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TRANSLATIONAL PHYSIOLOGY
2-adrenoceptors in human vascular smooth muscle cells
1Davis Heart and Lung Research Institute, Ohio State University, Columbus, Ohio 43210; 2Institut National de la Santé et de la Recherche Médicale U388, Institut Louis Bugnard, 31403 Toulouse Cedex 4, France; and 3Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118
Submitted 27 March 2003 ; accepted in final form 22 August 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-adrenoceptors (2-ARs) in human vascular smooth muscle cells (VSMs). Saphenous veins and dermal arterioles or VSMs cultured from them expressed high levels of 2-ARs (2C > 2A, via RNase protection assay) and responded to 2-AR stimulation [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK-14,304, 1 µM)] with constriction or calcium mobilization. In contrast, VSMs cultured from aorta did not express 2-ARs and neither cultured cells nor intact aorta responded to UK-14,304. Although 2-ARs (2C >> 2A) were detected in aortas, 2C-ARs were localized by immunohistochemistry to VSMs of adventitial arterioles and not aortic media. In contrast with aortas, aortic arterioles constricted in response to 2-AR stimulation. Reporter constructs demonstrated higher activities for 2A- and 2C-AR gene promoters in arteriolar compared with aortic VSMs. In arteriolar VSMs, serum increased expression of 2C-AR mRNA and protein but decreased expression of 2A-ARs. Serum induction of 2C-ARs was reduced by inhibition of p38 mitogen-activated protein kinase (MAPK) with 2 µM SB-202190 or dominant-negative p38 MAPK. UK-14,304 (1 µM) caused calcium mobilization in control and serum-stimulated cells: in control VSMs, the response was inhibited by the 2A-AR antagonist BRL-44408 (100 nM) but not by the 2C-AR antagonist MK-912 (1 nM), whereas after serum stimulation, MK-912 (1 nM) but not BRL-44408 (100 nM) inhibited the response. These results demonstrate site-specific expression of 2-ARs in human VSMs that reflects differential activity of 2-AR gene promoters; namely, high expression and function in venous and arteriolar VSMs but no detectable expression or function in aortic VSMs. We found that 2C-ARs can be dramatically and selectively induced via a p38 MAPK-dependent pathway. Therefore, altered expression of 2C-ARs may contribute to pathological changes in vascular function.
microcirculation; MK-912; BRL-44408; p38 mitogen-activated protein kinase
2-ADRENOCEPTORS (ARs) are classified as 2A, 2B, and 2C (12) based on molecular, biochemical, and pharmacological approaches. Within the human vascular system, the functional activity of constrictor 2-ARs located on vascular smooth muscle cells (VSMs) is prominent in small arteries, arterioles, and veins but not in large arteries (8, 26). An impressive increase in 2-AR activity is also found in cutaneous blood vessels, where these receptors act as thermoreceptors to mediate cold-induced vasoconstriction (8, 10). Analysis of the intact cardiovascular system in mice with targeted deletion or mutation for 2-AR subtypes suggested that 2A- and 2B-ARs mediated vasoconstriction to 2-AR stimulation, but that 2C-ARs were not involved in vascular regulation (19). However, the thermosensory function of 2-ARs in cutaneous arteries of the mouse tail is mediated solely by VSM 2C-ARs (3, 18). Abnormal activity of 2C-ARs may therefore contribute to the cold-induced vasospasm of Raynaud's phenomenon (3, 18).
Although VSM 2-ARs may contribute to vascular disease, there have been no studies to analyze their regulation in humans. The present study was therefore undertaken to analyze the expression and regulation of 2-AR subtypes in VSMs derived from human blood vessels that display 2-AR contractile activity (saphenous veins and dermal arterioles) or lack 2-AR responsiveness (aortas) (9, 14, 20).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human VSM culture. Arteriolar VSMs were cultured from dermal arterioles using explant techniques (upper-arm skin-punch biopsies, 4–6 mm) and were grown in Ham's growth medium that contained a 50:50 DMEM/F-12 solution, 10% FBS, L-glutamine, and antibiotics/antimycotic. The four donors were Caucasians and included 23- and 72-yr-old males and 23- and 44-yr-old females. Aortic VSMs were also obtained by explant techniques using segments obtained from heart-transplant donors. All four donors were Caucasian and included 16- and 44-yr-old males and 47- and 54-yr-old females. These aortic VSMs were grown in Ham's growth medium. Aortic VSMs were also obtained from Clonetics (Walkersville, MD) and were cultured in Clonetics smooth muscle growth medium according to the manufacturer's instructions. Clonetics donors included a 2-yr-old Caucasian male, an 18-yr-old Caucasian female, and a 7-mo-old African-American male. There were no differences in 2-AR expression between the different sources of aortic cells. Saphenous vein VSMs were kindly provided by Dr. Peter Libby (Brigham and Women's Hospital) and were grown in DMEM as previously described (2). Cells from passages 9–12 were generally used. Using lower passage numbers of cells (down to passage 4, the lowest available) did not alter the expression of 2-ARs. All cultures were confirmed to be >99% pure by fluorescence-activated cell sorting analysis for expression of the VSM proteins -actin and basic calponin (23). VSMs (75–85% confluence) were harvested as proliferating cells or were made quiescent by serum starvation (0 or 0.5% FBS) for 3 days. For serum stimulation, cells were cultured in Ham's quiescent medium for 48–72 h and then stimulated with 10% FBS for different time periods. When pharmacological inhibitors were used, VSMs were treated with inhibitors for 30 min before and during exposure to FBS.
RNase protection assays. Riboprobes were generated from previously described constructs (1). The 2A-fragment in plasmid pSP72 was removed by digestion with BglII, filled in with dNTPs and Klenow fragment, and then digested with HindIII. This fragment was ligated into pBlueScript II SK+ digested with SmaI/HindIII. The 2C-fragment in pGEM3Z was removed by digestion with EcoRI and HincII and ligation into pBlueScript II KS+ digested with EcoRI/SmaI. The digested vectors and respective 2-AR fragments were gel purified (QIAquick, Qiagen; Santa Clarita, CA) before ligation. The orientation of these constructs was confirmed by diagnostic restriction-enzyme digestions and by sequencing. Runoff transcripts were generated by linearization of the plasmids with SpeI for 2A, HindIII for 2B (in pGEM-3Z), and EcoRI for 2C. A 780-bp PstI-XbaI human GAPDH cDNA fragment (25) was subcloned in pBlueScript KS+ and used to generate an antisense riboprobe. With the use of in vitro transcription, 32P-labeled 2A-, 2B-, and 2C-antisense riboprobes were generated to yield protected fragments for 2A (324 nt, spanning transmembrane domains 3–5), 2B (333 nt, spanning initial 3' untranslated region), and 2C (348 nt, spanning the third transmembrane domain and initial third intracellular loop). RNA was isolated using guanidine thiocyanate and cesium chloride and was subsequently analyzed by quantitative RNase protection assays (RPA) as previously described (2). The total RNA used in the assays was 2–5 µg. Aortas and saphenous veins were denuded of endothelium before RNA isolation. Gels were quantitated using a Molecular Imager system (Bio-Rad; Hercules, CA). Riboprobe specificity for 2-AR subtypes was examined by testing total RNA obtained from transiently transfected COS-7 cells. COS-7 cells were transfected with expression plasmids that encoded the three human 2-AR subtypes. Figure 1 reveals the effectiveness and lack of cross-reactivity of riboprobes.
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Calcium mobilization. Cells cultured on glass coverslips were equilibrated in Krebs-bicarbonate solution (KBS) for 60 min and then incubated with 5 µM fura-2 AM (Molecular Probes, Eugene, OR) for 30 min. The fura-2 AM was removed from the cells 20 min before imaging. Coverslips were incorporated into a chamber (Warner Instruments; Hamden, CT) and superfused (2 ml/min) with warm (37°C) and gassed (16% O2 and 5% CO2) KBS. Cells were visualized using a Zeiss inverted microscope equipped with an AttoFluor Ratiovision system (Atto Bioscience; Rockville, MD). Cells were alternately excited at 334 and 380 nm, and coordinated emission was detected at 510 nm using a charge-coupled device camera. Calcium mobilization in the cells was detected as an increase in the 334-nm signal, a decrease in the 380-nm signal, and an associated increase in the 334:380-nm ratio. Data are presented as changes in the ratiometric signal.
Contractile activity. Human aortas and saphenous veins were obtained from specimens discarded after transplantation or coronary artery bypass surgery, respectively. Rings of blood vessel were suspended for isometric tension recording in chambers filled with KBS as previously described (10). Aortic and venous rings were initially stretched to passive tension values of 7 and 2.5 g, respectively. Arterioles were isolated from human skin biopsies or from human aorta and were cannulated for vasomotor analysis as previously described (3, 9). Contractile responses were expressed as a percentage of the response to 60 mM KCl.
Promoter analysis. The 2A-promoter constructs in pCAT enhancer have previously been described (13). Three of these constructs (–1,066/+928, –1,066/+291, and –193/+291) were recloned in pGL3-basic enhancerless vector (Promega; Madison, WI). The –1,066/+928 promoter fragment in DH68 as well as the –1,066/+291 promoter fragment in DH123 were removed by digestion with BamHI and EcoRV and were directionally cloned into BamHI-EcoRV of pBlueScript II SK+; the –193/+291 promoter fragment in DH72 was removed by digestion with PstI and EcoRV and cloned into PstI-EcoRV of pBlueScript II SK+. The three promoter fragments were subsequently removed by digestion with BamHI and HindIII and directionally cloned into BglII-HindIII of pGL3-basic luciferase reporter vector. The orientation of each construct was confirmed by diagnostic restriction-enzyme digestion and sequencing.
The 2C-promoter fragments were in pGL3-basic luciferase reporter vector (Promega) as previously described (22). The pGL3Control (SV40 promoter plus enhancer), pGL3Promoter (SV40 promoter), and promoterless pGL3Basic (Promega; 5 µg of each vector) were used as controls.
VSMs were transfected under optimal conditions using Tfx-50 reagent (Promega): 2.5 x 105 cells were transfected with 5 µg of total DNA (2:1 reagent-DNA ratio) in aortic and arteriolar VSMs for 45 and 30 min, respectively, in serum-free medium. Cells were cotransfected with pRL-CMV (Renilla luciferase gene-CMV enhancer/promoter) that served as an internal control. Cells were washed twice with serum-free medium before transfection. After transfection, cells were washed once with growth medium and replaced with growth medium. Cells were harvested in passive lysis buffer 48 h after transfection and received one medium change. Reporter activity was measured using a luminometer (Lumat LB9507, EG&G Berthold) in 20 µl of lysate with 100 µl of firefly luciferase assay substrate, which was followed by 100 µl of Renilla assay substrate (Stop & Glow). Preliminary pRL-CMV titration experiments were performed to determine the ratio of firefly luciferase reporter to Renilla internal control (1:50 to 1:1,000) that gave reporter activity with firefly luciferase promoter constructs without interference of internal control. These experiments showed the optimum ratio to be 1:1,000, which also gave Renilla activity above the background value. Luciferase reporter (4 µg) was cotransfected with pRL-CMV (0.005 µg); this was adjusted to a total of 5 µg with pGL3-basic vector. The data was expressed as the ratio of firefly luciferase to Renilla activity.
Adenovirus transduction. Adenovirus transduction in arteriolar VSMs was initially optimized using a different number of virus particles per cell (multiplicity of infection) that ranged from 100 to 1,000 and was visualized with 
-galactosidase staining. The cells transduced with the selected multiplicity of infection of 750 were further analyzed for green fluorescent protein (GFP) gene expression using fluorescence-activated cell sorting analysis (FACSCalibur, Becton Dickinson; San Jose, CA) and showed nearly complete transduction. Cells were plated (six wells, 220 cells/mm2) 1 day before transduction. Cells were washed twice with PBS (calcium and magnesium free) and incubated with virus in 0.6 ml of serum-free Ham's growth medium (SF Ham's). Transduction was carried out on a rocking platform for 2 h; after this, the volume was brought up to 1 ml with SF Ham's, and transduction was then allowed to continue for an additional 13 h without rocking. The medium was subsequently replaced with 3 ml of fresh SF Ham's, and the cells were allowed to recover for an additional 25 h before serum stimulation.
Adenovirus vectors that express the dominant-negative isoform of p38 (p38-DN), constitutively active form of mitogen-activated protein kinase (MAPK) kinase 6b (MKK6-CA), or control vector that drives the expression of GFP were kindly provided by Jiahuai Han (Scripps Research Institute; Ref. 15).
Western blots. The influence of serum on the phosphorylation status of p38 MAPK was examined by harvesting VSMs before and at various times after serum stimulation. Cells were harvested by cold trypsinization and were collected by centrifugation (466 g). Cell pellets were washed in PBS that contained antiproteases [15.7 µg/ml each of chymostatin, antipain, and pepstatin; 57.7 µg/ml leupeptin; and 250 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride], snap-frozen on dry ice, and stored at –80°C until used. Pellets were lysed (2% SDS, 25% glycerol, and 60 mM Tris, pH 6.8), and total protein concentration was determined (BCA, Pierce; Rockford, IL). Total cell lysates (28 µg) were separated using 10% SDS-PAGE, and immunoblots were subsequently performed with affinity-purified rabbit polyclonal antibody specific for phospho-p38 MAPK (1:1,000 dilution for 1 h at room temperature; PhosphoPlus p38 MAPK antibody kit, New England Biolabs; Beverly, MA). This antibody recognizes the dual phosphorylated (Thr180 and Tyr182, TY) form of p38. Also, the level of the protein was determined by using another rabbit polyclonal antibody specific for p38 MAPK that did not distinguish phosphorylated and unphosphorylated forms. For 2-ARs, 5 µg of human embryonic kidney (HEK)-293 lysate protein (transfected with 2A- or 2C-ARs; Ref. 18) or 10 µg of VSM lysates were analyzed as previously described using affinity-purified rabbit polyclonal antibodies (3, 4, 18) directed against the 2-ARs. Western blots were developed using ECL and were quantitated by densitometry (Personal Densitometer, Molecular Dynamics).
Immunohistochemistry. Formalin-fixed paraffin-embedded tissue sections (4 µm) were deparaffinized in xylene and rehydrated through grades of ethanol (100 to 0%). Sections were treated with 3% H2O2 and then blocked with 2% goat serum, 0.1% BSA, and 0.05% Tween 20 (blocking buffer). Sections were then incubated with blocking buffer plus primary antibody (1:100 dilution): affinity-purified rabbit polyclonal 2C-antibody (3) or monoclonal mouse anti-human smooth muscle -actin (Dako; Carpinteria, CA). Biotinylated secondary antibody was subsequently used in combination with Vectastain Elite ABC reagent with nickel enhancement (Vector Laboratories; Burlingame, CA) to visualize the signal.
Statistical analysis. Statistical evaluation of the data was performed using Student's t-test for either paired or unpaired observations. When more than two means were compared, ANOVA was used. If a significant F value was found, Scheffé's test for multiple comparisons was employed to identify differences among groups. Values were considered statistically different when P was <0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-ARs in human VSMs. RPA analysis of cultured proliferating VSMs from different blood vessels demonstrated remarkable differences in 2-AR expression. For 2C-mRNA, there was high expression in dermal arteriolar VSMs, lower expression in saphenous vein VSMs, and no detectable expression in aortic VSMs (Fig. 2, A and B). For 2A-AR mRNA, there was low expression in dermal arteriolar VSMs and saphenous vein VSMs with no detectable expression in aortic VSMs (Fig. 2, A and B). There was no detectable expression of 2B-AR subtype in any VSMs (Fig. 2, A and B). Consistent with receptor expression, the 2-AR agonist UK-14,304 caused calcium mobilization in proliferating VSMs from dermal arterioles and saphenous veins but not from aortas (Fig. 2C and data not shown).
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The pattern of expression and activity of 2-ARs in cultured cells paralleled the contractile response to 2-AR stimulation in the native blood vessels: response was greatest in dermal arterioles and absent in human aortas (Fig. 3). Despite the absence of functional 2-ARs in aorta or in aortic VSMs and the lack of detectable 2-AR transcripts in cultured aortic VSMs, RPA analysis of whole aorta demonstrated high expression of 2C-AR mRNA with lower expression of 2A-ARs and no detectable expression of 2B-ARs (Table 1). Expression of 2C-ARs was similar to that observed in saphenous vein (Table 1). Immunohistochemical analysis of aorta demonstrated 2C-ARs in VSMs of vasa vasorum, but these were not detected in medial VSMs. Staining in vasa vasorum was similar to that observed in dermal arterioles (Fig. 3). Indeed, arterioles isolated from aortic vasa vasorum constricted in response to 2-AR stimulation in a similar manner as dermal arterioles (Fig. 3).
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Differential activity of 2-AR promoters in human VSMs. To determine whether the difference in expression of 2-ARs between aortic and dermal arteriolar VSMs resulted from differences in gene activation, transient transfections with promoter constructs were performed in cultured proliferating dermal arteriolar and aortic VSMs. Initially, relative activity levels of pGL3Basic, pGL3Control, and pGL3Promoter reporters were assessed in arteriolar and aortic VSMs and were comparable (Fig. 4A, inset).
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For 2A-ARs, higher promoter activity was observed in arteriolar compared with aortic VSMs for all promoter fragments studied: the –193/+291, –1,066/+928, and –1,066/+291 fragments (transcription start site taken as +1) had 9.7-, 5.5-, and 2.3-fold higher activity levels, respectively (Fig. 4A). Higher activity was also observed for all 2C-AR promoter fragments in arteriolar compared with aortic VSMs with the –236/+5 fragment showing sixfold higher activity in arteriolar compared with aortic VSMs (Fig. 4B). Additional deletion of 83 bp (–153/+5 fragment) decreased activity in arteriolar but not aortic VSMs, which suggests loss of a specific site for arteriolar activation (Fig. 4B).
Differential regulation of 2-AR subtype in human arteriolar VSMs. Serum increased 2C-AR mRNA in arteriolar VSMs (Fig. 5), which peaked after 12 h (667.8 ± 81.2% increase, n = 19; P < 0.001) and declined thereafter. The influence of serum was also evident in proliferating VSMs, which had a higher level of 2C-AR transcript and protein than quiescent cells (Fig. 6). In contrast, although serum caused a small, transient increase in 2A-AR message, this reversed to a significant decrease after 12 h (69.0 ± 19.7% decrease, n = 4; P < 0.05; Fig. 5). This inhibitory effect of serum was also evident in proliferating arteriolar VSMs, which had lower levels of 2A-AR transcript and protein than quiescent cells (Fig. 6). Similar results were obtained in saphenous vein VSMs (data not shown). In aortic VSMs, serum caused a small transient increase in 2C-AR message above detectable levels only after 12 h (data not shown).
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Role of p38 MAPK in serum induction of 2C-ARs. Serum activated p38 MAPK in dermal arteriolar VSMs. This was demonstrated by a significant increase in dual phosphorylation of p38 MAPK (Thr180/Tyr182), which peaked 5–10 min after serum stimulation (3.1 ± 0.5-fold increase in phosphorylation, n = 4; P < 0.05). Inhibition of p38 MAPK by pharmacological (2 µM SB-202190) or molecular (adenoviral transduction with dominant-negative p38 MAPK) intervention dramatically reduced 2C-AR induction in response to serum by 83.9 ± 1.3% (n = 5, Fig. 7A) and 81.2 ± 2.3% (n = 4, Fig. 7B), respectively. However, adenoviral transduction of VSMs with constitutively active MKK6, which stimulated p38 MAPK (4.6 ± 0.4-fold increase in phosphorylation, n = 3; P < 0.05) had no effect on 2C-AR mRNA (Fig. 7B). The serum-induced increase in 2C-AR message was completely abolished by the transcription inhibitor actinomycin D (2 µg/ml; Fig. 7A). Similar results were observed in saphenous vein VSMs (data not shown).
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Function of 2-AR subtype in human arteriolar VSMs. Activation of 2-ARs by 1 µM UK-14,304 stimulated calcium mobilization in control and serum-stimulated arteriolar VSMs (Fig. 8). In control cells, the response to UK-14,304 was inhibited by the selective 2A-AR antagonist BRL-44408 (100 nM) but was not affected by the 2C-AR antagonist MK-912 (1 nM), which suggests that the response was mediated predominantly by 2A-ARs (Fig. 8A). In contrast, after serum stimulation (24 h), the response to UK-14,304 was inhibited by 1 nM MK-912 but not by 100 nM BRL-44408, which suggests that it was mediated predominantly by 2C-ARs (Fig. 8B).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-AR subtypes in human vasculature. Unlike the ubiquitous nature of 1-ARs, 2-ARs mediate constriction of small arteries and arterioles and veins but not large arteries (see Fig. 3; Refs. 8, 10, 11, 26). In the present study, functional constraint on 2-ARs was also observed in VSMs cultured from different blood vessels: 2-AR stimulation caused calcium mobilization in VSMs from dermal arterioles and saphenous veins but not aortas. This constraint was caused by differences in 2-AR expression between these VSMs, which in turn reflected differences in the transcriptional activity of 2-AR gene promoters. The present study also demonstrates that when 2-AR subtypes are expressed in VSMs, they are subject to differential regulation. In arteriolar and venous VSMs, 2C-ARs were dramatically induced by serum stimulation, whereas 2A-AR expression was depressed, which resulted in a switch in functional activity of these receptor subtypes. Stress or injury to the vascular wall may alter 2-AR expression and lead to pathological changes in vascular function.
VSMs cultured from dermal arterioles and saphenous veins expressed high levels of 2A- and 2C-ARs but did not express detectable levels of 2B-ARs. This confirms that 2C-ARs are indeed "vascular" receptors in contrast to the original proposal that only 2A- and 2B-ARs were present and functional in the vascular system (19). In fact, the expression of 2C-ARs in these human VSMs far exceeds that of HepG2 and SK-N-MC cells (data not shown), which were previously proposed as suitable models to study regulation of endogenous human 2C-ARs (21). In contrast to dermal and saphenous VSMs, VSMs cultured from human aortas did not express detectable levels of 2-ARs and did not respond to 2-AR stimulation. Lack of 2-AR expression was confirmed using aortic cells from seven different donors including cultures obtained from commercial sources (see MATERIALS AND METHODS). This site-specific variation in 2-AR expression paralleled the functional activity of 2-ARs in the native blood vessels. Therefore, although human dermal arterioles and saphenous veins constrict in response to 2-AR stimulation, human aorta does not respond (see Fig. 3; Refs. 9, 14, 20). Somewhat surprisingly, aortas express significant levels of 2A- and 2C-ARs (see also Ref. 1) with expression of 2C-ARs similar to that observed in saphenous veins. Divergence between expression and function of 2-ARs in aorta likely reflects discrete localization of 2-ARs. Indeed, 2C-ARs, the most abundant 2-AR subtype expressed in aorta, were not detected in the medial VSMs of aorta but were expressed in VSMs of vasa vasorum. Indeed, in contrast to intact aorta, arterioles isolated from vasa vasorum of aorta constricted in response to 2-AR stimulation. Therefore, the disparate expression and function of 2-ARs between proximal and distal vessels in the arterial system is also evident within the same blood vessel, namely, aorta. The absence of 2-AR expression and function in VSMs cultured from aorta likely reflects expansion of cells from the media rather than vasa vasorum. However, culture of human VSMs was associated with a diminution in expression of 2-ARs. Compared with native saphenous vein, cultured VSMs derived from it had an 
10-fold reduction in 2A- and 2C-ARs and decreased expression of 2B-ARs below detectable levels (see Fig. 2B and Table 1). However, the relative expression of the 2-AR subtypes was maintained after culture, which suggests that cultured VSMs represent an important model to analyze regulation of these receptors. With regard to culture-induced alterations in receptor expression, Faber et al. (7) detected low-level expression of 2A-AR mRNA (no expression of 2B- or 2C-mRNA) in native rat aorta and at markedly lower levels in VSMs cultured from aorta.
Analysis of 2-AR promoter activity following transient transfection of proliferating VSMs demonstrated that the differential expression of 2-AR subtypes in human VSMs reflected differential activities of the 2-AR gene promoters. Both the 2A- and 2C-promoters were transcriptionally active in arteriolar VSMs but only weakly active in aortic VSMs. Our results support the notion of interplay between positive and negative factors in the transcriptional modulation of 2-AR gene expression in different VSM cell types. The 5' truncations of the promoters led to increased activity for the promoter regions specifically in arteriolar VSM but not aortic VSM. For 2C-ARs, positive elements regulating specific transcriptional activity in arteriolar VSM were narrowed down to an 83-bp promoter region between –236 and –153. The promoter fragment containing this region (–236 to +5) differs from the regions responsible for highest promoter activity in SK-N-MC (–1,184 to +5) and HepG2 cells (–742 to +5; Ref. 22) and confers arteriolar VSM-specific activity. The cis element(s) responsible for this activity remains to be identified. Previous reports have suggested that transcriptional activity of VSM genes may be regulated by methylation at CpG islands located in the promoter region with methylation leading to silencing or decreased transcriptional activity of the gene (27). Although the 5' untranslated region of the 2C-gene (+5 to +892) contains a CpG island, this mechanism does not appear to be responsible for low transcriptional activity of the 2C-gene in aortic VSMs because deletion of the region (–1,915 to +5) showed an unremarkable effect on promoter activity. For the 2A-promoter, positive elements that regulate specific transcriptional activity in arteriolar VSMs were narrowed down to the –193/+291 promoter region. This region conferred cell specific promoter activity and displayed highest activity in arteriolar compared with aortic VSMs. This region also showed highest promoter activity in HT29 cells (13). Important regulatory sites in this region include –92 to –105 (Sp1 site) and –70 to –87 (a palindromic sequence that contains an upstream stimulatory factor site). However, whether these elements are specifically involved in arteriolar VSM expression remains to be determined.
The expression of individual 2-AR subtypes was differentially regulated by the presence of serum. Whereas serum caused a rapid, large, and sustained induction of 2C-AR mRNA and protein, it actually suppressed expression of 2A-ARs. This modulation of 2A-ARs is consistent with a previous report that demonstrated an inhibitory effect of serum components on 2A-transcription in HT29 cells (5). However, it contrasts with a lack of effect of serum on the expression of 2-ARs in rat aortic VSMs (7). Serum induction of 2C-ARs in human VSMs was markedly reduced after inhibition of the p38 MAPK pathway (either by SB-202190 or a p38-DN mutant), which indicates a crucial role for this signaling pathway in the response. However, activation of the p38 MAPK pathway by a constitutively active MKK6 mutant had no effect on 2C-AR expression. Therefore, p38 MAPK signaling may not be solely responsible for the induction and may interact with an additional (serum-stimulated) signaling pathway(s) to increase 2C-AR expression. Alternatively, distinct patterns of p38 MAPK activation by serum and MKK6-CA may have initiated differing responses. Our results also indicate that the serum-induced increase in 2C-AR mRNA level likely involved a transcriptional response as inclusion of actinomycin D completely abolished the serum effect.
Serum induction of 2C-ARs and suppression of 2A-ARs were associated with a switch in the functional activity of these receptors. In control arteriolar VSMs, 2-AR stimulation caused calcium mobilization that was inhibited by the selective 2A-AR antagonist BRL-44408 but not by the selective 2C-AR antagonist MK-912, which indicates that the response was mediated predominantly by 2A-ARs. In contrast, after serum stimulation, the response to 2-AR stimulation was inhibited by MK-912 but not BRL-44408, which indicates that it was mediated predominantly by 2C-ARs. A similar switch in receptor function was observed in cutaneous blood vessels during cooling. Although 2A-AR activity predominated at warm temperatures, cold-induced vasoconstriction in mouse cutaneous arteries was mediated by 2C-ARs (3). Culture of VSMs in serum-free media induces a more differentiated phenotype in the cells, whereas exposure to serum mimics an injury or stress response to the cells (23). Therefore, 2C-ARs appear to be subdued in VSMs under normal conditions and become functionally active only under stressful conditions (e.g., cold, injury). Unlike traditional G protein-coupled receptors including 2A-ARs, 2C-ARs are less sensitive to desensitization (6, 16, 17) and can support prolonged activation of signaling pathways leading to changes in cell phenotype (24). Because of these unique signaling properties, 2C-ARs may function as a "stress receptor" for the vascular sympathetic system.
The results of the present study demonstrate that functional constraint on 2-ARs results from remarkable site-specific variation in expression of 2-ARs in human VSMs, namely, high expression in venous and arteriolar VSMs and undetectable expression in aortic medial VSMs. This in turn reflects site-specific differences in activation of the 2-AR gene promoters. When 2-AR subtypes are expressed in VSMs, they are subject to differential regulation. In arteriolar and venous VSMs, 2C-ARs were dramatically induced by serum stimulation, whereas 2A-AR expression was depressed, which resulted in a switch in the functional activity of these receptor subtypes. Stress or injury to the vascular wall may alter 2-AR expression and lead to pathological changes in vascular function.
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ACKNOWLEDGMENTS |
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2A-, 2B-, and 2C-AR riboprobes; Peter Libby and Marysia Muszynski (Brigham and Women's Hospital) for providing human saphenous vein smooth muscle cells; and Jiahuai Han (Scripps Research Institute) for providing Ad-p38-DN, Ad-MKK6-CA, and Ad-GFP. The authors also thank Merck (West Point, PA) and SmithKline Beecham (Harlow, UK) for the generous gift of 2-AR antagonists MK-912 and BRL-44408, respectively. GRANTS
This work was supported by National Institutes of Health Grants AR-46126 and HL-56091 (to N. A. Flavahan), a Scleroderma Research Foundation grant (to N. A. Flavahan), and an American Heart Association, Ohio Valley Affiliate grant (to M. A. Chotani).
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FOOTNOTES |
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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.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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