Introduction

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₁-Adrenoceptors (ARs) belong to the superfamily of G-protein-coupled receptors (GPCR) that mediates the functions of catecholamines. Once activated by binding, ₁-ARs initiate the cellular pathways leading to the regulation of physiological effects, including blood pressure maintenance, glucose metabolism, renal sodium reabsorption, and cardiac inotropy (Michelotti et al., 2000; Piascik and Perez, 2001). Three ₁-AR subtypes (_1A-, _1B-, and _1D-AR) have been cloned and characterized pharmacologically (Cotecchia et al., 1988; Lomasney et al., 1991; Perez et al., 1991). All three ₁-AR subtypes display comparable binding affinities to catecholamines and show prevalence for coupling to the G_q family of G proteins, thereby activating IP₃-mediated increases in calcium. Despite having similar binding affinities for the endogenous ligands and predominance toward G_q signaling, all three ₁-AR subtypes are expressed in most tissues, thus raising the question of whether or not ₁-AR subtypes carry out redundant functions.

Discerning the physiological roles of ₁-AR subtypes in vivo has proven difficult, mainly because of the lack of probes with sufficient subtype selectivity (i.e., ligands and low avidity antibodies). This issue has been partly addressed by the use of cell lines transfected with individual ₁-AR subtypes, and transgenic animal models in which ₁-AR subtypes have been either knocked out (Cavalli et al., 1997; Rokosh and Simpson, 2002; Tanoue et al., 2002) or overexpressed (Milano et al., 1994; Zuscik et al., 2000; Lin et al., 2001).

Besides vascular effects, ₁-ARs are known to stimulate hypertrophy of cardiac myocytes by activating established signaling pathways (reviewed in Varma and Deng, 2000). This effect of ₁-ARs may be relevant during the pathogenesis of congestive heart failure, because plasma concentrations of the sympathetic neurotransmitter norepinephrine are elevated during the onset of heart failure. In addition to increased cardiac sympathetic drive, circulating levels of the proinflammatory cytokine IL-6 (and IL-6 family members LIF and cardiotrophin-1) are also elevated in patients with heart failure (Kanda et al., 2000; Eiken et al., 2001; Ng et al., 2002). A role for IL-6 in the development of heart failure has been proposed based upon evidence that mice overexpressing IL-6 or its receptor develop ventricular hypertrophy (Hirota et al., 1995), whereas IL-6 receptor or gp-130 knockout mice develop thin ventricular walls (Taga et al., 1996) or a massive apoptosis before the onset of heart failure (Hirota et al., 1999). Recent evidence indicates that other well-known cardiac stimuli, including angiotensin II (Sano et al., 2000) and activation of -ARs (Murray et al., 2000), increase expression of IL-6 in the heart, indicating a correlation between neuroendocrine signaling and cytokine release.

₁-AR subtype-specific signaling has been suggested previously, particularly in studies of ₁-AR stimulation of MAPK pathways (Zhong and Minneman, 1999), which integrate extracellular signals that regulate cell growth, proliferation, and fate. In the present study, we employed oligonucleotide microarrays to provide new insights, and a more sensitive assay of ₁-AR subtype signaling. We compared the profiles of gene expression after short-term (60-min) epinephrine-stimulation of Rat-1 fibroblasts stably transfected with individual ₁-AR subtypes and compare these with stimulated nontransfected cells. Our studies indicate that within the same cellular context, ₁-AR subtypes can stimulate or inhibit identical profiles of expression, despite differences in IP efficacy, as well as profiles that are subtype- and/or pathway-specific. We report that ₁-ARs activate the secretion of IL-6 and that there are synergistic effects on STAT3 activation, suggesting that ₁-AR mediation of STAT3 is through non-IL-6 effects, which was confirmed through neutralizing antibodies. Interestingly, the _1B-AR was not synergistic or as synergistic with IL-6 activation. This could be because the _1B-AR displays constitutive down-regulation of gp-130 protein levels. Our data also suggests that _1B-AR activation can stimulate the dimerization-dependent phosphorylation of Tyr705 on STAT3 but not the transcriptional-dependent phosphorylation of Ser727, the first report of this differential activation of STAT3. These results could be important in regulating pro-hypertrophic pathways and may be differentially regulated by the ₁-AR subtypes.

	Materials and Methods

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Materials. [¹²⁵-I]BE-2254, [myo-³H]inositol, and [-³²P]dCTP were obtained from PerkinElmer Life Sciences (Boston, MA). AG 1-X8 resin (formate form), 4 to 15% Ready-Gels, and Bio-Rad protein assay were purchased from Bio-Rad Laboratories (Hercules, CA). Full-length rat p21-cK-Ras and c-fos plasmids were obtained from American Type Culture Collection (Manassas, VA). M-PER lysis reagent and West-Pico enhanced chemiluminescent reagent were from Pierce (Rockford, IL). The gp-130 (M-20) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Both STAT3 and Phospho-STAT3 (Ser727 and Tyr705) antibodies were obtained from Cell Signaling Technology (Beverly, MA). IL-6 and IL-6 neutralizing antibodies were from R & D systems (Minneapolis, MN). Horseradish peroxidase-conjugated anti-rabbit IgG was from Jackson Laboratories (Bar Harbor, ME). ()-Epinephrine bitartrate, (S)-()-propranolol hydrochloride, rauwolscine hydrochloride, and prazosin hydrochloride were from Sigma-Aldrich (St. Louis, MO). Phentolamine mesylate was from RBI/Sigma (Natick, MA). Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, trypsin-EDTA, and Ca²⁺-Mg²⁺-free phosphate-buffered saline (PBS) were obtained from the Cleveland Clinic Foundation (CCF) house facility (Cleveland, OH). Fetal bovine serum was obtained from Cambrex Bio Science Walkersville, Inc. (Walkersville, MD). G418 was purchased from Calbiochem (La Jolla, CA).

Cell culture. Rat-1 fibroblasts stably transfected with human ₁-AR cDNAs corresponding to either the _1A-, _1B-, or _1D-AR subtype were a gift of GlaxoSmithKline (Uxbridge, UK). These cells were derived from clonal isolates. Cells were propagated in 75-cm² flasks in a humidified atmosphere (37°C) in DMEM containing 5% fetal bovine serum, 10 U/ml penicillin, 100 µg/ml streptomycin, and 500 µg/ml of the selection antibiotic G418. Cells were detached by trypsinization (0.05% trypsin, 0.53 mM EDTA) and subcultured at a ratio of 1:5 upon reaching confluence. Nontransfected Rat-1 fibroblasts were maintained under the same conditions but in the absence of G418.

Saturation Binding. Saturation binding experiments were performed using membranes from the Rat-1 cells stably transfected with the _1A-, _1B- or _1D-AR subtypes. Membrane isolations were performed as reported previously (Perez et al., 1991). Binding assays were performed in triplicate in a 0.25-ml assay volume with eight concentrations (0-400 pM) of ¹²⁵I-BE-2254 and 10 µg of membrane protein per tube. After equilibration for 30 min at 25°C, free and bound radioligand were separated by rapid filtration using a Brandel cell harvester and Whatman GF-C glass-fiber filters. Nonspecific binding was defined as the amount of radioactivity that remained bound to the filters in the presence of 10 µM phentolamine. B_max (maximum receptor density) and K_d (affinity) values were obtained using the nonlinear regression function of Prism (GraphPad, San Diego, CA).

Quantitation of Epinephrine-Stimulated Total IP Accumulation. Confluent cells expressing ₁-AR subtypes were grown for 24 h in the presence of 5 µCi of [³H]myo-inositol. Propranolol (1 µM) and rauwolscine (0.1 µM) were added to the growth media for 30 min to block putative - and ₂-ARs, respectively, followed by a 60-min incubation of 10 µM epinephrine in the presence of 10 mM LiCl. After drug incubations, the monolayers were washed with ice-cold PBS and the cells were lysed with 0.4 M perchloric acid. Lysates were scraped, collected, and neutralized by the addition of a 0.5-ml volume of a 0.72 N KOH/0.6 M KHCO₃ solution. Soluble [³H]IPs were isolated from the lysates by column chromatography using AG 1X-8 resin-packed columns. The columns were washed with 0.1 M formic acid, and the resin-bound [³H]IPs were displaced by elution with a 0.1 M formic acid solution containing 1.0 M ammonium formate. The eluant was collected in scintillation vials and the radioactivity was detected using a -counter (Beckman, Irvine, CA). EC₅₀ (potency) values and maximal epinephrine responses were compared using Prism (GraphPad).

Preparation of Biotin-Labeled cRNA and Hybridization to Oligonucleotide Arrays. Five pooled flasks (150 mm² each) of either nontransfected or ₁-AR subtype-transfected Rat-1 fibroblasts were used for individual hybridization to the oligonucleotide microarray. Two separate hybridizations from two separate RNA preparations were performed per cell group. Briefly, confluent monolayers were first incubated for 30 min with propranolol (1 µM) and rauwolscine (0.1 µM), followed by an additional 60-min incubation with 10 µM epinephrine. After incubations, the media was removed and the monolayers rinsed twice with ice-cold PBS. Poly(A)⁺ RNA was immediately isolated using the FastTrack 2.0 Kit from Invitrogen (Carlsbad, CA) and stored overnight at 70°C. Double-stranded cDNA was then synthesized from 1.0-µg aliquots of poly(A)⁺ RNA using the SuperScript Choice double-stranded cDNA synthesis kit from Invitrogen. From each sample, cDNA was purified by phenol/chloroform extraction and ethanol precipitation, and biotin-labeled cRNA was synthesized via an in vitro transcription reaction using the BioArray high-yield RNA transcript labeling kit (Enzo Diagnostics, Farmingdale, NY). cRNA transcripts were then purified from the in vitro transcription reaction using the RNeasy Mini kit from QIAGEN (Valencia, CA). The fragmentation of biotin-labeled cRNAs and hybridization of these fragments to the oligonucleotide arrays were both carried out by the Gene Expression Core Service at the CCF. To determine the quality of the mRNA preparations and the subsequent manipulations between test samples, an aliquot of the biotinylated cRNA fragments from each sample were hybridized to an Affymetrix (Santa Clara, CA) "test chip" before the rat genome U34A array was used. This test chip analyzes the percentage of genes that are present in the sample that hybridizes to the genes present in the test chip. The analysis also includes a measure of the amount of full-length cRNA transcribed as determined by the ratios of 3'/5' regions of both glyceraldehyde-3-phosphate dehydrogenase and -actin. Aliquots of biotin-labeled cRNA fragments were then hybridized to rat genome U34A Arrays (Affymetrix) containing 7000 known genes and expressed sequence tags from build 74 of the UniGene database. The hybridization signal was amplified by the Antibody Amplification Protocol as described in the Affymetrix GeneChip Expression Analysis Manual.

Northern Blot Analysis. Two genes that were identified via microarray to either increase or remain unchanged by activation of ₁-AR subtypes (c-fos and p21-cK-Ras) were examined by Northern analysis. Drug incubation protocols were kept identical to those in microarray experiments. Poly(A)⁺ RNA (10 µg) extracted with the Invitrogen FastTrack 2.0 kit were loaded onto 0.8% agarose-formaldehyde gels for subsequent transfer to nitrocellulose and hybridization. cDNA probes were derived from full-length plasmids obtained from American Type Culture Collection. Probes (c-fos, 5.0-kb EcoRI fragment; p21-cK-Ras, 4.4-kb EcoRI-HindIII fragment) were random-primed with [³²P]dCTP (6000 Ci/mmol) using the random primed DNA labeling kit from Roche (Indianapolis, IN). Labeled probes (1 × 10⁶ cpm/ml) were hybridized to the membranes overnight at 50°C. Membranes were each washed in 5× then 2× SSC containing 0.1% SDS for 10 min, and exposed to X-ray film for 3 (c-fos) or 48 h (p21-cK-Ras). Qualitative differences in band intensities were compared between ₁-AR subtype transfected and nontransfected Rat-1 cells using the software program NIH Image (http://rsb.info.nih.gov/nih-image/).

Measurement of IL-6 Secretion. Rat-1 fibroblasts stably expressing ₁-AR subtypes were incubated for 24, 48, and 72 h with 10 µM epinephrine in the presence of 1 µM propranolol and 0.1 µM rauwolscine. Additionally, experiments were performed in ₁-AR subtype expressing Rat-1 fibroblasts that were incubated for 48 h with epinephrine in the presence of -AR and ₂-AR antagonists and the ₁-AR antagonist prazosin (1 µM). Antagonists and epinephrine were replenished in the culture media every 12 h. After incubation, the culture medium was removed and concentrated by vacuum centrifugation. The IL-6 levels in the concentrate were determined by enzyme-linked immunosorbent assay (ELISA) using the rat IL-6 module set from Alexis Biochemicals (San Diego, CA) following the manufacturer's instructions. Sample IL-6 concentrations were extrapolated by spectrophotometric (405 nm) conversion of standard curves ranging in sensitivity from 31 to 2000 pg/ml. To account for IL-6 in serum, background absorbance values (DMEM containing serum and antibiotics) were subtracted from each sample.

Immunoblotting-Immunoprecipitation Studies. To investigate whether changes in transcription for gp-130 and STAT3 genes would be reflected in changes at the translational level, antibodies to gp-130 and STAT3 were employed in Western blotting experiments of ₁-AR subtype transfected cells treated with 10 µM epinephrine for 1 or 24 h. Nontransfected Rat-1 fibroblasts were also included in these experiments to exclude any effect of epinephrine incubation on gp-130 and/or STAT3 total protein levels. The effect of ₁-AR subtype stimulation on gp-130 total protein levels was studied after 15, 30, 60, 120, and 180 min of 10 µM epinephrine stimulation. After incubation, the monolayers were washed with ice-cold PBS, and the cells were scraped in M-PER lysis buffer (Pierce) containing protease inhibitors (1.0 mM phenylmethylsulfonyl fluoride, 1.0 mM EDTA, and 0.2 mM aprotinin). Total protein content in the lysates was determined using the Bio-Rad protein assay. Proteins were separated on 4 to 15% gradient gels by electrophoresis. The gels were transferred onto nitrocellulose membranes, blocked for 1 h, and incubated overnight with either STAT3 or gp-130 antibodies. Membranes were incubated with an anti-rabbit HRP-conjugated secondary antibody and visualized by enhanced chemiluminescence.

	Results

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Saturation Binding. The levels of ₁-AR subtype expression in Rat-1 fibroblasts were compared in membrane saturation binding assays using the radioligand ¹²⁵I-BE-2254. Rat-1 fibroblasts expressed similar densities and affinities of single-site binding curves. The B_max values for _1A-, _1B-, and _1D-AR transfected cells were 1.3 ± 0.1, 1.4 ± 0.4, and 2.6 ± 0.5 pmol/mg, respectively. The K_d values for ¹²⁵I-BE-2254 were also similar among subtypes: _1A-AR, 19.8 ± 1.5 pM; _1B-AR, 30.5 ± 6.5 pM; _1D-AR, 58 ± 6.5 pM. In all binding assays, nonspecific binding at the K_d ranged from 9.2 to 15% of the total radioligand bound, and no more than 10% of total radioactivity added was bound.

Quantitation of Epinephrine-Stimulated Total [ ³H]IP Accumulation. As a measure of ₁-AR subtype-G_q signaling, concentration responses to epinephrine stimulation of [³H]IP accumulation were performed in ₁-AR subtype transfected Rat-1 fibroblasts (Fig. 1). No significant differences in potency (log EC₅₀, molar) were noted for epinephrine stimulation of total [³H]IP accumulation among ₁-AR subtypes: _1A-AR, 7.16 ± 0.15; _1B-AR, 7.86 ± 0.24; _1D-AR, 7.72 ± 0.15. However, the maximal responses to epinephrine varied among subtypes, and were significantly higher in _1A-AR cells than those of _1B-AR (by 4.1-fold) and _1D-AR (by 2.8-fold) cells. The maximal responses to epinephrine in _1D-AR cells were modestly higher than those in _1B-AR cells (by 1.2-fold). There were no differences in basal or agonist-independent [ ³H]IP counts among ₁-AR subtype transfected fibroblasts (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   1-AR subtype-mediated total [3H]IP accumulation in Rat-1 fibroblasts expressing 1A-AR (), 1B-AR (), or 1D-AR () subtypes. Cells expressing 1-AR subtypes were incubated with increasing concentrations of epinephrine (1010-105 M) for 60 min in the presence of - and 2-AR blockers, and the soluble [3H]IPs were eluted by column chromatography and quantified by scintillation counting. Each point represents the mean ± S.E.M. of three independent experiments measured in counts per minute performed in triplicate. Despite equal receptor density as determined by ligand binding studies, the 1A-AR subtype displays greater efficacy in the IP response. EC50 values are not significant from one another, p > 0.23.

Comparison of mRNA Quality between Samples. The results of the "test chip" analysis between the samples used in the microarray study revealed that both hybridizations from separate RNA preparations had reproducible parameters of quality. All three parameters that determine the amount (percentage present) and quality of mRNA (3'/5' ratios) for both glyceraldehyde-3-phosphate dehydrogenase and -actin were comparable between samples and are considered excellent.

Genes commonly modified by ₁-AR subtypes. The gene expression profiles of Rat-1 fibroblasts stably expressing ₁-AR subtypes is shown in Table 1. Common to all three subtypes, gene expression changed for 29 genes by at least two-fold relative to nontransfected Rat-1 fibroblasts. These common gene expressions have been divided into four clusters, which include genes that code for: 1) secreted endogenous peptide ligands (cytokines and growth factors); 2) DNA binding proteins (transcription factors and immediate early genes); 3) signaling and/or catabolic enzymes; and 4) extracellular matrix proteins. In each cluster, genes have been arranged in decreasing order of magnitude of expression changes, starting with the highest fold-values of _1A-AR fibroblasts as reference. Thus, the strongest stimulations for each cluster were for the gp-130 related cytokine, IL-6, the immediate-early gene c-fos, the tyrosine phosphatase, CL100, and fibronectin, respectively. Figure 1 shows that even though the epinephrine-stimulated accumulation of total IPs was most efficacious in _1A-AR cells, all three subtypes changed the gene expressions of most of the genes in Table 1 by similar folds. In addition, some genes (GD-VEGF, CELF, rNFIL-6, Arc, RNR-1) were more strongly changed in their expression in _1B-AR and/or _1D-AR cells than _1A-AR cells.

View this table: [in this window] [in a new window]   TABLE 1 Increased gene expression changes in Rat-1 fibroblasts, common to all three 1-AR subtypes Data indicate the fold increase ± S.E.M. in gene expression with the indicated accession number. For each gene cluster, the genes are listed in decreasing order of magnitude, taking into account the 1A-AR fold values as reference point. Data are from two separate hybridizations from two separate RNA preparations and compared in a four-way analysis as described under Materials and Methods. Fold changes are similar among the subtypes.

View this table: [in this window] [in a new window]   TABLE 2 Decreased gene expression changes in Rat-1 fibroblasts, common to all three 1-AR subtypes Data indicate the fold decrease ± S.E.M. in the gene expression with the indicated accession number. The genes are listed in decreasing order of magnitude, taking into account the 1A-AR fold values as reference point. Data are from two separate hybridizations from two separate RNA preparations and compared in a four-way analysis as described under Materials and Methods. Fold changes are similar among the subtypes.

₁-AR subtype-unique changes in gene expression. Table 3 shows that compared with nontransfected Rat-1 fibroblasts, multiple changes in gene expression that were specific to each ₁-AR subtype existed. These included, for each subtype, both positive and negative changes, which varied in magnitude and number of genes modified. The _1B-AR modified the greatest number of genes (17), followed by the _1D-AR (12) and _1A-AR (6). We noted that in _1B-AR cells, a relatively large number of gene expressions modified have been associated with neurodegeneration and apoptosis, including tau and synuclein (Golbe, 2002), transforming growth factor 3 (Dunker et al., 2001), and caspase-6 (Mac-Lachlan and El-Deiry, 2002).

View this table: [in this window] [in a new window]   TABLE 3 1-AR subtype-unique changes in gene expression Data indicate the fold increase (positive values) or decrease (negative values) ± S.E.M. in gene expression with the indicated accession number. For each subtype-specific cluster, the genes are listed in decreasing order of magnitude. Data are from two separate hybridizations from two separate RNA preparations and compared in a four-way analysis as described under Materials and Methods.

Differential regulation of IL-6 signaling genes by ₁-AR subtypes. In addition to changes in gene expression that were common to all subtypes, and to subtype-specific changes, the expression of certain genes changed in a different way: stimulated by two, but not all three subtypes. Table 4 shows that this was true of genes that code for members of the IL-6 signaling pathway (gp-130, STAT3 and Ras). Table 4 shows that although all subtypes increased the gene expression of the cytokine, IL-6, only _1A-AR and _1D-AR subtypes were linked to increases in gene expression of gp-130 (the IL-6 high affinity receptor and signal transducer), and STAT3 (prototype IL-6-stimulated transcription factor). p21-Ras has been included in this pathway becauseit has been shown to relay IL-6/gp-130 signals in some cells (Taga and Kishimoto, 1997).

View this table: [in this window] [in a new window]   TABLE 4 1-AR subtype specific gene expression changes in IL-6 signaling Data indicate the fold increase ± S.E.M. in gene expression with the indicated accession number. Data are from two separate hybridizations from two separate RNA preparations and compared in a four-way analysis as described under Materials and Methods. Although both the 1A- and 1D-AR caused the gene expression to change in the downstream signals of this pathway, the 1B-AR did not, despite the stimulation of IL-6.

Northern blots. To validate some of the data obtained with the microarray technique, Northern blotting analyses were carried out for two different genes (c-fos and p21-cK-Ras) present in the microarray. Drug incubation protocols for obtaining northern and microarray data were kept identical. Figure 2 shows that in nontransfected cells epinephrine had no effect in the transcription of c-fos (NT lane). On the other hand, epinephrine robustly stimulated the transcription of c-fos in ₁-AR subtype transfected cells. The increase in c-fos transcription by northerns followed a similar pattern to those obtained by microarrays: _1A-AR (by 63.8 ± 23.4-fold), _1B-AR (43.5 ± 12.5-fold), and _1D-AR (61 ± 13-fold) cells relative to nontransfected controls. However, these changes were significantly higher in the microarray analysis, which is consistent with previous reports that the array is a more sensitive assay for detecting changes in mRNA levels (Yun et al., 2003).

View larger version (69K): [in this window] [in a new window]   Fig. 2.   Effect of 1-AR subtype-stimulation on steady-state c-fos and p21-cK-Ras mRNA levels in nontransfected Rat-1 fibroblasts (NT) or Rat-1 fibroblasts transfected with 1A-AR, 1B-AR or 1D-AR subtypes. Cells were exposed to 10 µM epinephrine for 60 min, in the presence of - and 2-AR blockers, then mRNA was prepared. Northern blot hybridizations were performed using 10 µg of poly(A)+ RNA per lane with c-fos and p21-cK-Ras cDNA probes. Hybridizations and washing conditions were performed as indicated in the text. These results are representative of two individual Northern blot experiments. c-fos mRNA seems to be equally stimulated by all three subtypes, with somewhat lower levels with the 1B-AR, consistent with the microarray results (Table 1). Fold-intensity differences (NIH Image) compared with control are 7.83, 6.13, and 7.58 for the 1A-, 1B-, and 1D-AR, respectively). With p21-cK-Ras mRNA, the 1B-AR does not stimulate, whereas both the 1A- and the 1D-AR do so, with a higher efficacy from the stimulation of the 1D-AR subtype, again consistent with the microarray results (Table 4). Fold-intensity differences (NIH Image) compared with control are 1.9, 1.1, and 2.20 for the 1A-, 1B-, and 1D-AR, respectively).

Epinephrine-Stimulated Secretion of IL-6 by ₁-AR Subtypes. Because IL-6 has been associated with cardiac hypertrophy and heart failure and was one of the most changed in gene expression, we decided to explore the IL-6 pathway in more detail. The concentration of secreted IL-6 in culture medium was measured in ELISA experiments. The first set of experiments showed that in Rat-1 fibroblasts, ₁-AR subtypes stimulated an increase in the concentration of IL-6 in culture medium after epinephrine incubation. The effect was time-dependent. No IL-6 could be detected in culture medium during the first 24 h of epinephrine incubation; however, longer incubation times (48 and 72 h) resulted in increased IL-6 levels (_1A-AR, 7.3- and 24-fold; _1B-AR, 5.5- and 12.5-fold; _1D-AR, 8.5 and 12.6-fold, respectively). This is probably a result of the sensitivity of the ELISA assay and is similar to previous reports that examined IL-6 secretion (Norris and Benveniste, 1993). Separate experiments summarized in Fig. 3B showed that at 48 h of epinephrine incubation, the accumulated IL-6 concentration in medium from ₁-AR subtype expressing cells was completely abolished by prazosin (1 µM, 48 h) in _1A- and _1B-AR expressing cells and mostly blocked in _1D-AR cells. No IL-6 protein accumulation was detected in nontransfected Rat-1 fibroblasts treated with identical epinephrine conditions (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   1-AR stimulation of IL-6 secretion in Rat-1 fibroblasts. A, in time-course studies, IL-6 was secreted into the culture medium of 1A-AR (), 1B-AR (), and 1D-AR () cells in a time-dependent fashion using a maximum concentration of epinephrine (10 µM). B, selecting the 48-h time point, IL-6 secretion can be stimulated by all three subtypes. IL-6 secretion can be blocked with the 1-AR antagonist prazosin (1 µM) incubated in the culture medium before and during the addition of epinephrine, demonstrating that the secretion of IL-6 is mediated through stimulation of the 1-AR receptor subtypes. IL-6 secretion was determined on the parental cells but was negligible.

Epinephrine-Stimulated Changes in STAT3 Protein and Ser727-Phosphorylation by ₁-AR Subtypes. Fig. 4A shows the effect of epinephrine incubation on STAT3 total protein levels in Rat-1 fibroblasts transfected with ₁-AR subtypes. After 1 h of incubation with 10 µM epinephrine, we observed no differences in STAT3 total protein levels between epinephrine-stimulated and nonstimulated subtype-transfected cells. Longer epinephrine exposure (24 h) resulted in substantially higher levels of STAT3 total protein, particularly for _1D-AR cells and, to a lesser extent, for _1A-AR expressing cells. We observed no differences in total STAT3 protein levels between epinephrine-stimulated and nonstimulated _1B-AR fibroblasts at either incubation time.

View larger version (58K): [in this window] [in a new window]   Fig. 4.   Time-course studies of 1-AR subtype stimulation on total STAT3 protein (A) and transcriptional function of STAT3 (Ser727-phosphorylation) (B) in Rat-1 fibroblasts. Total STAT3 protein levels were compared between subtype-stimulated (+) and nonstimulated () cells in Western blotting experiments with an antibody to STAT3. Total STAT3 levels increased upon 24-h incubation with the 1A- (1.70×) and 1D-AR subtypes (2.70×) but were unchanged for the 1B-AR (1.11×). Fold-changes in intensities are determined by NIH Image software using nonstimulated as the control. Active STAT3 was detected using an antibody that only recognizes the Ser727-phosphorylated form of STAT3 afterSTAT3 immunoprecipitation (IP). Both the 1A- (fold time-course intensities: 1, 1.65, 5.84, 3.93, and 1.42) and 1D-AR (fold time-course intensities: 1, 0.61, 1.25, 2.74, and 3.72) increase the transcriptionally active form of STAT3 upon stimulation. However, the 1B-AR (fold time-course intensities: 1, 0.47, 0.41, 0.33, and 0.28) decreases its formation.

₁-AR Subtype Regulation of gp-130 Protein Levels in Rat-1 Fibroblasts. The effect of ₁-AR subtype activation on total gp-130 protein levels was studied by Western blotting experiments with ₁-AR subtype-transfected and nontransfected fibroblasts that were incubated with epinephrine (10 µM) for 1 and 24 h (Fig. 6A). At 1 h incubation, we observed no differences in gp-130 protein levels between epinephrine-stimulated and nonstimulated fibroblasts (applies to both nontransfected and ₁-AR subtype-transfected fibroblasts). However, we observed that the _1B-AR reduced the levels of gp-130 total protein in an agonist-independent manner compared with nontransfected and _1A- and _1D- cells. This agonist-independent reduction in total gp-130 protein in _1B-AR cells was also apparent at 24 h of epinephrine incubation. Although epinephrine had little or no effect in _1A- and _1D-AR cells after 1 h of treatment, incubation for 24 h almost completely abolished the levels of gp-130 total protein relative to those of nonstimulated cells.

View larger version (57K): [in this window] [in a new window]   Fig. 5.   1-AR subtype regulation of gp-130 protein levels in Rat-1 fibroblasts. A, individual 1-AR subtypes were stimulated in the presence or absence of 10 µM epinephrine (with - and 2-AR blockers) for either 1 or 24 h. Although no changes were observed at 1 h, both the 1A- (fold-intensity change, 0.36) and the 1D-AR (fold-intensity change, 0.47) decreased gp-130 levels at 24 h, whereas the 1B-AR displayed neither basal nor stimulated levels of the protein. Fold changes in intensities are determined by NIH Image software using nonstimulated as the control. B, time course studies of epinephrine-stimulation revealed that although the 1B-AR stimulation of gp-130 levels (time-course intensity changes: 1, 0.91, 1.03, 1.02, 1, and 0.99) was not changed, the 1A- (time-course intensity changes: 1, 0.85, 0.67, 0.55, 0.36, and 0.30) and the 1D-AR subtype (time-course intensity changes: 1, 1.15, 1.26, 0.87, 0.44, and 0.54) displayed time-dependent decreases, consistent with A. The 1B-AR lane is overloaded by 5-fold for visualization of the protein. C, the decrease in gp-130 protein levels by 1A- and 1D-AR stimulation can be reversed with the 1-AR antagonist prazosin (1 µM), suggesting mediation through stimulation of the 1-AR.

IL-6 Regulation of STAT3 Activation and gp-130 Levels. To ensure that a correct dose of the neutralizing antibody was used, the IL-6-mediated phosphorylation of Tyr705 was first used in a dose-response to increasing concentrations of the neutralizing antibody (Fig. 6). Using a range of concentrations from 0.05 µg/ml to 5 µg/ml, only the 5 µg/ml dose provided sufficient blockage of the IL-6 response.

View larger version (57K): [in this window] [in a new window]   Fig. 6.   Dose-dependent antagonism of IL-6-mediated tyrosine phosphorylation of STAT3 with a neutralizing antibody against IL-6. IL-6 (40 ng/ml) was added to the culture medium of each 1-AR subtype cell line for 30 min in the presence or absence of an IL-6-neutralizing antibody at the indicated dose. Cell lysates were probed for STAT3 activation using an antibody that recognizes the Tyr705 phosphorylated form of STAT3. The blots were stripped and reprobed for total STAT3 protein levels. A level of 5 µg/ml of the neutralizing antibody was found to block IL-6 function.

View larger version (55K): [in this window] [in a new window]   Fig. 7.   1-AR- and IL-6-mediated activation of STAT3 tyrosine-phosphorylation. Epinephrine (10 µM) and/or IL-6 (40 ng/ml) were added to the culture medium of each of the three 1-AR subtype cell lines for 30 min in the presence or absence of the IL-6 neutralizing antibody (5 µg/ml). Cell lysates were probed for total STAT3 and Tyr705 phosphorylation status. All three 1-AR subtypes and IL-6 activated Tyr705 phosphorylation of STAT3. The neutralizing antibody only blocked the IL-6 activation. The combination of epinephrine and IL-6 was synergistic for only the 1A- and 1D-AR subtypes.

View larger version (39K): [in this window] [in a new window]   Fig. 8.   1-AR- and IL-6-mediated effects on gp-130 protein levels. Epinephrine (10 µM) and/or IL-6 (40 ng/ml) were added to the culture medium of each of the three 1-AR subtype cell lines for 3 h in the presence or absence of the IL-6 neutralizing antibody (5 µg/ml). Cell lysates were probed for gp-130 protein levels. The 1B-AR lane is overloaded by 5-fold for visualization of the protein. At 3 h of incubation, epinephrine caused the down-regulation of gp-130 protein levels for the 1A- and 1D-ARs but was constitutively down-regulated by the 1B-AR, as shown in Fig. 5. The IL-6 neutralizing antibody could not block this effect. However, the addition of IL-6 caused the up-regulation of gp-130, which was not blocked by the neutralizing antibody. The combination of epinephrine and IL-6 caused a synergistic effect on gp-130 by both the 1A- and 1D-AR subtypes but not the 1B-AR. The 1B-AR cell line was unresponsive to any treatment.

View larger version (54K): [in this window] [in a new window]   Fig. 9.   1-AR- and IL-6-mediated effects on STAT3 Ser727 phosphorylation levels. Epinephrine (10 µM) and/or IL-6 (40 ng/ml) were added to the culture medium of each of the three 1-AR subtype cell lines for 30 min. Cell lysates were probed for total STAT3 and Ser727 phosphorylation status. As before, the 1A- and 1D-AR activation increased Ser727 phosphorylation status, whereas 1B-AR activation did not. The addition of IL-6 also increased Ser727 phosphorylation but was not synergistic with the 1-AR.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Although much effort has focused on the regulation of ₁-AR subtype structure-function relationships and activation of common signaling pathways, no study has directly explored the comprehensive regulation of gene expression downstream of each subtype in the same cellular environment. This study represents the first microarray analysis of a GPCR in a transfected cell system. A common question raised in the GPCR field is whether each subtype can couple to different functions, or are they merely degenerate receptors? With the use of microarrays, we obtained the gene expression profiles generated by the ₁-AR subtypes (_1A-, _1B-, and _1D-) (epinephrine-treated) stably expressed in Rat-1 fibroblasts, and compared them with those of control cells (epinephrine-treated, nontransfected Rat-1 fibroblasts). We found that of 7000 genes in the microarray, epinephrine modified the expression of a total of 47, 55, and 53 genes by at least 2-fold relative to nontransfected controls for the _1A-, _1B-, and _1D-ARs, respectively. Of these, a profile was established that was highly similar to the three ₁-AR subtypes (38 genes; Tables 1 and 2). However, minor profiles that were unique to each ₁-AR subtype (Table 3) and a profile (IL-6 signaling genes) activated by two but not all three subtypes (Table 4) were also produced. The changes in gene expression, which were invoked by a 1-h activation protocol, are likely to be different from profiles generated from sustained activation. We chose the 1-h stimulation time point because many second-messenger studies use this same condition.

However, a limitation of our system is the use of a separate parental cell line that lacks the ₁-AR as the control instead of using each stably-transfected cell line as its own internal control of stimulated versus nonstimulated transcription. We did this for practical reasons. Therefore, with our experimental system, we cannot rule out gene expression changes that are caused by differences in cell line propagation. However, these should be rare events. Verification of the protein changes, however, used stimulated versus nonstimulated conditions within the same cell line. Along these same lines, we cannot rule out effects that are caused by subtype-specific but promiscuous couplings, because expression of the receptors is above physiological levels.

In these signaling studies, we used the endogenous ₁-AR ligand epinephrine and Rat-1 fibroblasts, a cell model commonly used in ₁-AR signaling studies (Garcia-Sainz et al., 1998; Chen et al., 1999), which we found to express similar densities of ₁-AR subtypes. Besides gene expression, another measure of ₁-AR function in Rat-1 fibroblasts included the activation of total IP accumulation by epinephrine. As expected, epinephrine stimulated the accumulation of IPs with higher efficacy in _1A-AR fibroblasts than the other two subtypes, consistent with previous studies of ₁-AR subtype-stimulated second messenger activation (reviewed in Zhong and Minneman, 1999). Interestingly, the gene expression changes in common between the three ₁-AR subtypes had similar fold changes, suggesting that the efficacy of the IP response does not influence the degree of expression changes. This could be because the IP effectors were saturated for all three subtypes, and the excess IP response may not be physiologically relevant. On the other hand, it may also suggest that a non-IP signaling molecule(s) may be responsible for the gene expression changes.

The largest profile generated by the ₁-AR subtypes was characterized by gene expressions commonly activated or repressed by all three ₁-AR subtypes (Tables 1 and 2), suggesting that a certain level of redundancy among ₁-AR subtypes exists. Several of these genes have been previously associated with ₁-AR activation in primary cell lines and tissues. For example, increases in mRNA levels for c-fos, an immediate-early gene that was highly up-regulated in the present study, have been reported after ₁-AR activation of cardiomyocytes (Deng et al., 1998), vascular smooth muscle cells (Okazaki et al., 1994), cerebral cortex (Shen and Gundlach, 2000), aortic rings (Carcillo and Hough, 1995), and hepatocytes (Im et al., 1998). ₁-AR activation in cardiomyocytes also results in activation of c-jun and egr-1 (Iwaki et al., 1990; Jin et al., 2000), both up-regulated in the present study. ₁-AR-mediated signaling in hepatocytes, renal tubular cells, and cardiomyocytes invoke protein tyrosine phosphatases and calcineurin (Aperia et al., 1992; Nguyen and Gao, 1999; Sugden, 2001). Furthermore, NF-1-X, a transcription factor up-regulated in this study, has been shown to bind to the _1B-AR promoter and regulate the expression of the _1B-AR gene (Gao and Kunos, 1998). Overall, evidence from the literature provides a certain measure of confirmation of the observed changes in the microarray and suggests that ₁-AR signals in Rat-1 fibroblasts can extend to other cell types and tissues. However, as an independent test of confirmation, we measured by Northern blotting the expression levels of two genes that showed significant changes in expression in the microarray. We found that the expression profiles of both c-fos and p21-cK-Ras were similar to actual mRNA levels (Fig. 2), but the increased sensitivity of the microarray analysis does lead to higher fold-changes.

A few additional genes that were commonly changed in expression by ₁-AR subtypes have never been associated with ₁-AR signaling (Table 1). These code for proteins that are primary regulators in cAMP-mediated responses (cAMP response element modulator protein and C/EBP-like factor), angiogenesis (VEGF and glioma-derived VEGF), fatty acid metabolism (stearoyl-CoA-desaturase 2), and formation of extracellular matrix (fibronectin, collagen III, and collagenase).

The existence of gene expression changes that were unique to each ₁-AR subtype suggests that nonredundant functions may exist (Table 3). One of the gene expressions that is the most robustly repressed in our study was synuclein (unique to the _1B-AR), which we have recently shown to be abnormally modified in transgenic mouse brains overexpressing this subtype (Papay et al., 2002). In addition, the modification of proapoptotic genes and those associated with neurodegeneration, including tau, synuclein, transforming growth factor 3, and caspase-6 by the _1B-AR subtype, is also supported by a neurodegenerative and apoptotic phenotype in the same transgenic mouse model (Zuscik et al., 2000), as well as a microarray analysis in transgenic brains (J. Yun, R. J. Gaivin, A. Boongird, Z. Ying, P. J. Gonzalez-Cabrera, R. S. Papay, I. Najm, and D. M. Perez, submitted), which contain caspase-3-mediated apoptosis, or in the heart, which contained terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive myocytes (Yun et al., 2003).

Gene expressions can be grouped into a third profile in Rat-1 fibroblasts through the activation of two but not three subtypes (Table 4) of the prototypic members of the IL-6 signaling pathway (STAT3 and gp-130). IL-6 is an endocrine cytokine that is secreted from fibroblasts, myocytes, and other cell types after inflammatory and stressful stimuli; its receptor mechanism includes the signal-transducing receptor gp-130. IL-6 signals through the JAK/STAT pathway induced by gp-130 (Taga and Kishimoto, 1997). Our results indicate that protein levels for gp-130 and STAT3 were differentially regulated by the three ₁-AR subtypes. Recent evidence suggests that increased protein levels of STAT1 by itself, which is not phosphorylated, is sufficient to drive the transcription of a subset of genes (Chatterjee-Kishore et al., 2000a,b), suggesting that ₁-AR-mediated increases in STAT3 protein may be physiologically relevant. Although the microarray studies successfully predicted changes in the protein levels for IL-6, gp130, and STAT3, the activational status for these proteins was more complicated. Our data showed that although all three subtypes stimulated the synthesis and secretion of IL-6 in Rat-1 fibroblasts (Fig. 3), only _1A- and _1D-AR subtypes increased both the protein level and the Ser727 phosphorylated form of STAT3 (Fig. 4), whereas _1B-AR did not. Figure 6 confirms that ₁-AR activation of STAT3 could be direct; at the very least, however, it occurs through non-IL-6 effects because a neutralizing antibody to IL-6 could not block it. Direct effects are also suggested by the synergistic stimulation of Tyr705 phosphorylation by both epinephrine and IL-6. However, this synergistic effect with IL-6 was weak or nonexistent with _1B-AR activation. The synergistic activation of STAT3 may have implications in heart failure, where there are elevations in both circulating catecholamines and IL-6. However, the most interesting effect was the _1B-AR activation of Tyr705 phosphorylation (Fig. 7) but not of Ser727 phosphorylation (Figs. 4 and 9). There is precedent for the differential phosphorylation of STATs. IL-1 has been shown to induce the phosphorylation of STAT1 on Ser727 but not Tyr 701 (H. Nguyen, M. Chatterjee-Kishore, Z. Jiang, Y. Qing, C. V. Ramana, J. Bayes, M. Commane, X. Li, and G. R. Stark, submitted).

STAT3 is activated by tyrosine phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding (Darnell et al., 1994). Transcriptional activation seems to be stimulated by serine phosphorylation at Ser727, apparently through MAPK or mammalian target of rapamycin (mTOR) pathways (Wen et al., 1995; Yokogami et al., 2000). Both phosphorylation events are not coupled but are independent of one another, although both are thought to be involved in transcriptional processes to achieve maximal effects (Wen et al., 1995). In the same Rat-1 fibroblasts that we used here, it has been shown previously that the ₁-AR subtypes can activate the MAPK pathways differentially with _1B-AR activation, resulting in increased p38 activity but not JNK activity, whereas the _1D-AR activated JNK but not p38 (Waldrop et al., 2002). In addition, _1B-AR-mediated inhibition of STAT3 in hepatocytes was found to be determined by p42/44 MAPK (Nguyen and Gao, 1999). These differences in MAPK activation could be the determining factor why the _1B-AR does not phosphorylate Ser727. Because the combination of epinephrine and IL-6 is not synergistic with Ser727 phosphorylation (Fig. 9), this also suggests that the two pathways converge at Ser727 phosphorylation, unlike Tyr705 phosphorylation. Nevertheless, the ability of the _1B-AR to suppress Ser727 phosphorylation is likely to produce differences in the transcriptional activity and eventual biological activity of STAT3.

Interestingly, whereas gp-130 protein levels were inhibited by long-term _1A- or _1D-AR stimulation (Fig. 5), they were constitutively down-regulated by the _1B-AR. Stimulation with IL-6 produced the opposite effect by increasing gp-130 protein levels (Fig. 8). The IL-6 neutralizing antibody could inhibit neither the epinephrine response nor the IL-6 response, suggesting that the IL-6-mediated changes in gp-130 protein levels are not a result of its biological function or signaling. However, the ₁-AR-mediated effects on gp-130 could be blocked with prazosin (Fig. 5C). The combination of both epinephrine and IL-6 were again synergistic, displaying even greater changes in gp-130 protein levels than either alone, suggesting independent events. The nonsignaling mechanism of the IL-6-mediated changes in gp-130 protein levels and the lack of ₁-AR-mediated IL-6 coupling, suggests that ₁-AR- and IL-6-mediated effects on gp-130 protein are different. ₁-AR-mediated changes are probably caused primarily by direct transcriptional events, as supported by the microarrays, and are not caused by activational paradigms with IL-6. However, the IL-6-mediated changes in gp-130 are probably caused by protein stability through complex formation (i.e., with IL-6/IL-6R/gp-130 trimer), which does not require IL-6 activation.

IL-6 is reported to increase in astrocytes and hepatocytes upon ₁-AR stimulation (Norris and Benveniste, 1993; Jung et al., 2000), confirming that our microarray data can be translated to endogenous systems. In addition, the _1B-AR transgenic mouse model also constitutively down-regulated gp-130 protein levels in the heart (Yun et al., 2003), again confirming the fibroblast results. Confirmatory for the _1A-AR-mediated increases in STAT3, Ser727 phosphorylation is also seen in the normal rat myocyte (P. J. Gonzalez-Cabrera, J. Yun, B. R. Rorabaugh, D. F. McCune, and D. M. Perez, in preparation), which is composed of both the _1A- and _1B-AR subtypes, but heart function is thought to be mainly driven by the _1A-AR (Lin et al., 2001). ₁-ARs as well as the Angiotension AT1 receptor have been previously linked to JAK/STAT activation (McWhinney et al., 1997; Zhong et al., 2000). Although there is controversy about whether this is caused by a direct coupling of the receptors to JAK/STAT, our data suggest at least a non-IL-6-mediated mechanism, because the IL-6-neutralizing antibody could not block STAT3 activation.

One of the most striking features of gene expression by ₁-AR subtypes in the present study was the appearance of numerous genes with known roles in the processes relevant to cellular growth or hypertrophy. A major effect of ₁-AR stimulation in the heart is the activation of growth-promoting pathways that lead to cardiac hypertrophy or vascular cell proliferation (Varma and Deng, 2000). Activation of all three ₁-AR subtypes increased the transcription of three potent inducers of myocyte hypertrophy (IL-6, LIF, and calcineurin), whereas the _1B-AR seems to inhibit STAT3 Ser727 phosphorylation and possibly gp-130-mediated pathways. The inhibition of both STAT3 and gp-130 protein levels has been implicated in wall thickening, heart failure, and apoptosis (Hirota et al., 1999). This suggests that the _1B-AR may not be as potent an inducer of hypertrophy as the other two ₁-AR subtypes. This was also suggested in Yun et al. (2003), in which many genes/proteins associated with hypertrophy were down-regulated in the heart by the _1B-AR in a transgenic mouse model. Although pharmacological studies have implicated the _1A-AR subtype in mediating the growth of rat myocytes (Rokosh et al., 1996), _1A-AR transgenic mice models have discarded this subtype as the sole mediator of hypertrophy (Lin et al., 2001; Rokosh and Simpson, 2002). Other studies have shown that overexpressed _1B-AR constitutively active mutants manifest a mild hypertrophic phenotype (Milano et al., 1994; Zuscik et al., 2001), whereas no evidence exists for the _1D-AR subtype in mediating any heart function (Tanoue et al., 2002). One study has even suggested that both the _1A- and _1B-AR subtypes need to be coactivated to achieve the previously reported robust hypertrophy phenotype seen in cultured cell models (McWhinney et al., 2000). Thus, although the subtypes may be differentially coupled to blood pressure homeostasis and induction of cardiac hypertrophy, it still seems that more than one ₁-AR subtype might be needed to mediate the same function.

Recent evidence indicates that vascular fibroblasts that make up the adventitial layer in blood vessels express functional ₁-AR subtypes (Faber et al., 2001; Zhang et al., 2002). These fibroblasts respond to catecholamines by activating growth and migratory pathways (vascular remodeling) as seen in vascular injury models. Here, too, gene expression modified by ₁-ARs in Rat-1 fibroblasts may be part of a coordinated regulation of groups of genes (i.e., IL-6, collagenase, VEGF, and fibronectin) whose products act in the remodeling (apoptosis, migration, extracellular matrix changes) process of vessels. Another gene cluster modified by the ₁-AR subtypes not reported here includes cell-cycle regulatory genes (i.e., cyclins, cyclin-dependent kinases, cyclin-dependent kinase inhibitors, etc.) that may be involved in differential ₁-AR subtype coupling to proliferative responses. In this cluster, we observe that the _1B-AR subtype signals differently from _1A- and _1D-AR cells, which display identical profiles with respect to this cluster (P. J. Gonzalez-Cabrera, J. Yun, B. R. Rorabaugh, D. F. McCune, and D. M. Perez, in preparation).

In conclusion, we show an in-depth comprehensive comparison of the gene expression changes caused by the ₁-AR subtypes. Although most of the gene expression changes were similar among the subtypes, some subtype-specific profiles were generated. This was verified at the protein and/or activational level for IL-6, gp-130, and STAT3 and suggests ₁-AR subtype-dependent differential coupling to pathways that can mediate hypertrophy and vascular remodeling.