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Am J Physiol Heart Circ Physiol 288: H69-H76, 2005. First published September 2, 2004; doi:10.1152/ajpheart.01223.2003
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TRANSLATIONAL PHYSIOLOGY

Distinct cAMP signaling pathways differentially regulate {alpha}2C-adrenoceptor expression: role in serum induction in human arteriolar smooth muscle cells

Maqsood A. Chotani, Srabani Mitra, Ali H. Eid, Seon A. Han, and Nicholas A. Flavahan

Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio

Submitted 30 December 2003 ; accepted in final form 19 August 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The physiological role of {alpha}2-adrenoceptors ({alpha}2-ARs) in cutaneous, arteriolar, vascular smooth muscle cells (VSMs) is to mediate cold-induced constriction. In VSMs cultured from human cutaneous arterioles, there is a selective increase in {alpha}2C-AR expression after serum stimulation. In the present study, we examined the cellular mechanisms contributing to this response. Serum induction of {alpha}2C-ARs was paralleled by increased expression of cyclooxygenase-2 (COX-2), increased release of prostaglandins, and increased intracellular concentration of cAMP. Inhibition of COX-2 by acetyl salicylic acid (1 mM), NS-398 (5 µM), or celecoxib (3 µM) abolished the increase in cAMP and markedly reduced {alpha}2C-AR induction in response to serum stimulation. The cAMP agonists, forskolin (10 µM), isoproterenol (10 µM), and cholera toxin (0.1 µg/ml) each dramatically increased expression of {alpha}2C-ARs in human cutaneous VSMs. The A-kinase inhibitor H-89 (2 µM) inhibited phosphorylation of cAMP response element binding protein, but not the increase in {alpha}2C-AR expression in response to these agonists. cAMP-dependent but A-kinase independent signaling can involve activation of guanine nucleotide exchange factors for the GTP-binding protein, Rap. Indeed, pull-down assays demonstrated Rap1 activation by serum and forskolin in VSM. Transient transfections using {alpha}2C-AR promoter-luciferase reporter construct demonstrated that Rap1 increased reporter activity, whereas the A-kinase catalytic subunit decreased reporter activity. These results indicate that cAMP signaling can have dual effects in cutaneous VSMs:activation of {alpha}2C-AR transcription mediated by Rap1 GTPase and suppression mediated by A-kinase. The former effect predominates in serum-stimulated VSMs leading to a COX-2, cAMP, and Rap 1-dependent increase in {alpha}2C-AR expression. Such increased expression of {alpha}2C-ARs may contribute to enhanced cold-induced vasoconstriction and Raynaud's phenomenon.

microcirculation; cyclooxygenase-2; Rap GTPase; A kinase

THE HEPTAHELICAL {alpha}2-adrenoceptors ({alpha}2-ARs), comprising subtypes 2A, 2B, and 2C, are a subfamily of G protein-coupled receptors that mediate a variety of physiological responses to the catecholamines epinephrine and norepinephrine (28). {alpha}2-ARs mediate constriction of cutaneous arterioles and veins, and vascular smooth muscle cells (VSMs) from these vessels demonstrate high expression of {alpha}2C-ARs (8). These cutaneous {alpha}2C-ARs have a unique function and role, mediating physiological constriction in response to cooling (7, 19). Furthermore, in VSMs from human cutaneous arterioles, serum stimulation selectively increases {alpha}2C-AR expression and function (8). Indeed, such abnormal expression of {alpha}2C-ARs may enable increased cold-induced vasoconstriction as seen in Raynaud's phenomenon.

The aim of the present study was to determine the subcellular signaling mechanisms underlying the serum-induced increase in {alpha}2C-AR expression in human cutaneous arteriolar VSMs.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Studies performed on human tissues were approved by the Biomedical Sciences Institutional Review Board of the Ohio State University.

Human VSM culture. Dermal arteriolar VSMs were cultured from arterioles isolated from upper arm skin punch biopsies as previously reported (8). VSMs were grown in Ham's growth medium composed of DMEM:F12 [50:50], 10% FBS plus L-glutamine, and antibiotics/antimycotic. Cells between passages 9 and 12 were used. VSMs (75%-85% confluence) were harvested as proliferating cells or made quiescent by serum deprivation (0.5% FBS) for 3 days. For serum stimulation, cells were cultured in quiescent medium for 72 h and then stimulated with 10% FBS for different time periods. Quiescent VSM were also treated with forskolin (10 µM, Sigma-Aldrich; St. Louis, MO), isoproterenol (10 µM, Calbiochem; San Diego, CA), or cholera toxin (0.1 µg/ml, List Biological Laboratories; Campbell, CA) for different time periods. Pharmacological inhibitors, including acetyl salicylic acid (aspirin, Sigma), NS-398 (Calbiochem), and celecoxib (kindly provided by Pharmacia; St. Louis, MO), were used at indicated concentrations. VSM were treated with inhibitors for 30 min before and during exposure to FBS, forskolin, isoproterenol, or cholera toxin.

RNase protection assays. Quantitative assays were performed as previously described (6, 8). The {alpha}2C-AR subtype-specific riboprobe gives a protected fragment of 348 nucleotides and the internal control GAPDH riboprobe a protected fragment of 433 nucleotides.

cAMP assays. Intracellular cAMP accumulation was measured by using a 125I-cAMP radioimmunoassay kit (Biomedical Technologies; Stoughton, MA). Cells were pretreated with 1 mM 3-isobutyl-1-methylxanthine 30 min before being harvested. Cells were placed on ice and washed twice with ice-cold phosphate-buffered saline, and lysed with 10 mM HCl in ethanol for 30 min. Samples were centrifuged and the supernatant was collected for measurement of intracellular cAMP production according to the manufacturer's instructions (nonacetylated protocol).

Prostaglandin assays. Prostaglandin E2 (PGE2) and 6-keto-prostaglandin F1{alpha} were measured using immunoassay kits (R&D Systems; Minneapolis, MN). Cells were treated with 10% FBS, and cell-culture supernatant removed at various time intervals (0–5 h), and stored in aliquots at –20°C. The assays were performed according to the manufacturer's instructions.

Transient transfections. Transient transfections were performed in proliferating VSMs using Tfx-50 (Promega; Madison, WI), and optimized for reagent:DNA ratio of 2:1, for 30 min in serum-free medium, as previously described (8). Cotransfections were performed with pRL-CMV [Renilla luciferase gene driven by the cytomegalovirus (CMV) immediate-early enhancer/promoter] that served as an internal control, along with firefly luciferase reporter. The reporters used were cAMP response element (4 copies, Stratagene; La Jolla, CA) driven luciferase reporter and {alpha}2C-AR promoter (–1,915/+5, relative to the transcription start site +1, kindly provided by Dr. Hervé Paris, Toulouse, France; 29) driven luciferase reporter. Also, effector molecules were examined in transfections and included the catalytic subunit of A-kinase (Stratagene), or constitutively active Rap1A GTPase [amino acid number 63 glutamine (Q) mutated to glutamic acid (E); Rap 1A-63E; kindly provided by LA Quilliam, Indiana University School of Medicine; 22].

For serum induction studies, VSM (~0.5 x 106) were cotransfected with 3.5 µg of firefly luciferase reporter plasmid ({alpha}2C-AR promoter or {alpha}2A-AR/–1,066/+928 promoter, kindly provided by Dr. Diane E. Handy, Boston, MA; Ref. 8), 0.001 µg of pRL-CMV and 0.499 µg of pGL3Basic (for a total of 4 µg) using Amaxa nucleofection (Amaxa; Gaithersburg, MD). After transfection, VSM were allowed to recover in Ham's growth medium and then placed in serum-free medium (0% FBS Ham's medium) for 48 h. VSM were treated with Ham's growth medium for 12 h, and cells harvested for analysis.

Western blot analysis. For COX analysis, VSMs were lysed in buffer (2% SDS, 25% glycerol, and 60 mM Tris, pH 6.8), and total protein concentration determined (bicinchoninic acid method; Pierce; Rockford, IL). Twenty micrograms of total cell lysates were separated on a 10% SDS-PAGE and immunoblotted with antibody specific for COX-1 or COX-2 (1:1,000 dilution for 1 h, at room temperature; Cayman Chemical; Ann Arbor, MI). The influence of forskolin, isoproterenol, and cholera toxin on the A-kinase substrate cAMP response element binding protein (CREB) was assessed by examining the phosphorylation status of CREB as well as total CREB (1:1,000 dilution for 1 h, at room temperature); using the PhosphoPlus CREB (Ser133) antibody kit (Cell Signaling Technology, Beverly, MA).

Rap GTPase activation was assessed by Rap pull-down assays (30). These assays were performed with 200 µg of cell lysate using the activation-specific probe corresponding to 97 amino acids of human Ral GDS-rap-binding domain (Upstate; Lake Placid, NY). Total Rap protein in arteriolar VSM was also examined using 30 µg of the same cell lysate. Antibodies specific for Rap 1 (1:500 dilution for 1 h, at room temperature, Upstate), and Rap 2 (1:2,500 dilution for 1 h, at room temperature, BD Transduction Laboratories; Lexington, KY) were used to analyze activated as well as total Rap. Blots were developed by ECL Western blot detection reagents (Amersham Biosciences; Piscataway, NJ) and quantitated by densitometry (Personal Densitometer, Molecular Dynamics; Sunnyvale, CA).

Statistical analysis. Mean values are expressed as means ± SE. Statistical analysis of the data was performed by Student's t-test for either paired or unpaired observations. When more than two means were compared, analysis of variance was used. If a significant F value was found, Bonferroni or Dunnett's test for multiple comparisons were employed to identify differences among groups. The analyses were performed using Prism version 3.00 for Windows (GraphPad Software; San Diego, CA). Values were considered statistically different when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Role of COX-2 in {alpha}2C-AR expression. Dermal arteriolar VSM {alpha}2C-AR expression was dramatically increased by serum (6.68 ± 0.81-fold increase in message after 12 h of serum stimulation [n = 19], P < 0.001; Ref. 8). The increase in {alpha}2C-ARs was paralleled by a serum-induced increase in COX-2 protein expression, which peaked between 4 and 8 h after initiation of serum stimulation (Fig. 1A; n = 3). This serum effect was not observed for COX-1, which showed constitutive expression (Fig. 1, and data not shown). The robust increase in COX-2 was associated with release of PGE2 (1.47 ± 0.25 ng/ml at 5 h, n = 8) and 6-keto-PGF1{alpha} (1.26 ± 0.11 ng/ml at 5 h, n = 8), the stable metabolite of prostacyclin (Fig. 1B). The nonselective COX inhibitor acetyl salicyclic acid (1 mM) reduced serum-induced {alpha}2C-AR message by 81.2 ± 3.8% (n = 7, P < 0.01; Fig. 2). To further distinguish between COX-1- and COX-2-mediated effects, selective COX-2 inhibitors NS-398 and celecoxib were utilized. NS-398 (5 µM) and celecoxib (3 µM) each showed effects similar to acetyl salicylic acid, reducing serum-induced {alpha}2C-AR by 76.0 ± 4.3% (n = 5, P < 0.01) and 75.1 ± 2.9% (n = 4, P < 0.01), respectively. These results suggest that COX-2 plays a critical role in serum-mediated increase in {alpha}2C-ARs in arteriolar VSM.



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  		Fig. 1. Cyclooxygenase (COX)-2 and {alpha}2C-adrenoceptors ({alpha}2C-ARs). A: COX-2, but not COX-1, is highly inducible by fetal bovine serum (FBS) in arteriolar vascular smooth muscle (VSM) (n = 3). The highest level of COX-2 protein was noted at 8 h after serum treatment, and this value was taken as 100 (left y-axis, bars). Representative profile of {alpha}2C-AR message at various times after serum exposure (0, 0.5, 1–3, 6, 12, 24 h, taken from Ref. 8). The level of {alpha}2C-AR message was determined by quantitative RNase protection assay. The highest level of {alpha}2C-AR message was noted at 12 h, and this value was taken as 100 (right y-axis, line graph). B: serum treatment (0–5 h) of arteriolar VSM leads to increased release of PGE2 and prostacyclin (PGI2; monitored by measuring the stable 6-keto-PGF1{alpha}), n = 8.

 


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  		Fig. 2. Effect of COX-2 inhibitors on {alpha}2C-AR induction. Representative autoradiograph illustrating serum induction of {alpha}2C-ARs in the absence or presence of the COX-inhibitor aspirin (acetyl salicylic acid; ASA). Yeast tRNA denotes control lane for background signal. Suppression of COX-2 activity by pharmacological inhibitors significantly reduced {alpha}2C-AR serum induction (ASA, 1 mM; 81.2 ± 3.8% inhibition, n = 7, P < 0.01, compared with FBS), NS-398 (5 µM; 76.0 ± 4.3% inhibition, n = 5, P < 0.01, compared with FBS), and celecoxib (3 µM; 75.1 ± 2.9% inhibition, n = 4, P < 0.01, compared with FBS). Q, quiescent cells; FBS, serum-induced cells (12 h).

 
COX-2, cAMP, and {alpha}2C-ARs. Serum stimulation of quiescent arteriolar VSM caused a time-dependent robust increase in intracellular concentration of cAMP, which paralleled the expression of COX-2, and prostaglandin release (Fig. 3). The serum-stimulated increase in cAMP was abolished by acetyl salicylic acid (1 mM, n = 6), NS-398 (5 µM, n = 4), or celecoxib (3 µM, n = 4; Fig. 3, P < 0.0001 for control vs. treated groups).



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  		Fig. 3. COX-2 and cAMP. Intracellular concentration of cAMP was measured over various time intervals (0 to 24 h) of serum (FBS) treatment, in the absence (control, n = 6), or presence of pharmacological inhibitors for COX, ASA (1 mM, n = 6), NS-398 (5 µM, n = 4), and celecoxib (3 µM, n = 4). The concentration of cAMP in the control samples was significantly higher than the concentrations in the treated groups (P < 0.0001).

 
To directly examine the role of cAMP in {alpha}2C-AR expression, VSMs were treated with forskolin (10 µM; n = 4), isoproterenol (agonist for the Gs coupled {beta}-ARs, 10 µM; n = 3–4), or cholera toxin (activator of Gs, 0.1 µg/ml; n = 3). Each stimulus increased {alpha}2C-AR message (Figs. 4 and 5, A and B). Forskolin and cholera toxin had the greatest effect, causing 12.65 ± 1.38-fold and 16.82 ± 4.15-fold increases in {alpha}2C-AR message, respectively, after 6 h of treatment (Figs. 4 and 5B). The profile of {alpha}2C-AR induction with isoproterenol was distinct, with peak message observed after 3 h (5.62 ± 1.16-fold increase; Fig. 5A), with the effect rapidly declining thereafter, falling below baseline level after 12 h.



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  		Fig. 4. Role of cAMP in {alpha}2C-AR expression. Representative autoradiographs illustrating the effect on {alpha}2C-AR expression on direct activation of adenylyl cyclase by forskolin (FSK; 10 µM, 0, 1, 2, 3, 6, 12, and 24 h, n = 4) in the absence or presence of the A-kinase inhibitor H-89 [2 µM, n = 4, P = not significant (NS)]. DMSO was used as a solvent control for H-89. Serum-induction of {alpha}2C-AR mRNA peaked at 12 h (6.68 ± 0.81-fold increase, n = 19), and is shown for comparison.

 


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  		Fig. 5. Role of cAMP and A-kinase in {alpha}2C-AR expression. A: activation of {beta}-ARs by isoproterenol (10 µM, 0, 2, 3, 6, 12 h, n = 3–4), in the absence or presence of H-89 (2 µM, 6 h, n = 3, P = NS). DMSO was used as a solvent control for H-89. B: ADP-ribosylation of G{alpha}s by cholera toxin (0.1 µg/ml, 0, 3, 6, and 12 h, n = 3), in the absence or presence of H-89 (2 µM, 6 h, n = 3–4, P = NS). Higher concentration of H-89 (5 µM) was also tested with isoproterenol and cholera toxin and gave similar results.

 
The selective A-kinase inhibitor H-89 (5, 29, 31), at a concentration of 2 µM (~42-fold above the inhibitor constant; Figs. 4 and 5, A and B), did not affect the increase in {alpha}2C-AR expression evoked by forskolin, isoproterenol, or cholera toxin. However, phosphorylation of the A-kinase substrate CREB was increased by forskolin (10 µM, n = 4, P < 0.01) or cholera toxin (0.1 µg/ml, n = 4; P < 0.01), and this effect was markedly inhibited by H-89 (2 µM; Fig. 6). These results suggest that in arteriolar VSMs, the increase in {alpha}2C-AR expression is not mediated by A-kinase-dependent signaling.



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  		Fig. 6. Effect of the A-kinase inhibitor H-89 (2 µM) on cAMP response element (CRE) binding protein (CREB) phosphorylation in forskolin (10 µM, 10 min, n = 4) or cholera toxin (CTX; 0.1 µg/ml, 10 min, n = 4) treated arteriolar VSM. H-89 inhibited phosphorylation of CREB in response to forskolin, or cholera toxin. Q, quiescent VSM. Forskolin caused a 4.2 ± 0.7-fold increase in phosphorylation, n = 4, P < 0.01, and cholera toxin a 2.2 ± 0.3-fold increase, n = 4, P < 0.01.

 
Rap GTPase and {alpha}2C-ARs. cAMP can also signal through Rap1 and Rap2, members of the Ras GTP superfamily binding proteins (12, 20). Western blot analyses demonstrated expression of both Rap1 and Rap2 in arteriolar VSMs. Rap pull-down assays were performed to assess the activation state of Rap proteins on forskolin treatment of quiescent VSM. Indeed, Rap1 was activated within 5 min after forskolin exposure (Fig. 7; 260.9 ± 23.3% increase, n = 3, P < 0.05). In contrast, Rap2 showed constitutive activity, and was not affected by forskolin [Fig. 7; 127 ± 34%, n = 3, P = not significant (NS)]. Serum also activated Rap1, but with a slower time course compared with forskolin (peaked after 1 h of serum stimulation, Fig. 8A, 358.7 ± 58.8% increase, n = 3; P < 0.05), consistent with the slower increase in {alpha}2C-AR expression. There was no significant change in Rap2 activation by serum (Fig. 8B).



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  		Fig. 7. cAMP and Rap GTPase activation in arteriolar VSM. Activation of small GTPases Rap1 and Rap2 was examined by pull-down assays (0, 5, 10, 30 min, n = 3). Forskolin activated Rap1 (A; maximum activation at 5 min, P < 0.05), whereas Rap2 (B) showed constitutive activity in arteriolar VSM, and was not affected by forskolin; P = NS.

 


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  		Fig. 8. Time course (0, 0.5, 1, 2, 3, 4, 5, 6 h, n = 3–4) of activation of Rap GTPases by serum. Serum activated Rap1 (A), which peaked at 1 h (P < 0.05), whereas Rap2 (B) did not show a significant response to serum (P = NS).

 
Transient cotransfections were performed in arteriolar VSM to assess the role of serum in transcriptional regulation of {alpha}2C-ARs. Indeed, serum increased {alpha}2C-AR-promoter-driven reporter activity by 3.5 ± 0.3-fold (n = 8; P < 0.001) at 12 h of serum stimulation. This effect of serum was not seen with the {alpha}2A-AR-promoter driven reporter (0.8 ± 0.1-fold, n = 4, P = NS). Experiments were performed to examine the role of A-kinase and Rap GTPase in transcriptional regulation of {alpha}2C-ARs. The catalytic subunit of A-kinase transcriptionally activated a luciferase reporter driven by four copies of CRE that responds to the A-kinase substrate CREB (increasing reporter activity from 28.7 ± 4 x 10–3 to 97.25 ± 21.3 x 10–3 firefly/Renilla luciferase ratio, P < 0.004; Fig. 9). In contrast, the A-kinase catalytic subunit suppressed {alpha}2C-AR promoter-driven reporter activity (decreasing from 66.05 ± 11 x 10–3 to 7.1 ± 0.9 x 10–3 firefly/Renilla luciferase ratio, P < 0.0014; Fig. 9). The constitutively active Rap 1 (Rap 1-CA) did not activate the CRE luciferase reporter (P = NS) but activated the {alpha}2C-AR promoter-driven luciferase reporter (increasing reporter activity from 66.05 ± 11 x 10–3 to 149.6 ± 37.2 x 10–3 firefly/Renilla luciferase ratio, P < 0.04; Fig. 9).



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  		Fig. 9. Role of A-kinase and Rap1 GTPase in transcriptional activation of {alpha}2C-AR promoter. The data are expressed as ratio of firefly luciferase reporter activity to the internal control Renilla luciferase activity and allow normalization for transfection efficiency. The empty cytomegalovirus (CMV)-driven expression plasmid (10–20 ng) was cotransfected with CRE and {alpha}2C-AR luciferase reporters, giving baseline reporter activity. The catalytic subunit of A-kinase (20 ng) activated the luciferase reporter gene driven by four copies of CRE (n = 6, P < 0.004) but inhibited basal {alpha}2C-AR (–1,915/+5, relative to the transcription start site +1) reporter activity (n = 6, P < 0.0014). The constitutively active Rap1–63E (Rap1-CA, 20 ng) increased {alpha}2C-AR reporter activity (n = 6, P < 0.04) but showed no effect on CRE reporter activity (n = 6, P = NS).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
{alpha}2C-ARs, expressed on VSMs, contribute to cold-induced vasoconstriction in the cutaneous circulation (7). Indeed, within the human circulation, {alpha}2C-ARs have high expression in cutaneous VSMs but are not expressed by aortic medial VSMs (8). Furthermore, {alpha}2C-AR expression in human cutaneous VSMs is dramatically and selectively increased by serum stimulation (8). The present study identifies an unusual signaling role for cAMP, independent of the classic A-kinase signaling cascade, in mediating this serum response. Indeed, although A-kinase inhibits transcription of the human {alpha}2C-AR (29, this study), the alternate cAMP-responsive Rap1 signaling pathway actually increased {alpha}2C-AR transcription. The present results suggest that serum increases {alpha}2C-AR expression in human cutaneous VSMs by a COX-2, cAMP, and Rap1-dependent increase in transcription. Pathological activation of this pathway could contribute to the cold-induced vasospasm occurring in Raynaud's phenomenon.

A crucial step in the response to serum was increased expression of COX-2. This enzyme converts arachidonic acid to prostaglandin H2 leading to the subsequent production of the prostaglandins thromboxane and prostacyclin (3). Indeed, serum stimulation caused a slow increase in cAMP accumulation in VSMs that paralleled induction of the COX-2 protein and prostaglandin release. Inhibition of COX-2 activity by acetyl salicylic acid, NS-398, celecoxib, markedly reduced the serum-induced increases in cAMP and {alpha}2C-AR expression. Likely candidates for coupling COX-2 to elevations in cAMP are the Gs coupled, E-prostanoid (EP) receptors (subtypes 2 and 4) and prostacyclin (IP) receptor (3, 4). This COX-2:cAMP signaling pathway was primarily responsible for serum induction of {alpha}2C-ARs and could be mimicked by elevating cAMP levels in the absence of serum using: forskolin, a direct stimulant of adenylyl cyclase, isoproterenol, a {beta}-AR agonist, or by cholera toxin, a direct activator of the Gs protein. Each of these stimuli caused dramatic increases in expression of {alpha}2C-AR, which occurred more rapidly than the response to serum. This would be consistent with the slow COX-2-dependent increase in cAMP in response to serum stimulation. Furthermore, with forskolin and with cholera toxin, the level of {alpha}2C-AR expression was over twofold that achieved after serum stimulation. All of the stimuli examined in the present study had a transient effect on {alpha}2C-AR expression, which mimicked the induction caused by serum (8). The transient nature of induction was especially notable with isoproterenol. Unlike the response to foskolin or cholera toxin, which peaked at ~6 h, the response to isoproterenol reached a maximum at ~3 h and had declined to basal levels by 12 h. Consistent with this abbreviated time course, the maximal effect of isoproterenol was significantly less than that achieved with forskolin or cholera toxin. The magnitude and kinetics of {alpha}2C-AR induction by isoproterenol are consistent with desensitization of {beta}-ARs, and degradation of cAMP by {beta}-arrestin-mediated recruitment of phosphodiesterases (27).

The powerful effect of cAMP agonists on the expression of {alpha}2C-ARs suggests that increases in cAMP are not only necessary but also sufficient for serum-induction of the receptor. This was somewhat surprising considering that cAMP, through the A-kinase signaling pathway, is known to inhibit the expression of human {alpha}2C-ARs. Schaak et al. (29) demonstrated an inhibitory role for cAMP and A-kinase signaling on transcription of {alpha}2C-ARs in the human hepatocarcinoma cell line HepG2. Indeed, in the present study, the A-kinase inhibitor H-89 did not affect the increase in {alpha}2C-AR expression evoked by forskolin, cholera toxin, or isoproterenol. Therefore, the classic cAMP-dependent A-kinase signaling pathway does not mediate the increase in {alpha}2C-AR expression in response to these stimuli in human cutaneous VSM. When the A-kinase catalytic subunit was expressed in human cutaneous VSMs, it inhibited the transcriptional activity of the {alpha}2C-AR promoter. This is consistent with the report by Schaak et al. (29) and indicates that A-kinase has the potential to inhibit {alpha}2C-AR expression. cAMP can also activate A-kinase independent signaling pathways. For example, the cAMP-responsive guanine nucleotide exchange factors Epac1 and -2 activate Rap1 and Rap2 (11, 26). This family of small GTPases includes Rap1A and 1B, which share >90% sequence homology (referred to as Rap1), and Rap2A and 2B (referred to as Rap2), which are 70% homologous to Rap1. Rap1 and Rap2 members differ by one amino acid residue in the effector domains implying similar effector interactions (2, 11). Results from the present study demonstrated that both Rap1 and Rap2 are expressed in cutaneous VSM. However, although the cAMP stimulus forskolin activated Rap1, Rap2 had constitutive activity and was unresponsive to the agonist. Transient transfections confirmed a direct role of Rap1 in transcriptional regulation of {alpha}2C-ARs. These results suggest that the Rap1 subtype may have a discrete role in {alpha}2C-AR expression in human cutaneous VSM. Altogether, these findings indicate a new and distinct role of cAMP-Rap1 signaling pathway in facilitating {alpha}2C-AR expression in cutaneous VSMs.

Therefore, cAMP can apparently have opposite effects on the transcriptional activity and expression of human {alpha}2C-ARs: inhibition, mediated by A-kinase-dependent signaling, and augmentation, mediated by Rap1-dependent signaling. The former effect was dominant in HepG2 cells (29), whereas the latter effect is dominant in cutaneous VSMs. Inhibition of A-kinase in VSMs did not enhance {alpha}2C-AR expression in response to cAMP stimuli. This suggests that the effect of cAMP in these cells is dominated by Rap1 signaling and is not restrained by the inhibitory effects of A-kinase-dependent signaling.

Although no functional effects of endogenous A-kinase could be observed on {alpha}2C-AR expression, overexpression of the catalytic subunit of A-kinase markedly reduced the transcriptional activity of the {alpha}2C-AR gene. Furthermore, the A-kinase substrate protein CREB (23) was phosphorylated in response to cAMP stimuli in VSMs, and the A-kinase inhibitor H-89 inhibited this effect. Therefore, these results suggest that A-kinase was activated in the VSMs and had the potential to inhibit {alpha}2C-AR expression. This suggests that these divergent cAMP-signaling cascades may be tightly controlled within discrete signaling compartments. For example, A-kinase anchor proteins play an important role in cAMP:A-kinase signaling by targeting the A-kinase to specific subcellular compartments (18, 24). Therefore, A-kinase may be targeted to a subcellular location that diminishes its inhibitory effect on {alpha}2C-AR expression. The mechanism(s) underlying the inhibitory effect of A-kinase on {alpha}2C-AR transcription has not been defined (29).

Previous studies (8) identified a crucial role of the p38MAPK in {alpha}2C-AR expression in human cutaneous VSMs. The p38 MAPK was necessary but not sufficient for {alpha}2C-AR expression. Indeed, p38 MAPK can mediate COX-2 induction and prostaglandin synthesis in non-VSMs (9, 10, 16, 21, 25). Similarly, in cutaneous VSMs, the p38 MAPK may augment the effect of COX-2 on {alpha}2C-AR expression.

In summary, the serum-mediated increase in {alpha}2C-AR expression by cutaneous VSMs involves COX-2-cAMP signaling. Increasing intracellular cAMP is sufficient to increase {alpha}2C-AR expression, and a cAMP-A-kinase-independent pathway preferentially triggers a robust increase in {alpha}2C-AR expression. The results identify an alternate, Rap1-dependent pathway for cAMP signaling in arteriolar VSM, facilitating acute {alpha}2C-AR expression. The {alpha}2-ARs have been implicated in mediating the cold-induced vasospasm of Raynaud's phenomenon (12), and selective increase in {alpha}2-AR responsiveness is observed in Raynaud's phenomenon (15) and in arterioles from scleroderma patients versus controls (14). Indeed, vasospastic attacks in Raynaud's phenomenon are believed to contribute to cycles of ischemia and reperfusion, known to involve COX-2 and postischemic inflammation, ultimately leading to local vascular injury (1, 13, 17). Conversely, vascular injury (vibration-induced mechanical, chemotherapeutic, or autoimmune disease related), may trigger an inflammatory response, leading to increased {alpha}2C-AR expression and secondary Raynaud's phenomenon (1). Signaling cascades identified in this study may facilitate increased expression of {alpha}2C-ARs, further contributing to the severity of the condition.


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TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants AR-46126 and HL-56091 from National Institutes of Health (to N. A. Flavahan), Scleroderma Research Foundation (to N. A. Flavahan), and American Heart Association, Ohio Valley Affiliate (to M. A. Chotani).


   ACKNOWLEDGMENTS
 
We sincerely thank Selvi C. Jeyaraj for assistance with cAMP assays, Dr. Hervé Paris (Institut National de la Santé et de la Recherche Médicale U388, Institut Louis Bugnard, Toulouse, France) for the {alpha}2C-(–1,915/+5) promoter-luciferase construct, Dr. Diane E. Handy (Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA) for the original {alpha}2A-(–1,066/+928) promoter construct, and Dr. Lawrence A. Quilliam (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN) for human Rap1A-63E expression construct. We also thank Dr. David Saffen and Ju Young Kim (Departments of Pharmacology and Psychiatry, The Ohio State University) for discussions and helpful suggestions for Rap pull-down assays, and Pharmacia (St. Louis, MO) for celecoxib.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. A. Chotani, Davis Heart and Lung Research Institute, Rm. 505, 473 W. 12th Ave., Columbus, OH 43210 (E-mail: chotani-1{at}medctr.osu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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