A luciferase-engineered cell line for study of cAMP regulation in endothelial cells

J. Xavier-Neto, A. C. Pereira, A. H. Motoyama, and J. E. Krieger

Laboratório de Biologia Molecular, Instituto do Coração, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

cAREL is a cAMP-responsive endothelial cell line carrying a luciferase reporter gene introduced by stable transfection of a luciferase enhancer trap into rabbit aortic endothelial cells. Luciferase gene expression in cAREL was stimulated 233-fold by 8-BrcAMP. Treatment with the beta -adrenoceptor agonist isoproterenol induced a 7.0-fold increase in luciferase expression, which was partially blocked by either beta 1- or beta 2-adrenoceptor antagonists and totally blocked by propranolol and by a combination of beta 1- plus beta 2-adrenoceptor antagonists. Receptor stimulation was mimicked by cholera toxin, forskolin, 8-BrcAMP, and isobutylmethylxanthine but not by 8BrcGMP, dexamethasone, or phorbol 12-myristate 13-acetate. Stimulation by isoproterenol was completely blocked by H-89, a protein kinase A inhibitor. cAREL was also stimulated by A-23187, and this effect was abrogated by EGTA and H-89. cAREL is the first cAMP-sensitive endothelial cell line described, and it can be useful as a positive control, as a model for cAMP regulation, as a background to genetic introduction of receptors, as an indicator of intracellular pathway activation, and as a tool to investigate cAMP effects on other signaling pathways.

endothelium; adenosine 3',5'-cyclic monophosphate; calcium

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE INVOLVEMENT OF signaling pathways in cellular processes is traditionally probed with pharmacological tools and direct measurements of second messengers (19). Usually, intracellular concentrations of second messengers are measured by immunologic and chemical methods, and different assays are utilized when more than one signaling pathway is being investigated (19). However, information from signaling pathways can be obtained in a single cell type with a single assay. Such cells can be developed by fusing a minimal promoter with tandem repeats of nucleotide sequences containing consensus motifs for transcriptional activation by different signaling pathways. Subsequently, the altered promoter is placed upstream from a reporter gene and stably transfected into a cell type. Several independent indicator cell lines can be obtained in which the expression of the reporter gene is under the control of a given signaling pathway. This strategy was developed by Meinkoth et al. (16) to study cAMP responsive element (CRE), 12-O-tetradecanoylphorbol 13-acetate (also called phorbol 12-myristate 13-acetate; PMA) responsive element (TRE) and serum responsive element (SRE) signaling pathways in fibroblasts (16). This panel of indicator cell lines has been successfully utilized to investigate signal transduction at the single- cell level using the lacZ gene as a reporter (1, 5).

An alternative way to generate systems in which reporter genes are driven by responsive elements is the enhancer trap method (18). Enhancer traps are reporter genes contained in a vector structure, usually a transposable element, that is suitable to random insertion of the construct in the genome of a given species. Reporter genes are usually very sensitive to regulation by adjacent sequences. Random insertion in the genome frequently places the reporter gene near potent promoters and enhancers determining positional as well as temporal patterns of expression of a downstream reporter gene. Enhancer traps have been most utilized in Drosophila to construct several lineages in which reporter or other genes of interest are expressed in specific and determined patterns in the Drosophila embryo (9, 17).

The methods for construction of indicator cell lines are of general application, and the concept has been successfully exploited to study fundamental aspects of signal transduction in fibroblasts (1, 5). However, it is unlikely that the fibroblast system will be useful for the study of regulation of cell-specific genes in other cell types.

Endothelial cell biology permeates many fields of knowledge (4), and various endothelial cell lines are available for the study of cell-specific regulation. To maximize information from a single cell type, we sought to develop a system of endothelial indicator cell lines. Here we report a sensitive cAMP responsive endothelial cell line obtained by stable transfection of a luciferase enhancer trap into rabbit aortic endothelial cells (REC) (4). In this cell line, luciferase expression is remarkably stimulated by cAMP and by agents acting on relevant steps in the beta -adrenergic/cAMP signaling pathway.

    METHODS
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Introduction
Methods
Results
Discussion
References

Materials. Cell culture materials and media were obtained from GIBCO. Isoproterenol, isobutylmethylxanthine (IBMX), 8-BrcAMP, forskolin, cholera toxin, 8-BrcGMP, propranolol, Ca2+ ionophore A-23187, EGTA, PMA, and dexamethasone were purchased from Sigma. H-89 was purchased from Calbiochem. CGP-20712A and ICI-118551 were generous gifts from Drs. Louis A. Barker and Maria Helena Catelli Carvalho, respectively. Cell lysis buffers and the luciferase assay system were from Promega. The beta -galactosidase assay system was from Tropix.

Plasmids. Luciferase reporter plasmid constructions were made by subcloning promoter fragments into the backbone of pGL2 basic (promoterless, enhancerless) vector (Promega). A positive control for CRE stimulation (CRETK) was constructed by subcloning a Xba I/Bgl II fragment from pTKCAT4CRE containing four tandem repeats of CRE consensus (TGACGTCA) linked to the herpes simplex virus thymidine kinase promoter LS-115/-105 (15) into the Nhe I/Bgl II sites of the pGL2 basic vector. The adenovirus E1b TATA box enhancer trap-luciferase reporter construct (TATALUC) was constructed by subcloning a Xba I/BamH I fragment from BCAT (23) containing the adenovirus E1b TATA box into the Nhe I/Bgl II site of pGL2 basic vector. BCAT was a generous gift from Dr. Trevor Williams.

Transient transfections. REC endothelial markers were previously characterized by Buonassisi and Venter (4) and further confirmed in our laboratory by typical cobblestone morphology, angiotensin-converting enzyme (ACE) activity, ACE mRNA expression, and uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil)-labeled acetylated low-density lipoprotein.

REC were grown in 12-well plates on F-12 medium (F-12 Coon's modification) supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin). Before REC reached confluence, they were transfected (16-24 h) with 3.25 µg/well of luciferase reporter plasmids and 0.48 µg/well of a control plasmid (LTR-beta -galactosidase) by the calcium phosphate method (8).

Stable transfections. CRETK and TATALUC plasmids were stably transfected into REC to generate CRETK (25) and TATALUC cell lines. The reporter plasmids were cotransfected with pSV7-neo (2) and selected with G418 at a concentration of 250 µg/ml. In brief, transfection was carried out in 60-mm tissue culture dishes by the calcium phosphate method (8). The relationship between reporter construct and pSV7-neo was 20:1 µg of supercoiled DNA. Total DNA content was 10.2 µg/dish. Individual clones were separated with cloning rings, expanded, and frozen. All reported experiments were performed on CRETK cell line 2, cAREL, and TATALUC clones B, C, and D.

Drug treatments. Stably and transiently transfected cells were cultivated in 24- and 12-well plates, respectively, with F-12 medium (F-12 Coon's modification) supplemented with 10% FBS. Before stimulation, cells were serum starved by 16-24 h on F-12 medium supplemented with 0.5% FBS. Antagonists and blockers were given to cells 30 min before stimulations and were maintained throughout the experiments.

cAMP measurements. cAREL cells were grown to confluence on F-12 supplemented with 10% FBS in 100-mm dishes as well as in 24-well plates. After 16-24 h on F-12 supplemented with 0.5% FBS, cAREL was treated with 1.0 µM isoproterenol, 100 µM IBMX, 10 µM forskolin, or 1 µU/ml cholera toxin for 90 min (100-mm dishes) or 4 h (24-well plates), respectively. Cells in 100-mm dishes were homogenized with cold 6% TCA, centrifuged at 2,000 g for 15 min at 4°C, and extracted with water-saturated diethyl ether. The aqueous phase was lyophilized and kept at -20°C until analysis. Samples were reconstituted to 1.0 ml, and cAMP measurements were performed in duplicate using a nonacetylation cAMP enzyme immunoassay kit (Biotrak, Amersham, code RPN 225). cAMP levels in cell extracts were normalized for protein concentration. Cells in 24-well plates were extracted for luciferase activity. cAMP data are expressed in femtomoles per milligram of protein in cell extracts. Results are means ± SE from three independent experiments.

Data presentation. In stably transfected cell lines, luciferase activity was normalized for protein concentration. In transient transfection assays, luciferase activity was normalized for beta -galactosidase activity driven by a cotransfected LTR-beta -galactosidase reporter gene to correct for differences in transfection efficiency. Data are expressed as means ± SE. When appropriate, results were analyzed by two-tailed t-test or by two-way ANOVA with Bonferroni t-test as a post hoc test. Statistical significance was set at P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

TATALUC was stably transfected into REC. As anticipated, all TATALUC clones showed low basal luciferase activity (Fig. 1 and data not shown), with cAREL showing the highest basal level. Treatment with 5 mM 8-BrcAMP for 4 h induced a 233-fold increase in luciferase activity in cAREL (Fig. 1) but did not induce significant increases in the remaining TATALUC clones (Fig. 1 and data not shown).


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Fig. 1.   Basal expression and cAMP responsiveness in 4 independent clones of stably transfected adenovirus E1b TATA box-luciferase reporter construct (TATALUC). TATALUC was cotransfected with pSV7-neo into rabbit aortic endothelial cells (REC), and stable transformants were selected with G418. Transfection was carried out in 60-mm tissue culture dishes by the calcium phosphate method. Ratio between reporter construct and neor plasmid was 20:1. Total DNA content was 10.2 µg/dish. Individual clones were separated with cloning rings. cAMP responsive endothelial line (cAREL) and TATALUCb, TATALUCc, and TATALUCd clones (Tata B, C, and D) were grown to confluence on F-12 supplemented with 10% fetal bovine serum (FBS) in 24-well plates. After 16-24 h on F-12 supplemented with 0.5% FBS, clones were stimulated for 4 h with 5 mM 8-BrcAMP. Luciferase activity in cell extracts was normalized for protein concentration. Data are expressed as luciferase units/mg protein in cell extracts. Results are means ± SE from 3 independent experiments (n = 4 in each). * Significantly different from control, P < 0.05. ** Significantly different from control, P < 0.01.

cAMP induction of cAREL was remarkable because CRETK, a cAMP-positive control cell line, showed much lower stimulation by cAMP in REC (1.85-fold, P < 0.01; Fig. 2). The mechanism of cAMP induction of the stably transfected adenovirus E1b TATA box in cAREL may depend on the intrinsic properties of the TATA box and associated vector sequences (13), or alternatively it might be due to modulation by nucleotide sequences located near sites of genomic integration. To explore these two possibilities, we tested cAMP responsiveness of the adenovirus E1b TATA box in transient transfection assays.


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Fig. 2.   cAMP responsiveness of cAREL as related to another cAMP-positive control cell line. Stably transfected CRETK cell line 2 and cAREL were grown to confluence on F-12 supplemented with 10% FBS in 24-well plates. After 16-24 h on F-12 supplemented with 0.5% FBS, stably transfected cell lines were stimulated for 4 h with 5 mM 8-BrcAMP. Luciferase activity in cell extracts was normalized for protein concentration. Data are expressed as luciferase units/mg protein in cell extracts. Results are means ± SE from a representative experiment (n = 4). ** Significantly different from control, P < 0.01.

Figure 3 shows that 5 mM 8-BrcAMP induced a 2.7-fold increase in luciferase activity driven by the positive control for CRE (CRETK), whereas transcription by the adenovirus E1b TATA box was not changed. Importantly, the failure of 8-BrcAMP to stimulate luciferase production by the transiently transfected adenovirus E1b TATA box was not due to luciferase activity being too low to measure, since it was five times greater than background (not shown). These data indicate that cAREL sensitivity to cAMP cannot be attributed to adenovirus E1b TATA box or to cryptic CREs in the vector backbone (13).


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Fig. 3.   cAMP responsiveness of transiently transfected TATALUC. REC were grown to subconfluent levels in 12-well plates and transiently transfected by the calcium phosphate precipitation method (16-24 h) with TATALUC, CRETK, and LTR-beta -galactosidase. One day after transfection, medium was changed to F-12 supplemented with 0.5% FBS for 16-24 h and cells were stimulated for 4 h with 5.0 mmol/l 8-BrcAMP. Luciferase activity was normalized for beta -galactosidase expression. Data are expressed as percent of control. Results are means ± SE from 3 independent experiments (n = 4 in each). ** Significantly different from control, P < 0.01.

The beta -adrenergic signaling system was used as a model to characterize cellular events linking surface receptor stimulation to cAMP-dependent signaling pathways in cAREL. Figure 4A shows that isoproterenol induces cAREL luciferase activity in a concentration-dependent manner from 0.01 to 10.0 µM.


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Fig. 4.   beta -Adrenergic regulation of cAREL. A: cAREL cells were grown to confluence on F-12 supplemented with 10% FBS in 24-well plates. After 16-24 h on F-12 supplemented with 0.5% FBS, cAREL was stimulated for 4 h with increasing concentrations of isoproterenol (Iso). Luciferase activity in cell extracts was normalized for protein concentration. Data are expressed as percent of maximal stimulation in each assay. Results are means ± SE from 6 independent experiments (n = 4 in each). B: cAREL cells were grown to confluence on F-12 supplemented with 10% FBS in 24-well plates. After 16-24 h on F-12 supplemented with 0.5% FBS, cAREL was stimulated for 4 h with 0.1 µM isoproterenol, alone or in presence of beta -adrenoceptor antagonists. C, control; ICI, 0.01 µM ICI-118551 (beta 2- antagonist); CGP, 0.01 µM CGP-20712A, (beta 1-antagonist); Prop, 0.1 µM propranolol (unselective beta -adrenoceptor antagonist). beta -Adrenoceptor antagonists were given to cells 30 min before stimulations and were maintained throughout experiments. Luciferase activity in cell extracts was normalized for protein concentration. Data are expressed in relation to maximal stimulation. Results are means ± SE from 3 independent experiments (n = 6 in each). ** Significantly different from control, P < 0.01.

Figure 4B shows that induction of luciferase activity by isoproterenol was partially inhibited by treatment with a beta 1-blocker (0.01 µM CGP-20712A). Treatment with a beta 2-blocker (0.01 µM ICI-118551) brought luciferase activity to levels that were not statistically significant from control, whereas either combined treatment with beta 1- and beta 2-adrenoceptor antagonists or treatment with 0.1 µM propranolol completely abolished isoproterenol-induced stimulation.

Results from Fig. 4A indicated that beta 2-adrenoceptors are responsible for a substantial fraction of isoproterenol-induced stimulation in cAREL. However, the role of beta 1-adrenoceptors is less clear, since treatment with a beta 2-adrenoceptor antagonist alone was able to reduce luciferase activity to levels that were not statistically significant from those of control.

To further substantiate the presence of beta 1-adrenoceptors, we stimulated cAREL with 0.1 µM isoproterenol in the presence of increasing concentrations of CGP-20712A. As shown in Table 1, CGP-20712A induced a concentration-dependent inhibition of isoproterenol stimulation that reached maximal levels at 0.1 µM. This result indicates that beta 1-adrenoceptors are indeed present in cAREL.

                              
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Table 1.   beta 1-Adrenoceptors in cAREL

To characterize intracellular events connected with beta -adrenergic signaling, REC were treated with cholera toxin, forskolin, 8-BrcAMP, IBMX and H-89. Cholera toxin, which stimulates Gs proteins (11), activated cAREL luciferase production by 87.0-fold (Fig. 5A). Forskolin, which directly activates adenylyl cyclase, stimulated luciferase production by 37-fold. The cAMP analog 8-BrcAMP (5.0 mM) induced cAREL luciferase expression by 86-fold. In contrast, 5.0 mM 8-BrcGMP, 100 nM PMA, and 1.0 µM dexamethasone were ineffective. Blockade of phosphodiesterase activity by 100 µM IBMX produced a lesser degree of stimulation (13.0-fold); however, when combined with isoproterenol, it induced luciferase expression to levels (71-fold) that were not different from those obtained with 5.0 mM 8-BrcAMP or 1 µU/ml cholera toxin (87- and 86-fold, respectively). Effects of isoproterenol plus IBMX on cAREL were almost completely blocked by H-89 at 25 µM, a cyclic nucleotide protein kinase inhibitor. Although H-89 is at least 10 times more effective as a protein kinase A (PKA) inhibitor than as a protein kinase G inhibitor (6), selectivity may be compromised at the concentration utilized. However, isoproterenol-induced stimulation of the adenovirus E1b TATA box in cAREL can be mimicked by 5.0 mM 8-BrcAMP but not by 5.0 mM 8-BrcGMP (Fig. 5A). This observation indicates that, in our system, the effects of H-89 were due solely to the inhibition of PKA.


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Fig. 5.   A: profile of cAREL stimulation. cAREL cells were grown to confluence on F-12 supplemented with 10% FBS in 24-well plates. After 16-24 h on F-12 supplemented with 0.5% FBS, cAREL was treated for 4 h with isoproterenol, isobutylmethylxanthine (IBMX), isoproterenol + IBMX, H-89, isoproterenol + IBMX + H-89, cholera (Chol) toxin, forskolin (FK), 8-BrcAMP, 8-BrcGMP, phorbol 12-myristate 13-acetate (PMA), dexamethasone (Dexa), or a Ca2+ ionophore (A-23187). Luciferase activity in cell extracts was normalized for protein concentration. Data are expressed in relation to maximal stimulation. Results are means ± SE from 10 independent experiments (n = 4 in each). B: activation of cAREL by beta -adrenergic and Ca2+ signaling pathways. cAREL cells were grown to confluence on F-12 supplemented with 10% FBS in 24-well plates. After 16-24 h on F-12 supplemented with 0.5% FBS, cAREL was treated for 4 h with 1 µM isoproterenol, 1 µM isoproterenol + 25 µM H-89, 25 µM H-89, 0.5 µM Ca2+ ionophore (A-23187), 0.5 µM A-23187 + 25 µM H-89, 2 mM EGTA, or 0.5 µM A-23187 + 2 mM EGTA and with 5 mM 8-BrcAMP. Luciferase activity in cell extracts was normalized for protein concentration. Data are expressed in relation to maximal stimulation (by 8-BrcAMP, not shown). Results are means ± SE from 3 independent experiments (n = 4 in each). * Significantly different from control, P < 0.05. ** Significantly different from control, P < 0.01.

Results in Table 2 show that luciferase activities in cAREL cells treated with 1.0 µM isoproterenol, 100 µM IBMX, 10 µM forskolin, and 1 µU/ml cholera toxin correlate directly with cAMP levels. This observation attests that luciferase production by cAREL is a bona fide detector of cAMP and indicates that cAREL can be used as a model system to study cAMP regulation in endothelial cells.

                              
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Table 2.   Luciferase assay directly correlates with cAMP levels in cAREL

As shown in Fig. 5B, luciferase activity in cAREL is also induced by treatment with the Ca2+ ionophore A-23187. A-23187 induction was much smaller than that by 8-BrcAMP, cholera toxin, or forskolin but was of the same order of magnitude as isoproterenol-induced activation. A-23187 effects were potentiated by increases in external Ca2+ up to 4 mM (shown) and were completely blocked by 2 mM EGTA (Fig. 5B). Interestingly, H-89 blocked cAREL stimulation either by isoproterenol or by A-23187 (Fig. 5B), suggesting that Ca2+ and cAMP signaling pathways converge at the PKA level or at earlier steps.

In summary, cAREL is a highly sensitive detector of cAMP pathway induction with sensitivity to Ca2+. These properties suggest that cAREL can be used to study regulation of endothelium-specific genes by extracellular receptors linked to the cAMP pathway or by cAMP-controlled transcription factors and coactivators.

    DISCUSSION
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In this work we described a cell model for study of the regulation of endothelial gene expression by the cAMP pathway. cAREL was identified after stable transfection of an enhancer trap and screening for cAMP responsiveness. cAREL showed the highest basal activity and a remarkable sensitivity to cAMP pathway stimulation. The mechanisms of luciferase induction by cAMP in cAREL remain to be established but, presumably, could include 1) trapping of a cellular cAMP responsive enhancer, 2) clustering of cryptic plasmid cAMP responsive sequences at chromosomal integration sites (13), and 3) direct activation of adenovirus E1b TATA box by transcription factors or coactivators of the cAMP pathway (1, 5).

If cryptic cAMP responsive sequences were present, direct activation of adenovirus E1b TATA box should occur in transient transfection assays. Failure of TATALUC to be activated by cAMP in these assays indicates that hypotheses 2 and 3 are highly unlikely. Lack of significant cAMP-induced luciferase expression in other independent clones indicates that cAMP responsiveness is a special feature of cAREL. This result suggests that, in this particular clone, random insertion of TATALUC in the genome most likely placed the adenovirus E1b TATA box close to a potent CRE.

The remarkable activation of cAREL contrasts with modest activation of CRETK (Fig. 2). These data suggest that in cAREL a specific ordering of CRE-like sequences or a totally unrelated and as yet unidentified CRE imparts cAMP regulation to the stably transfected adenovirus E1b TATA box. However, high induction in cAREL can also be explained by its low basal luciferase activity rather than by fundamental differences between the four canonical CREs contained in the stably transfected CRETK construct (15) and the putative cellular CRE controlling luciferase expression in cAREL. Support for this hypothesis arises from results depicted in Fig. 2 showing that absolute increases in cAREL luciferase activity after 8-BrcAMP stimulation were of the same order of magnitude as those obtained in the CRETK cell line. Despite great differences in relative levels of induction between cAREL and CRETK, data in Fig. 2 suggest that sequences underlying cAMP responsiveness in cAREL have a potential for absolute luciferase production that is similar to that of four tandem repeats of CREs of somatostatin (15). However, it is clear that disclosure of cAMP responsive sequences driving expression in cAREL must await molecular cloning of sequences flanking the stably transfected adenovirus E1b TATA box.

cAREL is also sensitive to Ca2+ signaling. A-23187 induction of cAREL is potentiated by increases in external Ca2+ (not shown) and inhibited by EGTA (Fig. 5B), indicating that influx of extracellular Ca2+ triggers activation of Ca2+-sensitive pathways and activates luciferase transcription by the stably transfected adenovirus E1b TATA box.

There are several possible mechanisms linking Ca2+ entry to gene activation in cAREL. First, it is possible that Ca2+ sensitivity results from an additional event of TATALUC integration near a Ca2+-responsive enhancer. However, it is highly unlikely that such random events could happen in a single cell clone. It is more likely that Ca2+ sensitivity in cAREL is due to interactions between cAMP and Ca2+ signaling pathways, as observed in other cell models (14, 21, 22). Molecular substrates of cross talk between Ca2+ and cAMP pathways include Ca2+/calmodulin-sensitive adenylyl cyclases (14, 22) and Ca2+/calmodulin kinases (calmodulin kinases I and II) that phosphorylate CRE binding protein (CREB) at the critical serine-133 residue (21). Ca2+/calmodulin-sensitive adenylyl cyclases have been identified in brain tissue, where they are implicated in learning tasks (14), whereas calmodulin kinases I and II have been shown to mediate activation of the c-fos promoter by Ca2+ ionophore A-23187 in PC-12 pheochromocytoma cells (21).

Figure 5 shows that A-23187 activation of cAREL is completely inhibited by the PKA inhibitor H-89 (25 µM). This is consistent with a calmodulin-sensitive adenylyl cyclase rather than with phosphorylation of CREB by Ca2+/calmodulin kinases; however, no further experiments were done to test this hypothesis. It might be argued that H-89 inhibition of A-23187-induced activation of cAREL is due to its weak Ca2+/calmodulin kinase II inhibitory activity (4). However, this is unlikely, since the inhibition constant (Ki) for inhibition of Ca2+/calmodulin kinases by H-89 is three orders of magnitude higher than the Ki for PKA inhibition by H-89 (6). Besides, Chijiwa et al. (6) have utilized H-89 at up to 50 µM to block PKA without interfering with Ca2+/calmodulin kinases in PC-12 cells.

cAREL induction by agents acting on the cAMP pathway is sensitive enough to allow its utilization as an indicator cell line. We have anticipated several ways in which cAREL could be useful. In the simplest way, cAREL could be utilized as a sensitive positive control cell line for gene activation by beta -adrenergic and cAMP pathways.

cAREL could also be used to study patterns of gene activation by cAMP and by Ca2+ after complex stimuli such as ischemia and reperfusion. In this experimental paradigm, generation of intracellular cAMP reduces damage and vascular permeability in lung endothelial cells (12), where both cAMP and Ca2+ are thought to play important roles in the control of cell shape (20) and fluid transport across capillaries (12).

With luciferase as its reporter, cAREL qualifies as a low-background quantitative assay with which to study the basic principles of molecular regulation by cAMP either in cell populations or in individual cells through microinjection studies (16) followed by luciferase immunohistochemistry. As assessed in Fig. 4, beta -adrenergic stimulation in cAREL is due to a combination of beta 1- and beta 2-adrenoceptors. Isoproterenol stimulates luciferase production in a concentration-dependent fashion up to 10 µM. Higher concentrations of isoproterenol actually reduce luciferase production, a well-known characteristic of beta 2-adrenergic signal transduction, which is currently thought to involve multiple mechanisms such as phosphorylation of beta 2-adrenergic receptors by a specific kinase (3) and decreases in beta 2-adrenoceptor mRNA production and stability (10).

Our data on the beta 2-adrenergic system are a limited prospect of the potential of cAREL for the study of signal transduction. As demonstrated in Fig. 4, plasma membrane receptors in cAREL interact with an integral system of signal transduction leading from the cell membrane to gene regulation in the nucleus. This makes cAREL a potential tool for study of signal transduction by other receptors, either by transient or by stable introduction of receptors linked to the cAMP pathway. A transient transfection strategy could also take advantage of the low basal luciferase activity of cAREL to clone receptors and intracellular proteins interacting with the cAMP pathway.

Finally, cAREL could be employed to quantitatively study endothelium-specific gene regulation by cAMP, to study the role of the cAMP pathway on other signaling systems, or, conversely, to study the interactions of other signaling pathways with the cAMP system. In this sense, cAREL and further endothelial indicator cell lines could be an alternative model to test the surprising overlapping between Ca2+ (21), CRE, TRE, SRE (1), mitogen-activated protein kinase (25), and nuclear receptor (5) pathways detected in other cell types.

    ACKNOWLEDGEMENTS

We are indebted to Dr. Louis A. Barker (Dept. of Pharmacology and Experimental Therapeutics, Louisiana State University Medical Center, New Orleans, LA) for helpful comments and provision of CGP-20712A, to Dr. Maria Helena Catelli Carvalho (Departamento de Farmacologia do Instituto de Ciências Biomédicas da Universidade de São Paulo, São Paulo, Brazil) for the donation of ICI-118551, and to Dr. Jenny Carrero (Department of Surgery, Massachusetts General Hospital, Charlestown, MA) for assistance with cAMP measurements. We are also grateful to Dr. Nadia Rosenthal (Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA) and members of the Rosenthal laboratory for support during the final steps of this work.

    FOOTNOTES

This research was supported by Fundacao de Amparo a Pesquisa do Estado de São Paulo Grant 4668-6, Conselho Nacional de Desenvolvimento Cientifico e Tecnologico Grant 520696/95-6, and Financiadora de Estudos e Projetos Grant 66.93.0023.00.

Present address of J. Xavier-Neto: Cardiovascular Research Center, Massachusetts General Hospital, 149 13th St., Charlestown, MA 02129 (xavier{at}helix.mgh.harvard.edu).

Address for reprint requests and for requests for cells: J. Xavier-Neto, Laboratório de Biologia Molecular, Instituto do Coração, Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Eneas C. Aguiar 44, São Paulo 05403-000, Brazil.

Received 24 March 1997; accepted in final form 30 March 1998.

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Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(1):C75-C81
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