Analysis of the Human G Protein–Coupled Receptor Kinase 2 (GRK2) Gene Promoter
Regulation by Signal Transduction Systems in Aortic Smooth Muscle Cells
Background—Desensitization of G protein–coupled receptors (GPCR) is emerging as an important feature of several cardiovascular diseases. G protein–coupled receptor kinase 2 (GRK2) plays a key role in the regulation of a variety of these receptors, and its cardiac expression levels are altered in pathological situations such as chronic heart failure. However, very little is known about the signals and mechanisms that modulate GRK2 expression in cardiovascular cells.
Methods and Results—We have studied the transcriptional activity of the 1.6-kb-long proximal genomic region of the human GRK2 gene. In an aortic smooth muscle cell line, agents that lead to physiological vasoconstriction and hypertrophy, such as phorbol esters, increased GRK2 promoter activity. Activation of signaling pathways by cotransfected Gαq subunits or α1-adrenergic receptors also markedly enhanced the expression of the GRK2 promoter constructs. Conversely, proinflammatory cytokines, such as interleukin-1β, tumor necrosis factor-α, or interferon-γ, led to the opposite effect, decreasing the activity of the GRK2 promoter.
Conclusions—Our results suggest that the expression of GRK2 in vascular cells is tightly controlled at the transcriptional level by the interplay between several extracellular messengers, which may trigger alterations of normal GRK2 levels in some physiopathological circumstances, thus promoting changes in the efficacy of the GPCR signal transduction.
Gprotein–coupled receptors (GPCRs)1 are modulated by a family of specific G protein–coupled receptor kinases, or GRKs. Receptor phosphorylation by GRKs is followed by binding of uncoupling proteins called arrestins, leading to loss of receptor responsiveness, a process called desensitization.1 2 GRK2 is a ubiquitous member of the GRK family, able to phosphorylate a variety of GPCRs in an agonist-dependent manner, suggesting its participation in the modulation of multiple signaling pathways. Moreover, recent data indicate that, in addition to the uncoupling function, GRK2 and β-arrestin would also directly participate in β2-adrenergic receptor (β2AR) sequestration, thus providing the trigger for its resensitization (reviewed in References 3 and 43 4 ). The key role of GRK2 in GPCR desensitization mechanisms suggests that changes in the expression or function of this protein may affect the efficacy of signal transduction and underlie pathophysiological processes.
GRK2 displays a complex subcellular distribution, and its activity is modulated by a variety of mechanisms.1 2 4 However, very little is known about the modulation of the expression levels of GRK2. Recent data indicate that GRK2 levels change in some physiological circumstances, such as in the rat perinatal period5 or on lymphocyte activation,6 and are altered in pathological situations, such as in heart failure. Ventricular GRK2 levels are consistently higher in patients suffering dilated or ischemic cardiomyopathies7 and in related rat models,8 whereas GRK2 levels drop in the hearts of animals chronically treated with a β-adrenergic antagonist.9 The possibility that GRK2 plays an essential role in cardiovascular development and physiology is further emphasized by the fact that cardiac contractility can be modulated in vivo in transgenic animals overexpressing GRK2 or an inhibitory construct of this kinase10 and by the marked myocardial hypoplasia and embryonic death that occurs on disruption of the GRK2 gene in mice.11 In addition, GRK2 levels have been reported to increase in mouse models of cardiac hypertrophy12 and in certain hypertensive patients.13
The genomic organization of the human GRK2 gene was recently reported.14 However, to the best of our knowledge, no functional analysis of the GRK2 promoter has been reported to date. To contribute to a better knowledge of the signals and mechanisms governing cellular GRK2 levels, we have started to analyze the transcriptional regulation of the human GRK2 gene in cell lines of cardiovascular origin. Our results indicate that in aortic SMCs, the activity of the GRK2 promoter is stimulated by activation of the αq/protein kinase C (PKC) signaling pathway, whereas it is partially inhibited by certain cytokines.
The following cell lines from ATCC were used in this study: human U87 astrocytoma, human embryonic kidney-derived HEK-293, human Jurkat T lymphocyte, and A10 fetal rat aorta–derived SMC lines. Human or rat recombinant cytokines were obtained from Gibco BRL.
The pGL3 basic plasmid (Promega) was used as the expression vector where different fragments of the human GRK2 promoter gene were introduced. We used the SE3 and HBG7 fragments14 to generate a plasmid, called pBSPB4K, that encompasses ≈4 kb of genomic DNA corresponding to the promoter region of the human GRK2 gene. We then generated progressively shorter DNA fragments corresponding to the sequence immediately upstream of the GRK2 gene start point (see Results).
Cell Transfection and Treatments
Cells were maintained at 37°C in a 7% CO2 atmosphere in DMEM supplemented with antibiotics, 2 mmol/L glutamine, and 10% FCS (from Whittaker or BioIndustries, according to the different cell types). Jurkat cells were cultured in RPMI medium supplemented with 2 mmol/L glutamine, nonessential amino acids, and 5% FCS (Bio Industries). U87 and HEK-293 cells were transiently transfected by a standard calcium phosphate procedure, Jurkat cells by electroporation, and A10 cells with the lipofectamine reagent (Gibco BRL). The pSV–β-gal plasmid (Promega) constitutively expressing the β-galactosidase gene was cotransfected at a 1:3 ratio to normalize transfection efficiencies within individual experiments. When needed, a third expression plasmid coding for different G protein α-subunits (provided by Drs A. Aragay and M. Simon, Caltech, Pasadena, Calif) or the α1BAR (provided by Dr S. Cotecchia, Institut de Pharmacologie, Lausanne, Switzerland) was also cotransfected at a 1:2 ratio with respect to promoter-containing constructs. Overexpression of Gα subunits was checked by Western blot analysis using specific antibodies. After 24 to 40 hours, cells were washed in serum-free medium, and activators were then added for the times required at the concentrations indicated in the figure legends. Lysis was achieved with 200 μL of 1% Triton X-100 in an hypotonic buffer, 25 mmol/L Tris-phosphate pH 7.8, 2 mmol/L DTT, 2 mmol/L 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, and 10% glycerol. β-Galactosidase activity was determined in duplicate with purified β-galactosidase (Sigma) as standard, and luciferase activity was determined in a Monolight 2010 luminometer (Analytical Luminescence Laboratory). Data were obtained as relative light units and corrected according to β-galactosidase activity in each extract. The effect of treatments on GRK2 promoter activity was analyzed by comparison of luciferase expression between treated cells and controls consisting of transfected cells incubated in medium alone. Data were analyzed by ANOVA with Fisher’s post hoc least significant difference test (PLSD) by use of the Statview program.
Analysis of Endogenous GRK2 mRNA Levels
Total RNA was extracted from A10 cells by the method of Chomczynski and Sacchi.15 For RT-PCR analysis of GRK2 gene expression, we used primers (5′-TGGTCTTCTTACAGAAGTACC-3′, sense, and 5′-TGGATCTCTTCCATGGTCAGG-3′, antisense) corresponding to regions encoding the C-terminus sequence of rat GRK2. RNA (1 μg) was reverse-transcribed by use of the Gene Amp Kit (Perkin Elmer) according to the manufacturer’s instructions. Amplification of cDNA was achieved by 20 cycles (94°C, 35 seconds; 57°C, 35 seconds; 72°C, 90 seconds) followed by a 3-minute final extension at 72°C. Under these conditions, a linear quantification of amplified product was obtained by use of 0.2 to 1.4 μg of total RNA (data not shown). Controls were included to ensure that amplification did not derive from contaminating genomic DNA. The PCR products were visualized by ethidium bromide staining in agarose gels and evaluated by densitometry.
To analyze the structure and functionality of the human GRK2 gene promoter, we designed a series of plasmid constructs encompassing progressively shorter DNA pieces corresponding to genomic sequences located immediately upstream of the GRK2 transcription start site. These sequences were cloned in the pGL3–basic luciferase reporter system. Because the human GRK2 transcript has been reported to have an extensive 5′-untranslated region (5′-UTR), which may affect translation efficiency,14 we removed most of GRK2 5′-UTR in these constructs (178 of 245 bp). Five plasmids were thus generated with appropriate restriction enzyme digestions and were called pGL3-1.6, pGL3-0.9, pGL3-0.7, pGL3-0.3, and pGL3-0.02, according to the length in kilobases of the cloned insert (Figure 1A⇓).
Plasmid pGL3-1.6 contains most of the 5′-proximal genomic DNA sequenced thus far and was taken to be the “complete” promoter region. The activity of this promoter was analyzed by transient transfection experiments in different types of cells, such as human astrocytoma (U87 cell line), human embryonic kidney (HEK-293 cell line), rat embryonic aorta smooth muscle (A10 cell line), and human T lymphocytes (Jurkat cell line) (Figure 1B⇑). All 4 cell lines tested supported transcription mediated by this GRK2 promoter region, although with different efficiencies. Removal of the distal DNA pieces to pGL3-0.3 led to an ≈3-fold increase in transcription levels. Deletion of DNA to 50 bp downstream of the proposed distal transcription start site (pGL3-0.02 construct) resulted in a complete absence of promoter activity, indicating the loss of relevant signals supporting transcription. Because the pGL3-0.3 construct displays maximal promoter activity in all 4 cell lines, our data favor a model in which signals supporting basal activity of the GRK2 promoter are located in the short region of DNA included between positions −213 and +50. When relative levels of expression of the pGL3-1.6 or pGL3-0.3 plasmids are compared among the different cell lines, the order of expression is HEK-293 > Jurkat > A10 > U87 cells. Interestingly, the relative basal transcriptional activity of the GRK2 promoter in the human cell lines is consistent with the endogenous expression levels of GRK2 mRNA, as assessed by reverse transcription–polymerase chain reaction (RT-PCR) analysis using intronic primers (data not shown).
We next focused on the influence of cellular activation on GRK2 expression in the A10 vascular SMC line (A10 VSMC). As a first approach, we analyzed the modulation of GRK2 promoter either by protein kinase A (PKA)– or by PKC-mediated pathways. The results, shown in Figure 2A⇓, indicate that 10−8 mol/L of the PKC activator phorbol 12-myristate 13-acetate (PMA) is able to induce GRK2 expression (2- to 2.5-fold induction) in A10 VSMCs, whereas cAMP-mediated stimulation by the β-agonist isoproterenol or the adenylyl cyclase stimulator forskolin did not induce any significant effect. In all cases, long-lasting treatments of up to 18 hours were necessary to observe the induction effect.
The same experiments were performed in the U87 astrocytoma cell line for comparative purposes. The stimulatory effect of PMA was not observed in U87 cells (Figure 2A⇑) or in HEK-293 cells (77±10% and 80±23% compared with unstimulated cells for pGL3-1.6 and pGL3-0.3 plasmids, respectively), and only a slight decrease of ≈30% was observed in GRK2 promoter activity on incubation of U87 cells with forskolin. These data indicate that the effects of signal transduction modulators on GRK2 promoter activity are cell type–specific. A remarkable point is that the effects of phorbol esters were observed equally with either the completed promoter construct pGL3-1.6 or the “minimal” promoter region pGL3-0.3, indicating that signals responsible for both basal and modulated GRK2 transcription lay within the DNA region present in the short construct. To confirm the physiological significance of the observed effect, we also tested whether PMA treatment would modulate endogenous GRK2 mRNA levels. Figure 2B⇑ shows that on PMA treatment, GRK2 mRNA levels were increased 1.82±0.11-fold over control conditions (mean±SEM of 4 experiments), consistent with the promoter data. It could be argued that PMA may be affecting posttranscriptional events such as translational efficiency, perhaps affecting the phosphorylation and functionality of proteins of the translation machinery.16 However, transfection with plasmids when the 5′-UTR had not been deleted did not modify PMA inducibility in A10 cells (not shown), and low concentrations of actinomycin D abrogated PMA induction (not shown), indicating the need for some transcriptional event taking place. Therefore, these results suggest that PMA upregulates GRK2 expression in A10 cells by a process involving transcription-driven mechanisms.
A variety of vasoactive messengers are able to stimulate cellular cascades leading to PKC activation in VSMCs through Gq-coupled receptors.17 18 19 Therefore, we directly addressed the effect of expressing Gαq subunits or a constitutively active (αqR182C) mutant molecule.17 Overexpression of αq alone moderately increased the activity of the GRK2 promoter (50% increase, Figure 3A⇓), whereas the constitutively activated form showed a much stronger effect, resulting in a significant 4-fold increase in the expression of the GRK2 promoter. Whereas Gαo did not alter GRK2 construct expression (Figure 3A⇓), a constitutively active form (Q212L) of Gα16, another member of the αq family, showed a similar, though weaker, effect (1.5-fold induction, not shown). Interestingly, stimulation of cotransfected α1BARs also led to an increased transcriptional activity of the GRK2 promoter in A10 cells (Figure 3B⇓). Taken together, our data suggest that the stimulation of αq/PKC signaling pathways in VSMCs upregulates the transcription of the GRK2 promoter.
Inflammatory cytokines exert vasodilatory effects opposite to those of PKC-activating messengers (see References 19 and 2019 20 and references therein), so they were also tested for their ability to modulate the GRK2 promoter in A10 cells. Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) reduced the activity of the GRK2 promoter to ≈50% in transfected A10 cells (Figure 4⇓). Mixtures of 2 cytokines, such as TNF-α plus IL-1β or IFN-γ, showed a slightly more potent activity than single cytokines, although the effect was not additive. Tenfold lower concentrations of cytokines showed similar effects (not shown). Again, long-term treatments (18 hours) were needed to observe the effects of cytokines on GRK2 promoter–driven expression. A similar cytokine effect was found in the U87 cell line, suggesting that the mechanism of GRK2 regulation by cytokines might operate in different tissues and cell types. The inhibitory activity of the cytokines in A10 VSMCs was conserved regardless of the presence or absence of the full 5′-UTR (not shown), suggesting that this region is not the target of the cytokine actions.
Very little is known about the regulation of the expression of GRK2, a key regulatory enzyme of a variety of GPCRs involved in cardiovascular function. Using a series of plasmid constructs of the human GRK2 promoter, we have found that the signals necessary for basal transcription in 4 different cell types (astrocytoma, T lymphocytes, aortic smooth muscle, or embryonic kidney) are contained in a 213-bp-long proximal genomic DNA fragment and/or the initial 50 bp of the first exon. In addition, we also analyzed for the first time the regulation of the GRK2 promoter activity in cells of vascular origin and found that a variety of signal transduction modulators are able to control GRK2 transcription in a VSMC line.
Among the various agents that were tested for modulation of the GRK2 promoter, we found an increased promoter activity induced by phorbol esters and the opposite effect by several cytokines. The results concerning phorbol ester stimulation showed tissue specificity, because no effect is observed in some cells of noncardiovascular origin, such as the human U87 or HEK-293 cell lines. The stimulatory effect of PMA required rather long incubation periods and a transcriptional step, suggesting that PMA action on GRK2 promoter may require the synthesis of an intermediary factor. This is in agreement with the lack of clear consensus sequences responsive to PKC stimulation in the minimal GRK2 promoter region. The fact that PMA also promotes an increase in endogenous GRK2 mRNA levels in A10 cells strongly suggests a physiological role for this promoter regulation event.
Phorbol esters mimic the action of diacylglycerol, which is produced in cells on stimulation of phospholipase C. Several GPCRs that mediate the action of key cardiovascular messengers modulate phospholipase Cβ (PLCβ) activity through stimulation of G proteins of the αq family (Reference 2121 and references therein). Our data indicate that the expression and activation of αq subunits led to induction of GRK2 promoter activity similar to that observed with PMA. Our results clearly indicate that Gq signaling pathways are able to increase the transcriptional activity of the GRK2 promoter. Moreover, activation of α1BARs also led to GRK2 promoter stimulation. All these results suggest that in VSMCs, the activation cascade involving Gq-PLCβ-PKC stimulation is related to enhanced GRK2 expression. A similar induction of GRK2 activity and mRNA expression by PMA has been documented in T lymphocytes.6 Our results extend these observations to a different cell type and show that increased gene transcription is one of the mechanisms affected by PMA stimulation. Moreover, our data indicate a more physiological pathway for the modulation of GRK2 expression levels through GPCR activation of PKC.
Conversely, the proinflammatory cytokines IL-1β, TNF-α, and IFN-γ exert an opposite effect, downregulating the transcriptional activity of GRK2 promoter constructs. The long-term periods required to observe the reduction in luciferase activity by cytokines suggest again the need for intermediary molecules between cytokine actions and GRK2 promoter response. The mechanism for this effect of cytokines on GRK2 expression deserves further investigation; it is tempting to suggest that it may be related to the changes in NO synthase expression that mediate the vasodilatory actions of cytokines.19 20
Recent reports have shown that GRK2 function is of major relevance for cardiovascular physiology10 11 and that changes in GRK2 expression occur in the heart in some pathological or pharmacological situations.7 9 In VSMCs, vasoconstrictor messengers, such as endothelin, thrombin, or angiotensin, are coupled to PLCβ metabolism and the induction of mitogenesis or hypertrophy, as is PMA.3 18 19 21 22 Therefore, it might be hypothesized that hypertrophy-inducing messengers may affect GRK2 promoter transcription by activating the PLC/PKC pathway.
Interestingly, it was recently reported that pressure-overload cardiac hypertrophy leads to βAR desensitization and a 3-fold increase in GRK activity and GRK2 protein levels in the hearts of mice.12 The increase in GRK2 is not due to a general response to cellular hypertrophy and could be ascribed to neurohumoral mechanisms, such as increased adrenergic or angiotensin signaling.12 It is tempting to suggest that prolonged exposure to hypertrophic stimuli, such as norepinephrine (acting through α1AR), angiotensin II, or other messengers whose receptors are coupled to Gq proteins, would result in an increased expression of GRK2 in myocytes or VSMCs. Consistent with this hypothesis, we find that α1BAR activation increases GRK2 promoter activity in A10 cells; the fact that angiotensin II does not promote a similar effect might be related to the complement of receptor and/or receptor-regulatory proteins in these cells or to the need for a more persistent or strong activation of this signaling pathway. In this regard, recent data indicate that transgenic mice overexpressing α1BAR display enhanced PLC activity, βAR desensitization, and increased GRK2 activity in myocardial extracts.23 More interestingly, overexpression of Gαq in the heart in transgenic mice induces cardiac contractile failure and a marked decrease in βAR function without changes in βAR levels, strongly suggesting an alteration in GRK2 function and/or levels.21
In summary, the hypothesis that the in vivo activation of Gαq/PKC pathways could upregulate GRK2 expression in cardiovascular cells, thus contributing to the increased desensitization of β-adrenergic and other GPCR systems, is of potential physiological relevance. The study of the detailed mechanisms of regulation of GRK2 expression in different types of cells of the cardiovascular system, particularly in cardiac cell lines, and the determination of possible changes in GRK2 levels in VSMCs of patients with cardiovascular diseases are interesting fields for future research and may help to develop new therapeutic strategies based on the modulation of GRK2 expression.4
This study was supported by grants from the Ministerio de Educación y Ciencia (PM95-003 and PM98-0020), Comunidad de Madrid, and the European Union (BMH4-98-3566). The Centro de Biología Molecular receives an institutional grant from Fundación Ramón Areces. The authors thank Drs J.L. Benovic and A. Aragay for critical reading of the manuscript and M. Sanz and A. Morales for skillful secretarial assistance.
- Received July 23, 1999.
- Revision received October 29, 1999.
- Accepted December 2, 1999.
- Copyright © 2000 by American Heart Association
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