We compared DNA replication, protein biosynthesis, and mitogen-activated protein kinase (MAPK) activity in Rat 1 fibroblasts stably expressing either the α1B-adrenergic receptor (AR) or α1D-AR subtypes. Activation of both the α1B-AR and α1D-AR inhibited DNA synthesis (as assessed by [3H]thymidine incorporation). In contrast, both receptors stimulated protein biosynthesis (as measured by [35S]methionine incorporation) and activated extracellular signal-regulated kinase (ERK)1/2. Importantly, these responses were agonist-dependent for the α1B-AR, but were agonist-independent for the α1D-AR. Agonist activation of the α1B-AR resulted in increased p38 kinase activity, but not c-Jun NH2-terminal kinase (JNK) activity, whereas the α1D-AR activated JNK but not p38 kinase. Unlike ERK1/2, JNK activity was increased by agonist treatment in the α1D-AR cells. An ERK1/2-pathway inhibitor PD98059 had no effect on phenylephrine-mediated inhibition of DNA synthesis in either cell line but blocked protein biosynthesis mediated by both receptors. The p38 kinase inhibitor SB203580 blocked α1B-AR effects on [3H]thymidine and [35S]methionine incorporation in α1B-AR-expressing cells, but had no effect on α1D-AR-mediated growth responses, consistent with the inability of the α1D-AR to activate p38 kinase. Therefore, α1B- and α1D-ARs mediated similar growth responses but differ with respect to the MAPK family member involved and the requirement for agonist.
Three genes encoding unique α1-AR subtypes, α1A-, α1B-, or α1D-AR, have been cloned and pharmacologically characterized (Cotecchia et al., 1988; Schwinn et al., 1990; Lomasney et al., 1991; Perez et al., 1991; Hieble et al., 1995). All three α1-AR subtypes exhibit similar affinity for endogenous catecholamines (Schwinn et al., 1990; Lomasney et al., 1991;Perez et al., 1991); however, the cellular functions of these receptors have not been adequately defined. Our previous work showed that although all receptor subtypes are expressed in peripheral arteries, the α1A-AR or α1D-AR couple agonist binding to smooth muscle contraction in a given vessel (Guarino et al., 1996; Hrometz et al., 1999; Piascik and Perez, 2001). In addition to the acute regulation of blood pressure, catecholamines induce vascular smooth muscle cell growth (Johnson et al., 1983;deBlois et al., 1996; Fingerle et al., 1991; van Kleef et al., 1992;Chen et al., 1995). These observations suggest that expressed α1-AR subtypes may differentially activate signaling pathways and physiological responses.
Mitogen-activated protein kinases (MAPKs) are important mediators of cell growth, proliferation, differentiation, and survival. There are three major MAPK subtypes: the extracellular signal-regulated kinases (ERK1/2), c-Jun NH2-terminal kinases (JNKs), and p38 kinases (Widmann et al., 1999). Activated MAPKs translocate to the nucleus and phosphorylate multiple transcription factors to increase transcriptional activity (Widmann et al., 1999). ERK1/2 and p38 kinase also phosphorylate cytoplasmic substrates, including those involved in protein biosynthesis (Widmann et al., 1999). Recent studies suggest that α1-AR subtypes differentially activate MAPK family members and that the profile of MAPK activation uniquely impacts on cellular phenotype (Alexandrov et al., 1999; Zhong and Minneman, 1999; Keffel et al., 2000). For example, in PC12 cells, inducible expression and activation of the α1A-AR, but not the α1B-AR or the α1D-AR subtypes, leads to activation of ERK1/2, JNK, and p38 kinase and promotes neurite outgrowth (Zhong and Minneman, 1999).
Discernment of the role of α1-AR subtypes in long-term growth responses in tissues that express multiple receptor subtypes is hindered by the lack of α1-AR subtype-selective agonists. An added complexity in studying the specific role of α1-AR subtypes in long-term responses is the observation that endogenous α1-ARs are differentially regulated by chronic agonist exposure in myocardial and vascular smooth muscle cells (Chen et al., 1995; Rokosh et al., 1996). As an alternative approach to study the regulatory roles of α1-ARs, several laboratories have compared signaling properties of α1-AR receptor subtypes in heterologous expression systems. These studies have demonstrated intrinsic differences between the α1-AR subtypes within a cell line and cellular responses for a given α1-AR expressed in different host cell lines.
Endogenously expressed α1A-ARs mediate hypertrophic growth of myocardial cells (Varma and Deng, 2000). Furthermore, accumulating evidence suggests that α1A-ARs, and to a lesser extent α1D-ARs, regulate arterial blood pressure (Piascik and Perez, 2001). The functional roles of the α1B-AR and α1D-AR in cardiovascular tissues are not well understood. We previously reported divergent regulation of subcellular localization and acute signaling events by α1B-AR and α1D-AR expressed in Rat 1 fibroblasts (McCune et al., 2000). The α1B-AR exhibits properties of a typical G protein-coupled receptor because it is expressed primarily on the cell surface and demonstrates agonist-dependent internalization and ERK1/2 activation (McCune et al., 2000). In contrast, the α1D-AR localizes primarily in internalized subcellular compartments and shows evidence of enhanced ERK1/2 and phospholipase activity in the absence of agonist (McCune et al., 2000). In this report, we examined JNK and p38 kinase activation, cellular proliferation, and protein biosynthesis mediated by these receptor subtypes. Both receptors inhibited DNA synthesis and increased protein biosynthesis and ERK1/2 activity. Interestingly, ERK1/2 and protein biosynthesis were agonist-dependent for α1B-AR and agonist-independent for the α1D-AR, suggesting constitutive ERK1/2 activity was coupled to protein biosynthesis. We also demonstrated divergent regulation of JNK and p38 kinase by these receptor subtypes. For example, the α1B-AR activated p38 kinase but not JNK, whereas the α1D-AR was coupled to JNK but not p38 kinase activation. Finally, we show that the α1B-AR, but not α1D-AR, mediated growth effects through a p38 kinase-dependent pathway. Therefore, although the α1B-AR and α1D-AR mediate similar effects on growth responses, they exhibit different requirements for agonist activation and MAPK isoforms.
Materials and Methods
Rat 1 fibroblasts stably transfected with either the cloned human α1B- or α1D-AR (GlaxoSmithKline, Uxbridge, Middlesex, UK) were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma Chemical, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA), 1% penicillin-streptomycin mixture (Invitrogen), and Geneticin (250 μg/ml; Invitrogen) at 37°C in 5% CO2. Twenty-four hours after plating, cells were washed and then serum-deprived for 48 h before the addition of drugs.
Rat 1 fibroblasts plated on 24-well dishes at 1 × 104cells/well were treated with the indicated drugs for 24 h with 1 μCi [3H]thymidine (PerkinElmer Life Sciences, Boston, MA) included during the last 6 h of incubation. Kinase inhibitors were added 20 min before phenylephrine (PE). Cells were rinsed with phosphate-buffered saline (PBS), fixed for 10 min with ice-cold methanol, washed 3 × 5 min with ice-cold 10% trichloroacetic acid, and dissolved in 1 N sodium hydroxide. [3H]Thymidine incorporation was quantified using liquid scintillation counting and used as an index of DNA synthesis.
Incorporation of [35S]methionine in Rat 1 fibroblasts was performed as described by Xin et al. (1997) with modifications (Xin et al., 1997). Serum-deprived Rat 1 fibroblasts plated on 24-well dishes at 1 × 104 cells/well were washed twice with methionine-free DMEM and incubated for 30 min. Cells were then treated with the indicated drugs in low-methionine (2 mg/l) DMEM for 24 h. Kinase inhibitors were added 20 min before PE. [35S]Methionine (1 μCi; PerkinElmer Life Sciences) was added during the last 6 h of incubation. Sample processing was essentially the same as for thymidine incorporation assay. [35S]Methionine incorporation was quantified using liquid scintillation counting and served as an index of protein synthesis.
Preparation of GST-c-Jun(1–135).
Recombinant c-Jun [c-Jun(1–135)] was produced in Escherichia coli as a glutathione S-transferase fusion protein expressed from plasmid pGEX-c-Jun (Prasad et al., 1995). GST-c-Jun(1–135) fusion protein was purified by conjugation to glutathione-Sepharose beads (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and eluted with 20 mM glutathione in 100 mM Tris, pH 8. The eluate was dialyzed for 2 h at 4°C to remove glutathione.
ERK1/2 Activity Assay.
ERK1/2 activity was detected by the in-gel method as described previously (McCune et al., 2000). Cells were treated with the indicated drugs, washed with ice-cold PBS, and scraped into 1 ml of 250 mM ice-cold buffered sucrose. Cell pellets were resuspended in cold lysis buffer [20 mM Tris-HCl, pH 7.4, 250 mM NaCl, 2.5 mM EDTA, 3 mM EGTA, 20 mM β-glycerophosphate, 0.5% (v/v) Nonidet P-40, 100 μM Na3VO4, and protease inhibitors (Calbiochem, La Jolla, CA)] for 30 min, centrifuged (15 min; 15,000g; 4°C), and the supernatant collected. Protein was resolved on 10% SDS-polyacrylamide gels containing 0.5 mg/ml myelin basic protein (MBP). After electrophoresis, the gels were washed with 20% 2-propanol in 50 mM HEPES, pH 7.6, and then with 5 mM β-mercaptoethanol in HEPES buffer. Proteins were denatured in 6 M urea and gradually renatured in HEPES buffer containing 0.05% (v/v) Tween 20 and 5 mM β-mercaptoethanol (renaturation buffer) at 4°C. After overnight incubation in renaturation buffer at 4°C, gels were preincubated in 25 ml of cold kinase buffer (20 mM HEPES, 20 mM MgCl2, 2 mM dithiothreitol, 5 mM β-glycerophosphate, 100 μM Na3VO4, pH 7.6) for 30 min. Phosphorylation of MBP was performed in situ by soaking the gel in 25 ml of kinase buffer containing 20 μM ATP and 150 to 160 μCi of [γ-32P]ATP (New England Biolabs, Beverly, MA) for 90 to 120 min at 30°C. After extensive washing with 5% trichloroacetic acid/1% sodium pyrophosphate the gels were dried and exposed to film. 32P incorporation into MBP was determined by densitometric analysis.
JNK Activity Assay.
Activities of the 46- and 55-kDa isoforms of JNK were determined by in-gel activity assays essentially as described for ERK1/2, except 0.1 mg/ml GST-c-Jun(1–135) was used as substrate.
p38 Kinase Immunoblotting.
Cells were maintained in serum-free media or treated with PE (100 μM) or anisomyosin (50 ng/ml) for 15 min. Cells were washed twice with ice-cold PBS and lysed with 150 μl of SDS-sample buffer. The lysates were sonicated (5 × 2 s), boiled for 5 min, and cooled on ice. Equal volumes of lysate were resolved on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). Phosphorylated and total p38 kinase was detected by protein immunoblotting with a 1:1000 dilution of rabbit polyclonal phosphospecific (Thr180/Tyr192) or total p38 kinase antibodies (New England Biolabs). Primary antibody was detected with 1:2000 horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody (Amersham Pharmacia Biotech AB). Bands were visualized by chemiluminescence (ECL+; Amersham Pharmacia Biotech AB) and quantitated by phosphorimaging (Molecular Dynamics, Sunnyvale, CA).
Differences among treatment groups were detected by one- or two-way analysis of variance with repeated measures followed by Student-Newman-Keuls multiple comparison tests. In the absence of a significant treatment by treatment group interaction, the statistical significance of the main or overall effect of a particular treatment is reported. Differences between the cell lines were detected by unpaired, two-tailed Student's t test. All calculations were performed using the Statistica program (release 5.1; StatSoft, Tulsa, OK). A p < 0.05 was considered significant. Nonlinear regression analyses of concentration-response curves were performed using GraphPad Prism (version 2.01; GraphPad Software, San Diego, CA).
Agonist Activation of α1B- and α1D-AR Inhibits DNA Synthesis.
The studies presented here extend our previous report demonstrating differences in the regulatory properties of the α1B- and α1D-AR (McCune et al., 2000). To determine the role of α1B-AR and α1D-ARs in cellular proliferation, we examined PE-mediated [3H]thymidine incorporation in Rat 1 fibroblasts stably expressing these receptor subtypes. Cells treated for 24 h with various concentrations of the α1-AR agonist PE exhibited a concentration-dependent decrease in [3H]thymidine incorporation (Fig.1). Maximal percentage of inhibition was not different for the two cell lines (α1B, 40 ± 11%; α1D-AR, 19 ± 7%; N.S.). The observed decrease in [3H]thymidine incorporation after prolonged PE treatment was not associated with a loss of cell number or decreased cell viability (data not shown). Levels of [3H]thymidine incorporation were similar in the absence of agonist [α1B, 8.6 ± 3.2 cpm (× 103); α1D, 11.0 ± 2.7 cpm (× 103); N.S.] and nonlinear regression revealed similar −log IC50 values for both cell lines (α1B, 6.6 ± 0.3; α1D, 7.0 ± 0.2; N.S.).
Inhibition of [3H]Thymidine Incorporation Mediated by α1B- and α1D-AR Is Reversed by Prazosin.
To determine whether the effects of PE in α1B- and α1D-AR-expressing cells were mediated through α1-ARs, we examined the effect of various adrenoceptor antagonists on PE-mediated inhibition of [3H]thymidine incorporation (Table1). PE (1 μM) reduced DNA synthesis to 44 ± 2% of that observed in unstimulated α1B-AR cells and 17 ± 4% for the α1D-AR. The effect of PE on DNA synthesis was blocked by 1 μM prazosin (nonselective α1-AR antagonist) in both cell lines and not affected by either 1 μM yohimbine (α2-AR antagonist) or 1 μM propranolol (β-AR antagonist), suggesting that agonist-induced inhibition of DNA synthesis was mediated through the expressed α1-AR.
Coupling of α1B- and α1D-ARs to Protein Biosynthesis.
Because α1-ARs are known to regulate both proliferative and hypertrophic growth responses, we also examined [35S]methionine incorporation as an index of protein biosynthesis. Levels of basal [35S]methionine incorporation were greater in the α1D-AR compared with the α1B-AR [7.8 ± 1.0 cpm (× 103) for the α1B-AR and 30.8 ± 3.4 cpm (× 103) for the α1D-AR (p < 0.001)]. PE increased [35S]methionine incorporation in a concentration-dependent manner in α1B- and α1D-AR-expressing fibroblasts (Fig.2), with a greater maximal effect of PE in the α1B cell line (238 ± 16 versus 162 ± 29%; p < 0.05). Differences in agonist and basal [35S]methionine incorporation between the two cell lines were not due to differences in cell number (data not shown). The −log EC50 values for two receptors were similar for the two cell lines (α1B-AR, 7.2 ± 0.3; α1D-AR, 6.9 ± 0.5; N.S.).
Coupling of α1B- and α1D-ARs to ERK1/2.
We compared the ability of these receptor subtypes to regulate ERK1/2 activation (Fig. 3). In α1B-AR-expressing cells, PE (100 μM; 5 min) significantly increased ERK1/2 activity over basal levels (*p < 0.05). In contrast, we did not detect PE- or serum-induced increases in ERK1/2 activity for the α1D-AR. Basal ERK1/2 activity was 2-fold greater in the α1D-AR-expressing cells (α1B, 1.7 ± 0.8 IOD; α1D, 3.5 ± 0.9 IOD) and similar in magnitude to the α1B cell line treated with PE (α1B, 4.7 ± 1.8 IOD). We previously showed that 1 μM prazosin inhibited basal ERK1/2 activity by ∼50% in α1D-AR-expressing cells (McCune et al., 2000). High basal ERK1/2 activity may have precluded our ability to observe further increases in ERK1/2 activity induced by 10% fetal bovine serum or PE.
Coupling of α1B- and α1D-ARs to JNK.
We examined the ability of PE and anisomyosin (positive control) to activate JNK in α1B-AR- and α1D-AR-expressing fibroblasts (Fig.4). Although anisomyosin (50 ng/ml) stimulated JNK activity to a similar extent in both cell lines, PE (100 μM; 20 min) increased JNK activity only in α1D-AR fibroblasts (*p < 0.01). Basal JNK activity was similar in both α1B-AR- and α1D-AR-expressing fibroblasts (average basal activity of α1B relative to α1D was 75 ± 30%; n = 4; N.S.).
Coupling of α1B- and α1D-ARs to p38 Kinase.
We compared the ability of PE to activate p38 kinase in both cell lines by immunoblotting for phospho- and total p38 kinase in unstimulated, PE-, or anisomyosin-treated cell lines (Fig.5). PE (100 μM; 15 min) induced p38 kinase activity in α1B-AR, but not in α1D-AR-expressing fibroblasts (*p < 0.05).
PD98059 Blocks PE-Induced ERK1/2 Activation in α1B-AR Cells and Decreases Basal ERK1/2 Activity in α1D-AR Cells.
Activation of MAPK family members requires phosphorylation on both threonine and tyrosine by dual specificity kinases (Widmann et al., 1999). To assess the role of ERK1/2 and p38 kinase in α1-AR-mediated DNA and protein biosynthesis we used selective cell-permeable inhibitors of these MAPK isoforms. Rat 1 fibroblasts were pretreated for 20 min with DMSO (0.1% v/v; control), 10 μM PD98059, a selective inhibitor of ERK1/2 activation (Dudley et al., 1995), or the p38 kinase inhibitor SB203580. After 5 min of PE or 10% FBS (control for ERK1/2 activation) treatment, cell lysates were prepared and ERK1/2 activity was determined using in-gel kinase assays. As shown in Fig. 6, PE-induced ERK1/2 activation in α1B-AR-expressing cells was blocked by PD98059. Unlike a previous report demonstrating inhibition of ERK1/2 activity by p38 kinase in Rat 1 fibroblasts (Alexandrov et al., 1999), we did not detect differences in either basal or agonist-induced ERK1/2 activity in the presence of SB203580. Relative to the α1B-AR, α1D-AR-expressing cells displayed elevated ERK1/2 activity in the absence of agonist that was inhibited by PD98059.
SB203580 Blocks PE-Induced p38 Kinase Activation in α1B-AR Cells.
To demonstrate the inhibitory effect of p38 kinase inhibitor SB203850 on α1-AR-mediated responses, cells were preincubated for 20 min with DMSO (0.1% v/v) or 10 μM SB203580 before the addition of PE (100 μM; 20 min). Cells were harvested and equal volumes of cell lysates were separated by SDS-PAGE and immunoblotted for phospho- or total p38 kinase (Fig.7). In α1B-AR cells, PE-mediated increases in phospho-38 kinase were blocked by SB203580. In contrast, PE did not activate p38 kinase in the α1D-AR cell line.
Role of ERK1/2 in α1-AR-Mediated DNA and Protein Biosynthesis.
Both α1B-AR and α1D-ARs inhibited DNA synthesis in Rat 1 fibroblasts (Fig. 1). Cell cycle arrest in HepG2 cells overexpressing α1B-AR occurs through an ERK1/2-dependent pathway (Auer et al., 1998). To investigate the role of ERK1/2 in α1-AR-mediated alterations in DNA synthesis, we examined [3H]thymidine incorporation in the absence and presence of 10 μM PD98059. Although PD98059 blocked ERK1/2 activation in both cell lines (Fig. 6), PD98059 did not reverse PE-mediated decreases in [3H]thymidine incorporation in either cell line (Fig.8, A and C), indicating that these α1-ARs regulate DNA synthesis through an ERK1/2-independent pathway.
In contrast to its inhibitory effects on DNA biosynthesis, PE significantly increased protein synthesis in α1B-AR-expressing fibroblasts (Fig. 8B; ***p < 0.001). Agonist-mediated activation of protein biosynthesis was inhibited ∼50% by 10 μM PD98059 (Fig. 8B;++ p < 0.01 versus PE alone). PE increased [35S]methionine incorporation in α1D-AR fibroblasts (Fig. 8D), but the observed increase in this series of experiments did not attain statistical significance (Fig. 2). Interestingly, basal [35S]methionine incorporation was greater in α1D-AR-expressing cells compared with the α1B-AR-expressing cell line [α1B, 7.8 ± 1.0 cpm (× 103); α1D, 30.8 ± 3.4 cpm (× 103); p < 0.001] and was similar in magnitude to PE-stimulated levels in α1B-AR-expressing cells [2.1 ± 0.3 cpm (× 103)]. Thus, any further increase in protein biosynthesis induced by PE might be expected to be small.
Role of p38 Kinase in α1-AR-Induced DNA and Protein Biosynthesis.
In contrast to PD98059, 10 μM SB203580 reversed α1B-AR-mediated inhibition of DNA synthesis by ∼50% (Fig. 9A;++ p < 0.01 compared with PE alone), suggesting that inhibition of DNA replication occurs, in part, through a p38 kinase-dependent pathway. Agonist-induced protein biosynthesis in α1B-AR cells was also inhibited ∼50% by SB203580 (Fig. 9B; ++ p< 0.01 versus PE alone). In contrast, SB203580 had no effect on either [3H]thymidine incorporation (Fig. 9C) or [35S]methionine incorporation (Fig. 9D) in α1D-AR-expressing fibroblasts. The effect of SB203580 on unstimulated DNA synthesis was not significantly different between the two cell lines. Combined PD and SB treatment in α1B-AR cells completely blocked the effect of PE on protein synthesis [control, 7.8 ± 0.9 cpm (× 103); PE, 20.5 ± 2.6 cpm (× 103), p < 0.001; PD + SB, 6.7 ± 0.6 cpm (× 103), N.S.; PE + PD/SB, 8.0 ± 1.0 cpm (× 103), N.S.], indicating that the α1B-AR uses both ERK1/2 and p38 kinase cascades to promote protein biosynthesis.
α1-ARs are important mediators of arterial blood pressure, vascular smooth muscle contraction, and growth. Multiple α1-AR subtypes are expressed in peripheral arteries; however, whether the same α1-AR subtype regulates both contractile and growth responses in vivo and whether this growth represents hypertrophy, hyperplasia, or both have not been adequately defined. Definitive assessment of α1-AR-mediated growth responses in vivo is hindered by the lack of α1-AR subtype-selective compounds and differential regulation of receptor expression by prolonged agonist exposure (Chen et al., 1995; Rokosh et al., 1996). However, several laboratories have shown differential coupling of α1-AR subtypes to MAPK activation (Zhong and Minneman, 1999; Keffel et al., 2000; McCune et al., 2000) and [3H]thymidine incorporation (Keffel et al., 2000) in heterologous cell expression systems, suggesting that α1-AR subtypes might differentially activate cellular growth responses.
To further examine the regulatory functions of the α1B-AR and α1D-AR, we compared cell proliferation, protein biosynthesis, and the MAPK isoforms mediating these growth responses in Rat 1 fibroblasts stably expressing these receptor subtypes. Despite similar levels of receptor expression (between 5.5 and 10 pmol/mg of protein; McCune et al., 2000) and the overall similarity of effect on cell proliferation and protein biosynthesis, our results reveal differential requirements for agonist and MAPK activation between these receptor subtypes. Agonist treatment of α1B-AR-expressing fibroblasts induces protein biosynthesis (Fig. 2), ERK1/2 activity (Fig. 3; McCune et al., 2000), and p38 kinase activity (Fig. 5), but has no effect on JNK activity (Fig. 4) and inhibits DNA synthesis (Fig. 1). In contrast, protein biosynthesis and ERK1/2 activity are elevated in the absence of agonist in α1D-AR cells. Although ERK1/2 activity and protein biosynthesis are agonist-independent for the α1D-AR, JNK activity and inhibition of [3H]thymidine incorporation are agonist-dependent, suggesting that the α1D-AR is not constitutively linked to these responses in Rat 1 fibroblasts.
The overall pattern of MAPK activation and cellular growth responses observed in Rat 1 fibroblasts is distinct from α1-AR subtypes expressed in either CHO or PC12 cells. Similar to results obtained in PC12 cells, activation of α1B-AR in Rat 1 fibroblasts cells increases ERK1/2 and p38 kinase activity, but does not activate JNK. However, although α1B-AR mediates increased protein biosynthesis and inhibition of DNA replication in Rat 1 fibroblasts, this receptor subtype does not affect DNA replication when expressed in CHO cells (Keffel et al., 2000). Expression and activation of the α1D-AR stimulate DNA replication, p38 kinase, and JNK in CHO cells (Keffel et al., 2000) but activate only ERK1/2 in PC12 cells (Zhong and Minneman, 1999). In contrast, agonist activation of the α1D-AR inhibits DNA replication (Fig.1), activates JNK (Fig. 4), and has no effect on p38 kinase activity (Fig. 5) in Rat 1 fibroblasts. Furthermore, studies in PC12 and CHO cells failed to report constitutive regulation of MAPK isoforms or growth responses for α1D-AR, although this receptor constitutively activates calcium transients in Rat 1 fibroblasts (Garcia-Sainz and Torres-Padilla, 1999). Several reports have shown that the α1D-AR is weakly coupled to intracellular signals (Theroux et al., 1996; Ruan et al., 1998; Taguchi et al., 1998). An apparent lack of agonist-mediated, growth-related responses is consistent with constitutive or agonist-independent signaling by the α1D-AR. The overall conclusion from heterologous expression studies is that growth-related responses are dependent on α1-AR subtype and host cell. Whether α1-AR subtypes differentially regulate MAPK activity and growth of various peripheral arteries is the focus of our ongoing investigations.
Our results indicate that ERK1/2 activation is associated with increased protein biosynthesis rather than proliferative growth of Rat 1 fibroblasts. For example, the ERK1/2 pathway inhibitor PD98059 blocks ERK1/2 activity in α1B-AR- and α1D-AR-expressing cells (Fig. 6), but does not reverse agonist-mediated inhibition of [3H]thymidine incorporation in either cell line (Fig. 8, A and C), suggesting that ERK1/2 activation is not required for α1-AR-mediated inhibition of DNA synthesis. In contrast to its effects on PE-mediated DNA replication, PD98059 attenuates PE-mediated increases in [35S]methionine incorporation in α1B-AR-expressing cells (Fig. 8B), suggesting this receptor couples agonist binding to protein biosynthesis through ERK1/2. Basal [35S]methionine incorporation is elevated in α1D-AR- relative to α1B-AR-expressing fibroblasts (Fig. 8, compare B and D). The ERK1/2 pathway inhibitor PD98059 blocks basal ERK1/2 activity (Fig. 6) and [35S]methionine incorporation (Fig. 8D), suggesting that constitutive ERK1/2 activity is coupled to increases in protein biosynthesis in α1D-AR-expressing cells. The physiological significance of agonist-dependent increases in ERK1/2 and protein biosynthesis by the α1B-AR and constitutive activity of these responses by the α1D-AR are not known. We did not observe increases in cell size (hypertrophy) in unstimulated α1D-AR cells or in agonist-treated α1B-ARs (data not shown). It is possible that constitutive activation of ERK1/2 for α1D-ARs or agonist-mediated increases in ERK1/2 via α1B-ARs induces a differentiated or synthetic phenotype characterized by constitutive synthesis of extracellular matrix proteins in Rat 1 fibroblasts.
In contrast to the requirement for ERK1/2 activation in [35S]methionine incorporation, inhibition of DNA synthesis occurs through an ERK1/2-independent pathway for both α1-AR subtypes (Fig.10). Similar to a previous study examining DNA synthesis in CHO cells expressing the α1A-AR (Keffel et al., 2000), the p38 kinase inhibitor SB203850 reverses PE-mediated inhibition of [3H]thymidine incorporation in the α1B-AR cell line (Fig. 9A). The lack of effect of SB203580 on PE-mediated inhibition of [3H]thymidine incorporation and elevated basal [35S]methionine incorporation in the α1D-AR cell line is consistent with the inability of this receptor subtype to induce p38 kinase activation (Fig. 5). Therefore, inhibition of [3H]thymidine incorporation in response to α1D-AR activation may occur through JNK and/or other signaling cascades in this cell line.
As illustrated in Fig. 10, we propose that agonist activation of the α1B-AR induces p38 kinase and ERK1/2 activation and increases protein biosynthesis. In addition, our results indicate that the α1B-AR uses both ERK1/2 and p38 kinase-dependent pathways to induce protein biosynthesis. Use of two parallel pathways may explain the robust effect of agonist on protein synthesis in the α1B-AR relative to the α1D-AR cell line (Fig. 2). In contrast to the regulatory properties of α1B-ARs, increases in protein biosynthesis occur primarily through constitutive ERK1/2 activity for the α1D-AR. Furthermore, inhibition of DNA replication occurs through an ERK1/2-independent pathway in both cell lines, suggesting that the profile of MAPK activity (i.e., ERK1/2 versus JNK or p38 kinase) may differentiate growth-related responses in Rat 1 fibroblasts.
We thank Carol Swiderski for expert technical assistance; Drs. Steven Post, Martin Michel, and Dianne Perez for helpful discussions; and Dr. Tatyana Voyno-Yasentskaya for pGEX-cJun expression plasmid. We also thank Dr. Dianne Perez for Rat 1 fibroblasts expressing α1-AR subtypes used in these studies (made available to Dr. Perez from GlaxoSmithKline).
↵1 Current Address: Bernard J. Dunn School of Pharmacy, Shenandoah University, 1460 University Dr., Winchester, VA 22601. E-mail:
This study was supported by an American Heart Association Scientist Development grant and the University of Kentucky Medical Center Research Fund (to G.R.P.), American Foundation for Pharmaceutical Education predoctoral fellowship (to B.A.W.), National Institutes of Health HL-38120 (to M.T.P.), and American Heart Association Grant-in-Aid (to M.T.P. and G.R.P.).
- adrenergic receptor
- mitogen-activated protein kinase
- extracellular signal-regulated kinase
- c-Jun NH2-terminal kinase
- Dulbecco's modified Eagle's medium
- fetal bovine serum
- phosphate-buffered saline
- myelin basic protein
- integrated optical density
- dimethyl sulfoxide
- polyacrylamide gel electrophoresis
- Chinese hamster ovary
- Received May 8, 2001.
- Accepted June 20, 2001.
- The American Society for Pharmacology and Experimental Therapeutics