 to S cell cycle transition by forming an active complex with CDK4/6, which phosphorylates the retinoblastoma protein, thus releasing the transcription factor complex, DP1/EF2, resulting in transactivation of S phase genes (i.e. DNA polymerases). CCND1 is frequently amplified and overexpressed in a variety of human carcinomas, correlates with poor prognosis, and is considered an oncogene (28). Such dysregulation causes growth advantage and enhances tumorigenesis (29). FACS analysis of the {alpha}1B-AR expressing Rat-1 cells showed an unchecked cell cycle progression disallowing changes in the various phases of the cell cycle (Fig. 2Go), consistent with the protooncogenic potential of cyclin D1 and its transcriptional up-regulation by {alpha}1B-AR fibroblasts. Because functional activity of cyclin D1 can be measured through activity of its CDK-6, we show in Fig. 3Go that {alpha}1B-AR fibroblasts had high CDK-6 activity that was unresponsive to epinephrine stimulation and/or prazosin blockade, consistent with unchecked cell cycle progression. CDK-2 activity was also high and unresponsive to epinephrine stimulation and/or prazosin blockade, in addition to {alpha}1B-AR cells having high basal levels of CDK-2 activity, also consistent with unchecked cell cycle progression (Fig. 4Go).

To confirm that these effects on the cell cycle could be translated into a phenotypic outcome, we determined proliferation rates in dividing fibroblasts (Fig. 6Go). In the {alpha}1B-AR expressing cells, basal rates of proliferation were twice those of {alpha}1A- and/or {alpha}1D-AR-expressing lines. Whereas epinephrine stimulation of the {alpha}1A- and {alpha}1D-AR cell lines caused decreased proliferation, consistent with the cell cycle arrest, the {alpha}1B-AR cell line caused a modest increase in the proliferation rate. All of these effects could be reversed with prazosin except for the high basal proliferation rate in the {alpha}1B-AR cells. These results indicate that the {alpha}1B-AR fibroblasts can proceed unchecked through G1-S transition, resulting in a higher rate of proliferation.

Because unchecked cell cycle progression and increased cyclin D1 is associated with protooncogenes, we checked for the potential of the {alpha}1B-AR Rat-1 fibroblasts to form foci. As shown in Fig. 7Go, whereas the parental and the {alpha}1A-AR cell lines formed monolayers through contact inhibition, the {alpha}1B-AR cell line formed foci, which by definition had a loss of contact inhibition, increased cell packing, and disordered array. When epinephrine was added to the cells for 4 d, the parental cell line maintained the monolayer, but the {alpha}1A-AR cell line underwent apoptosis (our paper in preparation), consistent with a prolonged cell cycle arrest; however, the {alpha}1B-AR cell line increased cell density and formed colonies. This result is similar to the study (30) in which the {alpha}1B-AR was labeled a protooncogene and was capable of producing foci and tumors in nude mice. Our data confirm those results but go a step further to show that the effect is at the level of cell cycle regulation.

We also confirmed these results in another stably transfected cell line, the PC12, which is a neuronal cell isolated from a pheochromocytoma. This cell line was stably transfected with the individual {alpha}1-AR subtypes at lower receptor density (reported in Ref. 17) to be 240–268 fmol/mg, compared with the 1–2 pmol/mg expression in the Rat-1 fibroblasts (15). We confirmed through FACS analysis that stimulation of the {alpha}1A-AR-expressing PC12 cells also caused G1-S cell cycle arrest, whereas stimulation of the {alpha}1B-AR with epinephrine or serum starvation could not alter the cell cycle and caused unchecked progression through the cell cycle phases (Table 2Go). The low or zero percentage of the cells in S phase was not due to growth arrest but rather to the high rate of proliferation. Therefore, these cells spend very little time in S phase, which cannot be captured by the FACS analysis. This was confirmed by measured proliferation rates, which are also very high, similar to the Rat-1 {alpha}1B-AR cell line. These results confirm that the {alpha}1-AR subtypes can control the cell cycle differentially, independent of cell type, and not by promiscuous coupling of the receptors due to overexpression or to clonal variation.

Because the Rat-1 fibroblasts and the PC12 cells do not contain endogenous {alpha}1-AR subtypes, it can be argued that {alpha}1-AR cell cycle regulation is not a physiological function of the {alpha}1-ARs but is due to promiscuous couplings in these transfected cell types. We believe that the differences in cell cycle regulation are also not due to clonal variation because the phenotype is repeated in a different type of cell (i.e. fibroblast vs. neuronal) and is also independent of high receptor density (picomoles vs. ficomoles). To explore {alpha}1-AR mediated regulation of the cell cycle in an endogenous cell line, we used the extensively characterized cell line, DDT-MF2, a tumor smooth muscle cell derived from the hamster vas deferens. This cell line has been determined to contain predominantly the {alpha}1B-AR subtype, close to 100% (25). This is the only cell line that we know of that has a predominance of one {alpha}1-AR subtype. In a dose response to epinephrine, {alpha}1B-AR stimulation did not cause unchecked cell cycle progression but increased cellular proliferation (Fig. 8Go).

Unchecked cell cycle progression is often seen in cancer cell lines. We show that the {alpha}1B-AR loses its regulation of p27, the major checkpoint for G1/S (Fig. 5Go). Loss of cell cycle checkpoints (i.e. p27) is a hallmark of human cancers. Low concentrations of p27 are seen in aggressive breast cancer (31) and oncogene-dependent transformation (32). CCND1 and its encoded protein, cyclin D1, is frequently amplified and overexpressed in a variety of human carcinomas and is considered an oncogene (28). In certain melanomas, predisposing mutants are impaired in their ability to inhibit the catalytic activity of the cyclin D1/CDK4 and cyclin D1/CDK6 complexes in vitro, causing unchecked cell cycle progression (33). Interestingly, the {alpha}1B-AR subtype, and not the other subtypes, has been implicated as being a protooncogene, having a potential to induce tumorgenicity (30). Therefore, unchecked cell cycle regulation through increased expression of cyclin D1 and/or unchecked p27 levels may be the mechanism(s) of the {alpha}1B-AR’s protooncogenic potential. We believe that the ability of the {alpha}1B-AR to cause either an unchecked progression and foci formation or simply a cell cycle progression depends on the ease of the cell line to become transformed. Fibroblasts are known for their ability to become transformed and form foci because they were produced from transformed revertants and still contain Simian virus 40 components (34). However, they are used routinely in assessing transformation ability. Because studies from a systemically overexpressed mouse model of the {alpha}1B-AR, which we developed, does not induce tumors (our unpublished observations), the issue of ability of the {alpha}1B-AR to induce oncogenesis is more complex and mechanisms may be in place to control unchecked proliferation in a animal. Therefore, the primary conclusion of our studies is not that the {alpha}1B-AR can induce cancer but that the {alpha}1B-AR induces cell growth.

Our study represents a continuation of the first report that {alpha}1-ARs can transcriptionally regulate the cell cycle (35). However, other investigators have also previously suggested that {alpha}1-AR subtypes may differentially couple to growth responses but was not mechanistically determined. In transfected Chinese hamster ovary (CHO) cells, one study indicated that the {alpha}1A-AR caused growth inhibition, whereas the {alpha}1D-AR caused growth stimulation, using tritiated thymidine incorporation (36). However, in this study, the cell cycle and its regulators were not followed. In contrast, in another study that used transfected CHO cells, {alpha}1-AR subtypes were shown to differentially control the cell cycle but did so through a cAMP mechanism (37). No other cell types were used. In this latter study, the {alpha}1A- and {alpha}1B-AR caused a G1-S cell cycle arrest, whereas the {alpha}1D-AR subtype displayed no effects on the cell cycle. Therefore, two studies using CHO cells had opposing results. In addition, the Rat-1 fibroblasts do not have the robust {alpha}1-AR mediated cAMP release, which is typically seen in CHO cells, and this could be an explanation for the differences between our studies. In a study using Rat-1 fibroblasts, both the {alpha}1B- and {alpha}1D-AR were shown to inhibit DNA synthesis (38). However, in this study kinase inhibitors were added, which would have blocked effects due to the cell cycle. In Rat-1 fibroblasts transfected with the {alpha}1A-AR, stimulation decreases the activity of many mitogenic indices in rat-1 fibroblasts, including DNA synthesis, ERK, phosphatidylinositol 3-kinase, and Akt (39). Therefore, all of these above studies agreed that the {alpha}1A-AR causes growth inhibition; the differences are between the roles for the {alpha}1B- and {alpha}1D-AR subtypes.

The major question invoked by our studies is how can most tissues that contain all three {alpha}1-AR subtypes cause proliferative effects, when the {alpha}1A- and {alpha}1D-ARs cause cell cycle arrest, and only the {alpha}1B-AR causes cell cycle progression. It is commonly accepted that a particular {alpha}1-AR subtype predominates in any given response. For example, in blood pressure regulation, the {alpha}1A-AR is thought to play a major role in vascular contraction with a minor role for the {alpha}1D-AR (40). We propose that the {alpha}1B-AR subtype in these tissues may be involved in proliferative responses, especially in injury models in which vascular remodeling can occur. Interestingly, cell types or tissues known to express predominantly the {alpha}1B-AR such as liver hepatocytes have increased potential for cell cycle progression, which occurs after a partial hepatectomy. In the regenerating liver, stress from liver damage triggers a complex mechanism that activates a program of hepatocyte proliferation followed by inhibition after the liver regains its original mass, mediated by adrenergic agonists (41). Interestingly, c-Jun-N terminal kinase, activated after a partial hepatectomy, has been shown to increase expression of cyclin D1, which in turn was shown to drive hepatocytes through the G1-S transition (42). The liver is the only tissue known to express predominance of one {alpha}1-AR subtype; all other tissues are composed from all three subtypes, and thus, the ratio of subtypes could also be the deciding factor that determines whether catecholamines induce proliferation or arrest.

The signal transduction pathways that might be responsible for the differences in {alpha}1-AR mediated transcriptional regulation of the cell cycle genes is unknown. It is not due to a constitutive activity of the {alpha}1B-AR because the inositol phosphate response and other transcriptionally regulated genes were not different from the other two {alpha}1-AR subtypes (15). It is unlikely due to common transcription factors such as c-fos or c-jun because previous microarray analysis in these same cells reveal no potential differences in their regulation between the subtypes (15).

In summary, we conclude that in several cell types, the {alpha}1-AR subtypes play a direct role in regulating the cell cycle. However, this regulation may be subtype specific, with the {alpha}1A- and {alpha}1D-ARs causing cell cycle arrest, whereas the {alpha}1B-AR causes cell cycle progression.


   Acknowledgments
 
We thank Dr. Kenneth P. Minneman for his generous gift of the transfected and nontransfected PC12 cells and GlaxoSmithKline for the transfected Rat-1 fibroblasts. We also thank Erica DuPree in the laboratory of Dr. Alex Almasan for help in the CDK kinase assays, and Cathy Stanko and Dolly Klingman from the CCF Flow Cytometry Core facility.


   Footnotes
 
This work was supported by Grant RO1HL61438 (to D.M.P.), a T32 HL07914 training grant in Vascular Cell Biology (to J.Y., B.R.R.), a National Research Service Award (to D.F.M.), and a local American Heart Association fellowship (to P.J.G.-C.).

Abbreviations: AR, Adrenergic receptor; CDK, cyclin-dependent kinase; CDKI, CDK inhibitor; CHO, Chinese hamster ovary; FACS, fluorescence-activated cell sorter; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen.

Received June 8, 2004.

Accepted for publication July 28, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rokosh DG, Simpson PC 2002 Knockout of the {alpha}1A/C-adrenergic receptor subtype: the {alpha}1A/C is expressed in resistance arteries and is required to maintain arterial blood pressure. Proc Natl Acad Sci USA 99:9474–9479[Abstract/Free Full Text]
  2. Tanoue A, Nasa Y, Koshimizu T, Shinoura H, Oshikawa S, Kawai T, Sunada S, Takeo S, Tsujimoto G 2002 The {alpha}1D-adrenergic receptor directly regulates arterial blood pressure via vasoconstriction. J Clin Invest 109:765–775[CrossRef][Medline]
  3. Cavalli A, Lattion AL, Hummler E, Nenniger M, Pedrazzini T, Aubert JF, Michel MC, Yang M, Lembo G, Vecchione C, Mostardini M, Schmidt A, Beermann F, Cotecchia S 1997 Decreased blood pressure response in mice deficient of the {alpha}1B-adrenergic receptor. Proc Natl Acad Sci USA 94:11589–11594[Abstract/Free Full Text]
  4. Hoffman BB, Hu ZW 2000 {alpha}1-Adrenoceptors and vascular smooth muscle cell growth. Prostate Suppl 9:29–33[CrossRef][Medline]
  5. Siwik DA, Brown RD 1996 Regulation of protein synthesis by {alpha}1-adrenergic receptor subtypes in cultured rabbit aortic vascular smooth muscle cells. J Cardiovasc Pharmacol 27:508–518[CrossRef][Medline]
  6. Zhang H, Facemire CS, Banes AJ, Faber JE 2002 Long-term reduction of intimal hyperplasia by the selective {alpha}1 adrenergic antagonist doxazosin. Am J Physiol Heart Circ Physiol 282:H2364–H2370
  7. Vashisht R, Sian M, Franks PJ, O’Malley MK 1992 Long-term reduction of intimal hyperplasia by the selective {alpha}1-adrenergic antagonist doxazosin. Br J Surg 79:1285–1288[Medline]
  8. Nakaki T, Nakayama M, Yamamoto S, Kato R 1990 {alpha}1-Adrenergic stimulation and ß2-adrenergic inhibition of DNA synthesis in vascular smooth muscle cells. Mol Pharmacol 37:30–36[Abstract]
  9. Hu ZW, Shi XY, Lin RZ, Hoffman BB 1996 {alpha}1-Adrenergic receptor stimulation of mitogenesis in human vascular smooth muscle cells: role of tyrosine protein kinases and calcium in activation of mitogen-activated protein kinase. J Biol Chem 271:8977–8982[Abstract/Free Full Text]
  10. Faber JE, Yang N, Xin X 2001 Expression of {alpha}-adrenoceptor subtypes by smooth muscle cells and adventitial fibroblasts in rat aorta and in cell culture. J Pharmacol Exp Ther 298:441–452[Abstract/Free Full Text]
  11. Stewart ZA, Westfall MD, Pietenpol JA 2003 Cell-cycle dysregulation and anticancer therapy. Trends Pharmacol Sci 24:139–145[CrossRef][Medline]
  12. Tanner FC, Yang ZY, Ducker SE, Gordon D, Nabel GJ, Nabel EG 1998 Expression of cyclin-dependent kinase inhibitors in vascular disease. Circ Res 82:396–403