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(Circulation. 2000;102:793.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Overexpression of G Protein–Coupled Receptor Kinase-2 in Smooth Muscle Cells Attenuates Mitogenic Signaling via G Protein–Coupled and Platelet-Derived Growth Factor Receptors

Karsten Peppel, PhD; Anne Jacobson, BA; Xuewei Huang, BS; John P. Murray, BS; Martin Oppermann, MD; Neil J. Freedman, MD

From the Department of Medicine (Cardiology), Duke University Medical Center, Durham, NC, and the Department of Immunology, Universitätskliniken, Göttingen, Germany (M.O.).

Correspondence to Neil J. Freedman, MD, Box 3187, Duke University Medical Center, Durham, NC 27710. E-mail neil.freedman{at}duke.edu

	Abstract

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Background—Neointimal hyperplasia involves activation of smooth muscle cells (SMCs) by several G protein–coupled receptor (GPCR) agonists, including endothelin-1, angiotensin II, thrombin, and thromboxane A₂. Signaling of many GPCRs is diminished by GPCR kinase-2 (GRK2). We therefore tested whether overexpression of GRK2 in SMCs could diminish mitogenic signaling elicited by agonists implicated in the pathogenesis of neointimal hyperplasia.

Methods and Results—Overexpression of GRK2 was achieved in primary rabbit aortic SMCs with a recombinant adenovirus. Control SMCs were infected with an empty vector adenovirus. Inositol phosphate responses to endothelin-1, angiotensin II, thrombin agonist peptide, and platelet-derived growth factor (PDGF) were attenuated by 37% to 72% in GRK2-overexpressing cells (P<0.01), but the response to the thromboxane A₂ analogue U46619 was unaffected. GRK2 also inhibited SMC [³H]thymidine incorporation stimulated not only by these agonists (by 30% to 60%, P<0.01) but also by 10% FBS (by 35%, P<0.05). However, GRK2 overexpression had no effect on epidermal growth factor–induced [³H]thymidine incorporation. Agonist-induced tyrosine phosphorylation of the PDGF-ß receptor, but not the epidermal growth factor receptor, was reduced in GRK2-overexpressing SMCs. GRK2 overexpression also reduced SMC proliferation in response to endothelin-1, PDGF, and 10% FBS by 62%, 51%, and 29%, respectively (P<0.01), without any effect on SMC apoptosis.

Conclusions—GRK2 overexpression diminishes SMC mitogenic signaling and proliferation stimulated by PDGF or agonists for several GPCRs. Gene transfer of GRK2 may therefore be therapeutically useful for neointimal hyperplasia.

Key Words: muscle, smooth • cells • signal transduction • receptors

	Introduction

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Vascular injury results in endothelial dysfunction, mechanical stress, and growth factor– or cytokine-mediated activation of medial smooth muscle cells (SMCs), which consequently proliferate and migrate into the subintimal space.¹ This neointimal hyperplasia (NH) compromises venous bypass grafts² and percutaneous coronary interventions, despite intravascular stent implantation.³ Growth factors implicated in SMC activation include several agonists for G protein–coupled receptors (GPCRs): thrombin,⁴ endothelin-1 (ET-1),⁵ angiotensin II (Ang II),⁶ and thromboxane A₂ (TXA₂).⁷ Like many GPCRs, receptors for these agonists desensitize rapidly,^{8 9} in a process that appears to involve GPCR kinases (GRKs), a family of enzymes that phosphorylate agonist-occupied heptahelical receptors.⁹ GRK2, when overexpressed in model transfected cells, diminishes signaling by the endothelin A and B receptors, the Ang II type 1 receptor, and the thrombin receptor.⁸

The contribution of GPCR activation to experimental NH has been demonstrated by studies that used antagonists for the type 1 Ang II⁶ or endothelin receptors.⁵ However, inhibition of single GPCR or receptor tyrosine kinase¹⁰ signaling systems has invariably resulted in only partial inhibition of experimental NH. This incomplete efficacy of individual receptor antagonists suggests the desirability of strategies capable of targeting multiple receptor signaling systems simultaneously.

One candidate molecular strategy for reducing SMC mitogenesis is the cellular overexpression of GRK2, because of its ability to suppress signaling through a number of receptors coupled to phosphoinositide hydrolysis via G_q in model transfected cell systems.⁸ To test the ability of GRK2 overexpression to suppress mitogenic signaling in vascular SMCs, we used primary SMCs infected with recombinant adenoviruses encoding GRK2 or control constructs. In this model system, we assessed SMC responsiveness to the G_q-coupled receptor agonists ET-1, thrombin, Ang II, and TXA₂, and also—because of its apparent role in NH¹ —platelet-derived growth factor (PDGF).

	Methods

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Primary SMC Isolation and Culture
Thoracic aortas were harvested from euthanized male New Zealand White rabbits, and primary SMCs were obtained by the explant outgrowth technique¹¹ in the presence of mycoplasma removal agent (50 µg/mL, ICN Pharmaceuticals, Inc). Immunofluorescence for SMC -actin confirmed SMC identity.¹¹ SMCs were passaged in DMEM/10% FBS with antibiotics,⁸ split 1:4, and discarded after passage 7.

Adenovirus Production and Infection
The bovine GRK2 cDNA¹² was inserted into the plasmid pSKAC, as described previously.¹³ The GRK2ct adenovirus has been described.¹³

Subconfluent SMCs were infected in batches of 150-mm plates. A single plate was trypsinized and counted; subsequently, plates for infection were washed and exposed (37°C, 30 minutes) to 5 mL of infection medium (DMEM/2% FBS/25 mmol/L HEPES; pH 7.4) with or without virus (multiplicity of infection=100), with gentle agitation. Virus-containing medium was removed, and plates were incubated in fresh infection medium for 24 hours. Cells were then trypsinized and divided into aliquots in various dishes for assays, at either 2.6x10⁴ cells/cm² (phosphoinositide hydrolysis and [³H]thymidine {[³H]TdR} incorporation) or 1.3x10⁴ cells/cm² (proliferation, cell cycle, and immunoblotting assays).

Phosphoinositide Hydrolysis
SMCs were rendered quiescent by a 72-hour incubation in low-mitogen medium (DMEM supplemented with 20 mmol/L HEPES [pH 7.4], fatty acid–free BSA [1 mg/mL], 0.2% FBS, insulin [1.7 µmol/L], transferrin [5.5 µg/mL], sodium selenite [6.7 ng/mL], and antibiotics⁸ ). SMCs were labeled in this medium containing 2 µCi/mL of [³H]inositol for 12 to 18 hours, and then assayed for agonist-stimulated phosphoinositide (PI) hydrolysis, as described previously.⁸

[³H]TdR Incorporation
SMCs were plated in 24-well plates, rendered quiescent (as above), then challenged with agonist(s) in low-mitogen medium for a total of 24 hours. After 20 hours, [³H]TdR was added at 1 µCi/mL (final concentration) for the last 4 hours of the agonist challenge. After 24 hours, cells were washed 3 times with cold PBS and solubilized with 0.5 mL/well phenol : 4 mol/L guanidine thiocyanate (1:1, vol/vol), which was subjected to liquid scintillation spectrometry to determine [³H]TdR incorporation (dpm/well) into SMCs. Under the conditions we used, SMCs incorporated [³H]TdR into acid-precipitable macromolecules, 85% of which were digestible with DNAse I (data not shown).

Immunofluorescence
SMCs on culture slides were fixed for 2 minutes (25°C) in methanol : acetone (1:1), and then incubated with the indicated primary IgG (1 to 10 µg/mL) for 30 minutes (25°C) in PBS with 3% BSA. After 3 washes in PBS, slides were incubated as before, but with Alexa 488–conjugated anti-mouse IgG (Molecular Probes). After further washes, slides were imaged by fluorescence microscopy. To identify GRK2 and SMC -actin simultaneously, SMCs were incubated sequentially with C5/1 anti-GRK2 IgG,¹⁴ Alexa 488–conjugated anti-mouse IgG, and then cyanine 3–conjugated 1A4 anti–SMC -actin (Sigma), with washes between each incubation. To identify cell nuclei, the blue-fluorescing DNA-binding dye Hoechst 33258 (Molecular Probes) was added to the buffer (5 µg/mL) during the last antibody incubation.

Immunoprecipitation and Immunoblotting
The PDGF receptor (PDGFR) and the epidermal growth factor receptor (EGFR) were immunoprecipitated from SMCs as we have described previously,⁸ with rabbit IgG specific for cytoplasmic domains of each receptor (sc-432 and sc-03, Santa Cruz Biotechnology, Inc). Immunoprecipitates from equal masses of solubilized SMC protein were electrophoresed on 4% to 20% polyacrylamide gradient gels and transferred to nitrocellulose.

Immunoblotting was performed as described previously,^{8 12} with C5/1¹⁴ (anti-GRK2), sc-432 (anti-PDGFR), sc-03 (anti-EGFR), or PY20 (anti-phosphotyrosine, Transduction Laboratories). To visualize the GRK2 carboxyl terminal domain polypeptide (GRK2ct, amino acids 495 to 689),¹³ we used the monoclonal antibody E23/8 (IgG1/), raised (by standard protocols¹⁴ ) against a synthetic peptide comprising residues 658 to 689 of bovine GRK2.

Proliferation Assay
SMC proliferation over 12 days was assessed in response to the indicated agonists. SMCs were plated in 8-well chamber slides, rendered quiescent as described above, and treated on day 1 with the indicated stimulus diluted in low-mitogen medium. On day 6, SMCs were re-treated as on day 1. On day 12, SMCs were washed, incubated with Hoechst 33258 (5 µg/mL) for 30 minutes (25°C), washed with PBS, and visualized with a Chroma Blue GFP filter on a Leica fluorescence microscope. From each slide chamber, 4 images (at x10 magnification) were captured with Adobe Photoshop. Nuclei were counted with the UTHSCSA ImageTool program (developed at the University of Texas Health Science Center at San Antonio and available from the Internet by anonymous FTP from maxrad6.uthscsa.edu). At least 200 cells per well of each chamber slide were counted.

Cell Cycle Analysis
SMCs in low-mitogen medium were assayed flow-cytometrically for DNA content on days 6 and 12 of the proliferation assays.¹⁵ Analyses on days 6 and 12 were equivalent, so only those from day 12 are presented. To ensure that apoptotic or necrotic SMCs could be identified,¹⁵ we harvested and pelleted debris or floating SMCs in the medium and pooled this pellet with that from SMCs removed from the plate by trypsinization. SMCs were stained with Hoechst 33258, and 10⁴ cells per cell line were analyzed. The fraction of apoptotic cells increased 4- to 6-fold with a 4-hour treatment with 1 µmol/L staurosporine (Sigma) (data not shown).

Statistical Analysis
To facilitate pooling of results across several independent experiments, results within each experiment were normalized to values obtained from SMCs infected with the empty vector adenovirus. With Prism software (GraphPad), data from uninfected, vector-infected, and GRK2- or GRK2ct-virus–infected SMCs were analyzed by repeated-measures (ie, paired within individual experiments) 1-way ANOVA. Tukey’s multiple comparison test was used to compare the SMC groups with one another. Data are presented in the text as ±SD, and probability values are 2-tailed.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Overexpression of GRK2 in SMCs
Cells migrating out from de-endothelialized aortic explants were identified as SMCs by immunofluorescent staining for SMC -actin¹¹ (Figure 1A). The 90% prevalence of cells staining for SMC -actin was assessed by comparison of immunofluorescence for SMC -actin with fluorescent nuclear staining of all cells (data not shown).

View larger version (110K): [in this window] [in a new window]   Figure 1. Adenovirus-mediated overexpression of GRK2 in SMCs. SMCs infected with empty vector (A and C) or GRK2 adenovirus (B and D) were triple-stained, for GRK2, SMC -actin, and nuclear DNA, 4 days after infection. SMCs were imaged with 3 different fluorescence filter sets to identify SMC -actin (orange, A), GRK2 (green, B through D), and cell nuclei (blue, C and D) within a single microscopic field. A, SMC -actin stain, magnification x200. (This reagent produced no immunofluorescence in Rat 1 fibroblasts [data not shown].) B, GRK2-overexpressing SMC stained for GRK2, magnification x200. No detectable immunofluorescence was seen in absence of anti-GRK2 C5/114 (data not shown). C and D, Computerized overlay of nuclear- and GRK2-staining images, magnification x20. These panels are indistinguishable when imaged for SMC -actin (not shown). There was no significant "bleed-through" fluorescence in either blue, green, or orange fluorescence windows (not shown).

To achieve overexpression of GRK2 in these primary SMCs, we infected them with a recombinant adenovirus that effected GRK2 overexpression in nearly 100% of the SMCs (compare Figure 1C and 1D), with a predominantly cytoplasmic distribution of the GRK2 (Figure 1B). This GRK2 overexpression was 40-fold above endogenous levels, as assessed by immunoblotting (Figure 2). GRK2 overexpression persisted for 12 days in SMCs maintained in low-mitogen medium. Immunoblotting of solubilized SMC protein along with a purified GRK2 standard demonstrated that these GRK2-adenovirus–infected SMCs expressed 12 to 25 pmol GRK2/mg total cell protein. Uninfected and empty vector adenovirus–infected SMCs were indistinguishable with regard to GRK2 expression, and GRK2 overexpression did not alter SMC -actin expression (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 2. Persistence of GRK2 overexpression in SMCs. SMCs infected with empty vector (Empty) or GRK2 adenovirus were plated in low-mitogen medium. On indicated day after infection, cells were harvested and extracts prepared. Purified bovine GRK213 (Std, 20 ng, Mr 80 000) and 10 µg cell protein from each extract were subjected to SDS-PAGE and immunoblotting for GRK2. Immunoblot is representative of 3 performed.

Agonist-Induced SMC PI Hydrolysis and [³H]TdR Incorporation: Effects of GRK2 Overexpression
To assess SMC second-messenger signaling in response to agonists implicated in NH, we studied PI hydrolysis mediated by phospholipase C isoforms ß (stimulated by G_q-coupled, heptahelical receptors) and (stimulated by receptor tyrosine kinases, like the PDGFR) (Figure 3A). ET-1, Ang II, thrombin agonist peptide, and PDGF-BB each promoted a time-dependent cellular accumulation of inositol phosphates, ranging from 1.5±0.3- to 9±4-fold over basal at 15 minutes.

View larger version (20K): [in this window] [in a new window]   Figure 3. Agonist-stimulated PI hydrolysis in SMCs is attenuated by overexpression of GRK2. A, Time course of PI hydrolysis: SMCs were challenged for indicated times with either 100 nmol/L ET-1, 500 nmol/L Ang II, 100 µmol/L thrombin agonist peptide (SFLLRN-NH2), or 0.4 nmol/L (10 ng/mL) PDGF-BB (PDGF). Incorporation of 3H into inositol phosphates was normalized to that of unstimulated SMCs from same time point, to obtain % of basal: 100x (stimulated/unstimulated). Displayed are means±SD of a single experiment executed in triplicate, representative of 2 performed. Basal values for % incorporation of 3H into inositol phosphates were 1.0±0.1% (5 minutes) and 1.3±0.2% (20 minutes). B, SMCs infected with either no adenovirus, empty vector (control SMCs), or GRK2 adenovirus were stimulated (15 minutes, 37°C) with indicated agonist (as in A, plus 10 µmol/L of TXA2 analogue U46619). Signal above basal for each cell type was normalized to cognate value from control SMCs in same experiment and processed to obtain % inhibition: 100x[1-(GRK2/control)]. Shown are mean±SEM of 3 experiments performed in triplicate. Vector-infected SMCs were indistinguishable from uninfected SMCs. Basal values for % incorporation of 3H into inositol phosphates were 2.8±1.2%, 3.2±0.9%, or 2.3±0.5% in uninfected, vector-infected, or GRK2-infected cells, respectively. Agonist-stimulated PI hydrolysis (fold over basal) for vector-infected cells was 2.9±0.9, 2.7±0.9, 1.5±0.3, 8.6±3.6, and 3.8±0.3 for PDGF, SFLLRN, Ang II, ET-1, and U46619, respectively. *P<0.01 vs control SMCs.

Agonist-promoted PI hydrolysis was reduced by 37% to 72% in SMCs overexpressing GRK2 (Figure 3B). Interestingly, signaling inhibition by GRK2 appeared to be receptor-specific, because PI hydrolysis induced by the TXA₂ analogue U46619 (3.8±0.3-fold over basal) was unaffected by GRK2 overexpression. Thus, GRK2 overexpression attenuated second-messenger signaling promoted not only by several G_q-coupled receptors but also, surprisingly, by the PDGFRß¹⁶ of rabbit vascular SMCs.

To determine whether the GRK2-mediated reduction of second-messenger signaling in SMCs could lead to a corresponding reduction in agonist-promoted DNA synthesis, we assayed SMC [³H]TdR incorporation evoked by the agonists used in the preceding experiments (Figure 4). Compared with control cells, GRK2-overexpressing SMCs incorporated 35% less [³H]TdR in response to either PDGF or 10% FBS and 60% less [³H]TdR in response to either ET-1 or the TXA₂ analogue U46619. Synergistically, the combination of PDGF and ET-1 promoted SMC [³H]TdR incorporation to an extent 2- to 6-fold greater than any of the foregoing stimuli. Nonetheless, GRK2 overexpression reduced this effect by 30% (P<0.02), whereas it had no effect on the lesser degree of [³H]TdR incorporation induced by EGF. Thus, as we observed with PI hydrolysis, overexpression of GRK2 in SMCs attenuated [³H]TdR incorporation promoted by PDGF and various individual G_q-coupled receptor agonists in a receptor-specific manner. In addition, overexpression of GRK2 in SMCs reduced [³H]TdR incorporation promoted by agonist combinations, including 10% FBS, which may more accurately model stimuli encountered by SMCs in injured arteries or vein grafts.

View larger version (32K): [in this window] [in a new window]   Figure 4. Effects of GRK2 overexpression on stimulus-induced SMC [3H]thymidine incorporation. SMCs were infected as in Figure 3B and exposed to low-mitogen medium without (basal) or with stimuli, as in Figure 3. Additional stimuli: 10% FBS, 1.7 nmol/L human EGF, and 0.4 nmol/L PDGF-BB with 100 nmol/L ET-1 (PDGF/ET). Stimulus-induced [3H]TdR incorporation is plotted (mean±SEM) from 4 experiments performed in triplicate, as dpm above basal: [(stimulated cells)-(unstimulated cells)]. Incorporation of [3H]TdR by unstimulated cells (dpmx10-3) was 4±4, 9±5, and 14±8 for uninfected, GRK2-infected, and vector-infected cells, respectively. Uninfected and vector-infected SMCs showed indistinguishable stimulus-induced [3H]TdR incorporation. *P<0.01, **P<0.05 vs control SMCs.

Effects of GRK2 on PDGFR Signaling
Although GRK2 can phosphorylate a wide array of heptahelical GPCRs, it has yet to be implicated in the regulation of any receptor tyrosine kinase.⁹ We therefore found it surprising that overexpression of GRK2 attenuated both PI hydrolysis (Figure 3) and [³H]TdR incorporation (Figure 4) stimulated by the PDGFR(s) in primary SMCs. One potential explanation for these phenomena could lie in the ability of the GRK2 carboxyl-terminal domain to bind phosphatidylinositol-4,5-bisphosphate (PIP₂),⁹ the preferred substrate for 2 important PDGFR-stimulated effector enzymes: phospholipase C- and phosphatidylinositol 3'-kinase.¹⁷ Sequestration of PIP₂ by overexpressed GRK2 might therefore inhibit PDGF-stimulated PI hydrolysis as well as downstream events, such as DNA synthesis.¹⁷ Could attenuation of PDGF-promoted signaling by overexpressed GRK2 involve more than competition for binding of PIP₂? To address this question, we compared PDGF-promoted signaling in SMCs overexpressing either GRK2 or a polypeptide encompassing just the carboxyl-terminal 195 amino acids of GRK2 (GRK2ct) (Figure 5). The GRK2ct contains the pleckstrin homology (PIP₂-binding) and G protein ß–binding domains of GRK2 but lacks the central catalytic and amino-terminal targeting domains of GRK2.⁹ Because we performed these experiments with equivalent levels of GRK2 and GRK2ct expression (Figure 5A), differences between the effects of GRK2 and the GRK2ct could be ascribed to differences in protein activity, rather than to differences in intracellular protein concentration.

View larger version (29K): [in this window] [in a new window]   Figure 5. Comparative effects of GRK2 and GRK2ct overexpression on PDGFR signaling. SMCs infected with either no adenovirus (None), empty vector adenovirus (Empty, control), GRK2 adenovirus (GRK2), and GRK2ct adenovirus (GRK2ct) were studied in 3 ways: A, Immunoblotting: SMC extracts were subjected to SDS-PAGE on 4% to 20% gradient gels, then immunoblotted with IgG E23/8, which recognizes GRK2 and GRK2ct equivalently. Immunoblot represents 3 performed. B, PDGF-induced PI hydrolysis and [3H]thymidine incorporation: SMCs were subjected to these signaling assays without (basal) or with 0.4 nmol/L PDGF-BB. Signals (stimulated-basal) for each cell type were normalized to cognate values from control SMCs in same experiment and processed to obtain % inhibition: 100x{1-[(GRK2 polypeptide)/(control)]}, where GRK2 polypeptide = GRK2 or GRK2ct. Displayed are mean±SEM of 3 experiments performed in triplicate. For control, GRK2, and GRK2ct cells, respectively, basal values were 3.9±2.2, 2.4±1.8, and 2.4±1.8 (% conversion, PI hydrolysis); 25±17, 8±6, and 8±6 (dpmx10-3, [3H]thymidine). For control SMCs, agonist-stimulated signals (fold over basal) for inositol phosphates and [3H]TdR incorporation, respectively, were 3.8±2.5 and 5.2±3.5. *P<0.01, **P<0.001; , P<0.05 vs control SMCs. #P<0.05 vs GRK2ct-expressing SMCs.

PDGF-stimulated PI hydrolysis was inhibited by 38% in SMCs overexpressing GRK2 (Figure 5B). This degree of inhibition was more than twice that (P<0.05) effected by equivalent expression of the GRK2ct. Despite these discordant findings in PI hydrolysis, GRK2 overexpression inhibited PDGF-induced [³H]TdR incorporation only insignificantly more than GRK2ct. However, the more robust [³H]TdR incorporation promoted by the combination of PDGF and ET-1 (Figure 4) was reduced (by 32±3%, P<0.01) only in SMCs overexpressing GRK2, but not in those overexpressing GRK2ct (data not shown).

These observations suggest that overexpressed GRK2 may reduce PDGF-promoted signaling, in part, by binding to the ligands for its pleckstrin homology/G_ß-binding carboxyl terminal domain, as the GRK2ct does. However, the larger part of GRK2-mediated inhibition of PDGF-promoted signaling appears to operate through either the central catalytic domain, the amino-terminal targeting domain, or perhaps both of these domains.

If GRK2 interacted via its N-terminal or catalytic domain with the PDGFR, GRK2 might interfere with agonist-promoted tyrosine phosphorylation of the PDGFR. To test this possibility, we assessed the phosphotyrosine content of PDGFRßs immunoprecipitated from PDGF-challenged SMCs, which either did or did not overexpress GRK2 (Figure 6). GRK2 overexpression reduced agonist-induced tyrosine phosphorylation of the PDGFRß by 37±20% (P<0.01). However, GRK2 overexpression had no effect on agonist-promoted tyrosine phosphorylation of the SMC EGFR (Figure 6). Thus, the ability of GRK2 to attenuate agonist-promoted receptor tyrosine kinase activation appears to be receptor-specific. Moreover, the specificity of GRK2-mediated receptor tyrosine kinase inhibition is congruent at the level of receptor activation (Figure 6) and receptor-stimulated [³H]TdR incorporation (Figure 4).

View larger version (43K): [in this window] [in a new window]   Figure 6. GRK2 overexpression reduces PDGFRß but not EGFR activation. Empty vector– or GRK2 virus–infected SMCs were rendered quiescent as in Figure 3, then exposed to fresh medium lacking (-) or containing (+) either 0.4 nmol/L PDGF-BB or 1.7 nmol/L EGF (Agonist) for 10 minutes at 37°C. Detergent extracts of SMCs were then subjected to immunoprecipitation (IP) with indicated IgGs (- indicates nonimmune). Aliquots of immunoprecipitates were resolved as in Figure 5A and immunoblotted for either phosphotyrosine content (top) or total receptor (bottom). Results shown represent 3 independent experiments.

GRK2 Overexpression Reduces SMC Proliferation
Agonist-stimulated [³H]TdR incorporation may not indicate either an increase in DNA synthesis¹⁸ or ongoing cellular proliferation.¹⁹ We therefore sought to determine whether the GRK2 overexpression that inhibited agonist-induced [³H]TdR incorporation would also inhibit SMC proliferation. Accordingly, aliquots of the same SMCs as those subjected to assays for [³H]TdR incorporation were subjected to proliferation studies, presented in Figure 7. In response to either ET-1, PDGF-BB, or a combination of these agonists, the number of control SMCs increased 1.4±0.3-, 1.8±0.4-, or 2.0±0.8-fold in 12 days. The number of SMCs increased 2.7±0.7-fold over 4 days in response to 10% FBS. Compared with control cells, the proliferation of GRK2-overexpressing cells was reduced by 50% to 60% (P<0.01) in response to either ET-1, PDGF-BB, or the 2 agonists together (Figure 6). Likewise, proliferation of GRK2-overexpressing cells was reduced by 29% (P<0.01) in response to 10% FBS (Figure 7). This reduction in SMC proliferation by GRK2 overexpression was achieved without any evidence of GRK2 adenovirus-induced apoptosis in SMCs maintained in low-mitogen medium (Table). Thus, GRK2 overexpression in SMCs attenuates not only agonist-stimulated [³H]TdR incorporation but also agonist-stimulated SMC proliferation in vitro.

View larger version (52K): [in this window] [in a new window]   Figure 7. Attenuation of SMC proliferation by GRK2 overexpression. SMCs infected with empty vector or GRK2 adenovirus were exposed to low-mitogen medium containing either vehicle (basal), 100 nmol/L ET-1 (ET), 0.4 nmol/L PDGF-BB (PDGF), or combination of latter 2 (ET+PDGF) for 12 days. Alternatively, SMCs were exposed to low-mitogen medium without (basal) or with 10% FBS for 4 days. Plotted are mean±SEM for growth above basal for each stimulus: [(cells in stimulated slide chambers)-(cells in unstimulated slide chambers)] for 4 to 5 independent experiments. Basal SMC counts were 500±180, 470±160, and 380±130 for uninfected, vector-infected, and GRK2 virus–infected SMCs, respectively. Uninfected and vector-infected SMC counts from stimulated wells were indistinguishable. *P<0.01 vs control cells.

View this table: [in this window] [in a new window]   Table 1. Cell Cycle Analysis of SMCs

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In primary cultures of vascular SMCs, we have shown that overexpression of GRK2 diminishes signaling elicited via several receptors believed to be important in the pathogenesis of NH, including the PDGFR. GRK2 overexpression suppressed SMC signaling assessed either at the level of the second messenger or considerably downstream from the second messenger, at the level of SMC [³H]TdR incorporation. Moreover, GRK2 overexpression attenuated SMC proliferation without potentiating SMC apoptosis. The possibility that GRK2 overexpression may attenuate NH is suggested by the ability of GRK2 to diminish SMC [³H]TdR incorporation and proliferation promoted by the complex mixture of mitogens in 10% FBS.

GRK2 specifically binds and phosphorylates agonist-bound, or activated, heptahelical GPCRs.⁹ Expressed at physiological levels, GRK2 appears to initiate desensitization of GPCRs in a manner dependent on GRK2-mediated phosphorylation of the heptahelical receptor. When expressed at 40-fold above physiological levels, however, GRK2-mediated inhibition of receptor signaling involves primarily agonist-promoted association of the receptor with the GRK (a process that is both receptor- and GRK-specific).⁸ Overexpression of just the amino-terminal domain of GRK2 can attenuate G_q-coupled receptor signaling.⁸ This phenomenon may result from binding to either the heptahelical receptor or the G protein itself, because the GRK2 amino-terminal domain possesses homology with regulator of G-protein signaling (RGS) proteins.²⁰

Surprisingly, GRK2 overexpression inhibited signaling not only via SMC GPCRs but also via the SMC PDGFRß.¹⁶ By what mechanisms could overexpressed GRK2 suppress PDGFR-mediated signaling in SMCs? PIP₂ sequestration has been discussed above. In addition, overexpressed GRK2 may interfere with PDGFR signaling by sequestering G_ß subunits. The heterotrimeric protein G_i has been implicated in PDGFR-mediated stimulation of p42/p44 mitogen-activated protein kinases in airway SMCs.²¹ G_i can also mediate mitogen-activated protein kinase activation elicited through heptahelical receptors, and this process is inhibited by GRK2ct, presumably by sequestration of G_ß subunits.²² Thus, to the extent that PDGFRs signal via a G_ß-related mechanism in our vascular SMCs, GRK2ct and GRK2 itself should be expected to inhibit the signaling. Inhibition of PDGF-evoked mitogenesis through sequestration of G_ß and PIP₂ may, in part, underlie the efficacy of GRK2ct in attenuating NH in rabbit jugular vein bypass grafts.²³

If the cytoplasmic tail of the PDGFR can mimic heptahelical receptor cytoplasmic domains by activating G_i,²¹ perhaps it can also mimic heptahelical receptor cytoplasmic domains by activating GRK2, as the wasp venom peptide mastoparan can.²⁴ If the PDGFR cytoplasmic domain can activate GRK2, it may also be phosphorylated by GRK2, with a consequent impairment of downstream signaling. Indeed, our observation that overexpressed GRK2 reduces PDGFRß tyrosine phosphorylation is consistent with the possibility of GRK2-mediated PDGFRß phosphorylation. Recently, casein kinase I2 has been shown to phosphorylate the PDGFRß on serine(s) and consequently to reduce PDGF-promoted receptor tyrosine phosphorylation.²⁵ Intriguingly, sites phosphorylated by casein kinase I2 are strikingly similar to those that can be phosphorylated by GRK2.^{9 25} Whether GRK2 directly phosphorylates the PDGFRß remains to be determined.

Conceptually, inhibiting mitogenic signaling at the level of the receptor seems to be strategically advantageous in treating NH. Receptor-mediated signaling involves catalytic cascades that amplify signals as they propagate toward the cell nucleus.²⁶ Such signal amplification may at least partly explain the incomplete effectiveness in treating NH observed with molecular strategies targeting transcriptional or other cell cycle–regulatory proteins.²⁶ With GRK2 overexpression in SMCs, multiple signaling pathways are attenuated simultaneously at the level of cell surface receptors signaling through G_q-coupled and tyrosine kinase pathways. Our data regarding GRK2 overexpression therefore suggest that GRK2 overexpression may be therapeutically useful as a novel, plasma membrane–targeted treatment for NH.

	Acknowledgments

 
This work was supported by NIH grants HL-57432 and HL-03008 (Dr Freedman), an American Heart Association Grant-in-Aid (Dr Peppel), and Deutsche Forschungsgemeinschaft grant Op42/7-1 (Dr Oppermann).

Received February 11, 2000; revision received March 17, 2000; accepted March 22, 2000.

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