Role of RACK1 in the differential proliferative effects of neuropeptide Y_1–36 and peptide YY_1–36 in SHR vs. WKY preglomerular vascular smooth muscle cells

Abstract

Previous studies show that neuropeptide Y_1–36 (NPY_1–36) and peptide YY_1–36 (PYY_1–36), by engaging Y₁ receptors, stimulate proliferation of spontaneous hypertensive rat (SHR) preglomerular vascular smooth muscle cells (PGVSMCs). In contrast, these peptides have little effect on proliferation of Wistar-Kyoto (WKY) PGVSMCs. Why SHR and WKY PGVSMCs differ in this regard is unknown. Because receptor for activated C kinase 1 (RACK1) can modulate cell proliferation, we tested the hypothesis that differences in RACK1 levels/localization may explain the differential response of SHR vs. WKY PGVSMCs to NPY_1–36 and PYY_1–36. Western blotting for RACK1 in subcellular fractions of cultured SHR and WKY PGVSMCs demonstrated increased levels of RACK1 in the membrane and cytoskeletal subcellular fractions of SHR vs. WKY PGVSMCs. NPY_1–36 and PYY_1–36 stimulated proliferation of SHR PGVSMCs, and siRNA knockdown of RACK1 abrogated this effect. Neither NPY_1–36 nor PYY_1–36 stimulated the proliferation of WKY PGVSMCs. However, in WKY PGVSMCs treated with a RACK1 plasmid, both NPY_1–36 and PYY_1–36 stimulated proliferation. In SHR PGVSMCs, inhibitors of the G_i/phospholipase C/PKC pathway (a pathway known to be organized by RACK1) attenuated the ability of NPY_1–36 to stimulate the proliferation of SHR PGVSMCs. Our results suggest that RACK1 modulates the ability of PGVSMCs to respond to the proliferative actions of NPY_1–36 and PYY_1–36 and differences in RACK1 levels/localization account for, in part, differential proliferative responses to NPY_1–36 and PYY_1–36 in SHR vs. WKY PGVSMCs. Because dipeptidyl peptidase IV inhibitors increase NPY_1–36 and PYY_1–36 levels, our findings have implications for the use of such drugs in diabetic patients.

neuropeptide y_1–36 (npy_1–36) and peptide YY_1–36 (PYY_1–36) are members of the pancreatic polypeptide-fold (PP-fold) family (6). Importantly, the kidney is likely exposed to biologically active levels of PP-fold peptides because 1) renal sympathetic varicosities release NPY_1–36 in response to CNS-mediated activation of renal sympathetic nerves (13); 2) NPY_1–36 is made by renal epithelial cells and released into the renal interstitium (22); and 3) fatty meals stimulate endocrine L-cells in the GI tract to release PYY_1–36 into the systemic circulation, producing physiologically active levels of PYY_1–36 in plasma (3, 4, 19, 28, 34, 44, 53), and this PYY_1–36 would be promptly delivered to the kidney via the bloodstream.

The exposure of the kidney to pharmacologically active levels of NPY_1–36 or PYY_1–36 may have implications for renovascular structure and health. In this regard, our recent experiments indicate that both NPY_1–36 and PYY_1–36 stimulate the proliferation of preglomerular microvascular smooth muscle cells (PGVSMCs) obtained from spontaneously hypertensive rats (SHR) via a mechanism involving activation of Y₁ receptors (26). In contrast, NPY_1–36 and PYY_1–36 exert little or no effect on proliferation of PGVSMCs obtained from normotensive Wistar-Kyoto rats (WKY). Thus, endogenous PP-fold peptides may have adverse effects on renovascular structure in genetic hypertension. However, the reason that NPY_1–36 and PYY_1–36 robustly stimulate proliferation of PGVSMCs only in PGVSMCs obtained from genetically susceptible kidneys is unclear. Thus, the focus of the current study was to investigate why SHR, but not WKY, PGVSMCs are responsive to the proliferative effects of NPY_1–36 and PYY_1–36.

Our previously published work demonstrates that Y₁ receptor expression is not different in preglomerular microvessels from SHR vs. WKY (15), so receptor expression differences are unlikely to account for the differential proliferative responses of SHR vs. WKY to NPY_1–36 and PYY_1–36. On the other hand, our previously published work does show that signaling via G_i-linked receptors is enhanced in the renal vasculature of SHR vs. WKY (15). Inasmuch as Y₁ receptors are G_i-coupled receptors, the most likely reason that NPY_1–36 and PYY_1–36 stimulate growth more in SHR than WKY PGVSMCs is that postreceptor signaling via the G_i pathway is enhanced in SHR PGVSMCs.

Receptor for activated C kinase 1 (RACK1) is a seven-sided propeller protein with seven WD40 repeats, and RACK1 participates in cell signaling by functioning as a scaffolding protein that interacts with G protein βγ subunits, phospholipase C, and PKC (10, 49) and, therefore, organizes and modulates the G_i/phospholipase C/PKC pathway. Because Y₁ receptors are coupled to G_i (38) and because recent studies from our laboratory indicate that RACK1 participates in proliferation of PGVSMCs (11), we hypothesized that differences in RACK1 levels or distribution, via interacting with the G_i/phospholipase C/PKC pathway, may explain why SHR, but not WKY, PGVSMCs are sensitive to the proliferative effects of NPY_1–36 and PYY_1–36.

METHODS

Animals.

Studies utilized male SHR and WKY obtained from Taconic Farms (Germantown, NY). The Institutional Animal Care and Use Committee approved all procedures. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85–23, revised 1996).

Isolation and culture of PGVSMCs.

A complete description of our method for culturing and confirming the identity of rat PGVSMCs can be found in Mokkapatti et al. (39). All experiments employed cells in 3rd to 5th passage.

Levels of RACK1 in cellular fractions from SHR vs. WKY PGVSMCs.

Membrane, cytosolic, nuclear, and cytoskeletal fractions were isolated from SHR and WKY PGVSMCs using the BioVision FractionPREP Cell Fractionation System Kit (BioVision Research Products, Mountain View, CA). This cell fractionation system has been widely used in a variety of cell types, including vascular and airway smooth muscle cells and renal cells, and reliably separates membrane, cytosolic, nuclear, and cytoskeletal fractions (2, 32, 43, 54–56). Briefly, four separate batches (∼22.5 million cells per batch) of WKY and SHR PGVSMCs were treated with trypsin and suspended in PBS. The cells were centrifuged, and the supernatant was replaced with cytosol extraction buffer mix containing protease inhibitors, phosphatase inhibitors, and dithiothreitol (DTT). After 20 min, samples were centrifuged, and the supernatant was collected and used as the cytosolic fraction. The pellet was resuspended in membrane extraction buffer-A containing protease inhibitors, phosphatase inhibitors, and DTT and then treated with membrane extraction buffer-B solution. After 1 min, samples were centrifuged, and the supernatant was collected as the membrane fraction. The pellet was resuspended in nuclear extraction buffer mix containing protease inhibitors, phosphatase inhibitors, and DTT, and after 40 min, the samples were centrifuged, and the supernatant was collected as the nuclear fraction. The remaining pellet was dissolved in sample/lysis buffer and collected as the cytoskeletal fraction. Samples were stored at −80°C until analyzed.

Protein in each sample was determined by the BCA assay (Pierce Biotechnology, Rockford, IL), and then diluted with loading buffer containing β-mercaptoethanol, boiled for 10 min, cooled on ice for 5 min, and then centrifuged. SDS-polyacrylamide-gel electrophoresis was performed on each sample (16 μg of protein per sample). Samples were loaded along with Bio-Rad Precision Plus Protein Standard markers (Bio-Rad; Hercules, CA) onto Invitrogen NuPAGE Novex 12% Bis-Tris gels (1.0 mm, 12 wells) (Life Technologies; Carlsbad, CA). Electrophoresis was performed using NuPAGE MOPS SDS running buffer (Life Technologies), and proteins were transferred onto polyvinylidene difluoride membranes in NuPAGE Transfer Buffer (Life Technologies), and membranes were washed briefly in methanol and blocked with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) and incubated in Odyssey blocking buffer overnight with anti-RACK1 mouse monoclonal antibody (1:400 dilution; Santa Cruz Biotechnology; Santa Cruz, CA; expected size of signal band, 36 kDa) and anti-β-actin rabbit polyclonal antibody (1:10,000; Santa Cruz Biotechnology). Membranes were washed, incubated in Odyssey blocking buffer for 50 min in the dark at room temperature with IRDye 800CW goat anti-mouse IgG (1:10,000; LI-COR Biosciences), and IRDye 680 goat anti-rabbit IgG (1:10,000; LI-COR Biosciences). Membranes were again washed and placed on filter paper to air dry in the dark. Blots were analyzed using the Odyssey infrared imager (LI-COR Biosciences). For quantitative data analysis, rectangles were placed around the RACK1 (green) and β-actin (red) bands, and the integrated intensity values for both red and green channels were determined for each band. Each RACK1 band was normalized to the β-actin band. SHR and WKY samples were run on the same gel for each subcellular fraction.

Effect of RACK1 siRNA on cell growth induced by NPY_1–36 and PYY_1–36 in SHR PGVSMCs.

SHR PGVSMCs were cultured in DMEM/F12 medium containing 10% FCS, 20 units/ml penicillin, 20 μg/ml streptomycin, and 0.05 μg/ml amphotericin at 37°C with 5% CO₂. One day before transfection, 100,000 cells were plated in 500 μl of DMEM/F12 medium containing 2.5% FCS without antibiotics in a 24-well plate. On the day of transfection, 40 pmol of RACK1 siRNA pool or nontargeting siRNA pool (Dharmacon, Lafayette, CO) and 1.5 μl of DharmaFECT 1 (Dharmacon) were diluted to 50 μl in Opti-MEM I medium and incubated for 5 min at room temperature. Then the diluted siRNA and DharmaFECT 1 were combined and incubated for 20 min at room temperature to allow transfection complexes to form. DMEM/F12 medium containing platelet-derived growth factor-BB (PDGF-BB; 25 ng/ml) without antibiotics was added to the complexes (transfection mixture). In some experiments, the transfection mixture included NPY_1–36 (10 nM) plus sitagliptin (1 μM) or PYY_1–36 (10 nM) plus sitagliptin (1 μM). Sitagliptin was included because PGVSMCs express dipeptidyl peptidase IV (DPPIV), an enzyme that metabolizes and inactivates both NPY_1–36 and PYY_1–36, and sitagliptin, an inhibitor of DPPIV, augments and prolongs the proliferative effects of both NPY_1–36 and PYY_1–36 by blocking their metabolism (26). The growth medium was removed from the cells and replaced with the transfection mixture, and the cells were incubated with the transfection mixture at 37°C with 5% CO_2. After 72 h, the transfection mixture was replaced with fresh DMEM/F12 medium containing PDGF-BB and other treatments as noted, and at 96 h, cells were dislodged and counted on a Coulter counter.

Assessment of siRNA knockdown of RACK1 mRNA.

RNA was isolated (TRIzol Reagent; Life Technologies), and cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad). The RACK1 primers were forward, 5′-gtgctcttcgaggtcactcc-3′, reverse, 5′-cggttgtcagaggagaaagc-3′, and 184-bp amplification product. β-actin primers were forward, 5′-actcttccagccttccttc-3′, reverse, 5′-atctccttctgcatcctgtc-3′, and 171-bp amplification product. Real-time PCR analysis was performed using SYBR Green PCR Master Mix (Applied Biosystems; Foster City, CA) in the AB 7300 real-time PCR System (Applied Biosystems). Threshold cycle (Ct) for β-actin was subtracted from Ct for target to calculate 2^ΔCt.

Assessment of siRNA knockdown of RACK1 protein.

Protein was extracted (mammalian protein extraction reagent; Pierce Biotechnology), measured (BCA assay; Pierce) and boiled (5 min in Laemmli buffer). SDS-PAGE was performed on polyacrylamide gels (8–16%) with 40 μg of protein per lane. Proteins were transferred to PDVF membranes. Membranes were blocked in TBS-Tween-20 containing 5% milk and probed with an anti-RACK1 mouse monoclonal primary antibody (1:400; Santa Cruz Biotechnology). Membranes were exposed to horseradish-peroxidase-conjugated-goat anti-mouse antibody (1:4,000; Pierce) and visualized with the Bio-Rad VersaDoc Imaging System using luminal-based enhanced chemiluminescence substrate (Supersignal West Dura Extended Duration Substrate; Pierce).

Cloning of RACK1.

The cDNA fragment encoding rat RACK1 was obtained by PCR using rat PGVSMC cDNA as a template with primer set: sense, 5′-ctaagctatccggtgccatc-3′; antisense, 5′-gcgggtaccaatagtcacctg-3′. The RACK1 cDNA was then cloned into pcDNA 3.1/V5-His-TOPO vector by using pcDNA3.1/V5-His TOPO TA expression kit (Life Technologies) as per the manufacturer's instructions. The clone was verified by DNA sequencing.

Effect of RACK1 plasmid on cell growth induced by NPY_1–36 and PYY_1–36 in WKY PGVSMCs.

WKY PGVSMCs were cultured in DMEM/F12 medium containing 10% FCS, 20 unit/ml penicillin, 20 μg/ml streptomycin, and 0.05 μg/ml amphotericin at 37°C with 5% CO₂. On the day of transfection, 100,000 cells were plated in 500 μl of growth medium without antibiotics in a 24-well plate so that cells would be 60–80% confluent. After cells were attached (2 h), 0.45 μg of RACK1 plasmid DNA or vector control and 2 μl of Plus Reagent (Life Technologies) were diluted to 25 μl in Opti-MEM I medium and incubated for 15 min at room temperature. Two microliters of Lipofectamine (Life Technologies) were diluted to 25 μl in Opti-MEM I medium. Then, the diluted precomplexed plasmid DNA and diluted Lipofectamine were combined and incubated for 30 min at room temperature to allow transfection complexes to form. The complexes (brought to 250 μl with Opti-MEM I) were added into the wells of WKY PGVSMCs, and the cells were incubated for 3.5 h at 37°C with 5% CO₂. An equal volume (250 μl) of DMEM/F12 containing 2.5% FCS was added to each well, and the cells were incubated for another 18 h. Next, the transfection complexes were removed and replaced with 500 μl of DMEM/F12 containing PDGF-BB (25 ng/ml), and the cells were incubated for another 96 h in the absence or presence of NPY_1–36 (10 nM) plus sitagliptin (1 μM) or PYY_1–36 (10 nM) plus sitagliptin (1 μM). Cells were then dislodged and counted on a Coulter counter. RACK1 protein expression level in the transfected WKY PGVSMCs was evaluated by Western blotting (luminal based enhanced chemiluminescence substrate as described above) using a mouse anti-V5 primary antibody (1:1,000; Life Technologies) and a goat anti-mouse IgG, secondary antibody (1:2,500; Thermo Fisher Scientific, Waltham, MA).

Determination of the effects of inhibitors of the G_i/phospholipase C/PKC pathway and downstream signaling molecules on [³H]thymidine incorporation induced by NPY_1–36 in SHR PGVSMCs.

SHR PGVSMCs were plated at a density of 5,000 cells per well in 24-well tissue culture dishes and allowed to grow to subconfluence. Cells were growth arrested by feeding DMEM containing 0.4% albumin for 48 h. Next, PGVSMCs were stimulated with DMEM supplemented with PDGF-BB (25 ng/ml) in the presence or absence of various treatments as summarized below. After 20 h of incubation, treatments were repeated with freshly prepared solutions but supplemented with [³H]thymidine (1 μCi/ml). Four hours later, experiments were terminated by washing the cells twice with Dulbecco's PBS and twice with ice-cold trichloroacetic acid (10%). The precipitate was solubilized in 500 μl of 0.3 N NaOH and 0.1% sodium dodecylsulfate by incubating at 50°C for 2 h. Samples were mixed with 10 ml scintillation fluid and counted in a liquid scintillation counter. Treatment groups were as follows: No treatment, NPY_1–36 (10 nM), pertussis toxin (100 ng/ml; starting 24 h before the experiment), pertussis toxin (100 ng/ml; starting 24 h before the experiment) + NPY_1–36 (10 nM), U73122 (10 μM), U73122 (10 μM) + NPY_1–36 (10 nM), GF109203X (10 μM), GF109203X (10 μM) + NPY_1–36 (10 nM), PP1 (0.1 μM), PP1 (0.1 μM) + NPY_1–36 (10 nM), diphenyleneiodonium (DPI, 10 μM), DPI (10 μM) + NPY_1–36 (10 nM), Y27623 (10 μM), Y27623 (10 μM) + NPY_1–36 (10 nM), rapamycin (0.2 μM), rapamycin (0.2 μM) + NPY_1–36 (10 nM), PD98059 (10 μM), PD98059 (10 μM) + NPY_1–36 (10 nM), LY294002 (10 μM), LY294003 (10 μM) + NPY_1–36 (10 nM), SB203580 (10 μM), and SB203580 (10 μM) + NPY_1–36 (10 nM). Cells treated with NPY_1–36 were also cotreated with sitagliptin (1 μM) to block the metabolism and prolong the action of NPY_1–36.

Statistical analysis.

Data were analyzed either by two-factor analysis of variance (ANOVA; in which case if and only if the interaction term was statistically significant, post hoc tests were performed with a Fisher's LSD test to characterize the nature of the interaction) or by unpaired Student's t-tests. The criterion of significance was P < 0.05. Data are presented as means ± SE.

RESULTS

Levels of RACK1 in PGVSMC subcellular compartments.

To determine whether subcellular levels of RACK1 in PGVSMCs differ between SHR vs. WKY, subcellular compartments of SHR and WKY PGVSMCs were fractionated and examined for RACK1 expression. Western blotting for RACK1 revealed signals at 36 kDa in the membrane, cytosolic, nuclear and cytoskeletal fractions of PGVSMCs, indicating the presence of RACK1 in all of these subcellular compartments (Fig. 1A). The β-actin signal was not different between SHR and WKY for any of the subcellular fractions. Importantly, RACK1 levels were significantly greater in both SHR membrane fractions (2.5-fold; P < 0.0001) and SHR cytoskeletal fractions (1.8-fold; P = 0.0046) compared with the corresponding WKY fractions (Fig. 1, B and C, respectively); however, RACK1 levels were similar in the nuclear and cytosolic fractions of SHR vs. WKY PGVSMCs (Fig. 1, D and E, respectively).

Fig. 1.

A: Western blots depict subcellular localization of receptor for activated C kinase 1 (RACK1) (green bands; 36 kDa) expression in preglomerular vascular smooth muscle cells (PGVSMCs) from spontaneously hypertensive rats (SHR) vs. Wistar-Kyoto normotensive rats (WKY). Also shown are the signals for β-actin (red bands), which were used as loading controls. Subcellular fractions from SHR vs. WKY were processed on the same gel with the same amount of total protein. Bar graphs depict RACK1 expression in the membrane (B), cytoskeletal (C), nuclear (D), and cytosolic (E) fractions in SHR vs. WKY PGVSMCs. P values are from Student's unpaired t-test. Values are expressed as means ± SE.

Effects of NPY_1–36 and PYY_1–36 on cell proliferation in SHR PGVSMCs treated with RACK1 siRNA.

To determine whether RACK1 is a critical determinant of the proliferative response to NPY_1–36 and PYY_1–36 in SHR PGVSMCs, we next examined whether siRNA knockdown of RACK1 would attenuate the proliferative response to NPY_1–36 and PYY_1–36 in SHR PGV SMCs. These experiments were not performed in WKY PGVSMCs, since in these cells, there was no response to either NPY_1–36 or PYY_1–36 and, therefore, no response to inhibit. As reported previously in PGVSMCs (11), RACK1 siRNA decreased the expression of both RACK1 mRNA and RACK1 protein (Fig. 2, A and B, respectively). Two-factor analysis of variance (2F-ANOVA) indicated a significant (P = 0.0044) interaction between RACK1 siRNA and NPY_1–36 (10 nM) on proliferation of SHR PGVSMCs (Fig. 3A), and post hoc tests indicated that NPY_1–36 increased cell number in SHR PGVSMCs transfected with a nontargeting siRNA (control siRNA) but did not affect cell proliferation in SHR PGVSMCs transfected with targeting RACK1 mRNA. 2F-ANOVA also indicated a significant (P = 0.0328) interaction between RACK1 siRNA and PYY_1–36 (10 nM) on SHR PGVSMC proliferation (Fig. 3B), and post hoc tests indicated that PYY_1–36 increased cell number in SHR PGVSMCs transfected with a control siRNA, but it did not affect cell proliferation in PGVSMCs transfected with targeting RACK1 siRNA.

Fig. 2.

Bar graphs depict effects of RACK1 siRNA vs. negative control siRNA (control) on RACK1 mRNA (A) and protein (B) expression in PGVSMCs from SHR. For both negative control siRNA-treated cells (control) and RACK1 siRNA-treated cells (RACK1 siRNA), expression levels of RACK1 mRNA and protein were normalized to β-actin mRNA or β-actin protein, respectively. For mRNA, this was performed by subtracting the threshold cycle (Ct) for β-actin mRNA from the Ct for RACK1 mRNA to calculate 2^ΔCt. For protein, this was performed by dividing the optical density of the RACK1 band by the optical density of the β-actin band. For both mRNA and protein, the readings in the RACK1 siRNA cells were expressed as a % of the average reading for the control cells. Values are expressed as means ± SE, and P values are from unpaired Student's t-test.

Fig. 3.

Bar graphs illustrate the effects of either neuropeptide Y_1–36 (NPY_1–36; 10 nmol/l) (A) or peptide YY_1–36 (PYY_1–36; 10 nmol/l) (B) on cell number in PGVSMCs from SHR in the absence and presence of RACK1 siRNA. The PP-fold peptides were coadministered with sitagliptin (1 μM) to block their metabolism and inactivation. P values in panels are from two-factor ANOVA, and letters above bars (a, b, c, d) indicate significantly different [Fisher's least significant difference (LSD) test] from group without RACK1 siRNA and without NPY_1–36 or PYY_1–36 (a), group without RACK1 siRNA but with NPY_1–36 or PYY_1–36 (b), group with RACK1 siRNA but without NPY_1–36 or PYY_1–36 (c), or group with siRNA and with NPY_1–36 or PYY_1–36 (d). “Control siRNA” indicates nontargeting siRNA. Values are expressed as means ± SE.

Effects of NPY_1–36 and PYY_1–36 on cell proliferation in WKY PGVSMCs transfected with a RACK1 plasmid.

If RACK1 participates in the proliferative effects of NPY_1–36 and PYY_1–36 in SHR PGVSMCs, and if reduced levels of RACK1 are responsible for the lack of a proliferative response to these peptides in WKY PGVSMCs, then overexpression of RACK1 in WKY PGVSMCs should reproduce the SHR phenotype in WKY PGVSMCs. Western blot analysis confirmed that transfection with RACK1 plasmid was successful in WKY PGVSMCs (Fig. 4). 2F-ANOVA indicated a significant (P = 0.0160) interaction between RACK1 plasmid and NPY_1–36 (10 nM) on proliferation of WKY PGVSMCs (Fig. 5A), and post hoc tests indicated that NPY_1–36 increased cell number in WKY PGVSMCs transfected with RACK1 plasmid but did not affect cell proliferation in WKY PGVSMCs transfected with an empty plasmid (control plasmid). 2F-ANOVA also indicated a significant (P = 0.0157) interaction between RACK1 plasmid and PYY_1–36 (10 nM) on proliferation of WKY PGVSMCs (Fig. 5B), and post hoc tests indicated that PYY_1–36 increased cell number in WKY PGVSMCs transfected with RACK1 plasmid but did not affect cell proliferation in WKY PGVSMCs transfected with control plasmid.

Fig. 4.

The cDNA fragment encoding rat RACK1 was obtained by PCR using rat PGVSMC cDNA as a template with primer set: sense, 5′-ctaagctatccggtgccatc-3′ and antisense, 5′-gcgggtaccaatagtcacctg-3′. The RACK1 cDNA was then cloned into pcDNA 3.1/V5-His-TOPO vector by using pcDNA3.1/V5-His TOPO TA expression kit (Life Technologies) as per the manufacturer's instructions. The clone was verified by DNA sequencing. Western blot depicts expression of V5-tagged RACK1 in WKY PGVSMCs treated with RACK1-expressing plasmid vs. and empty (control) plasmid.

Fig. 5.

Bar graphs illustrate the effects of either NPY_1–36 (10 nmol/l; A) or peptide YY_1–36 (PYY_1–36; 10 nmol/l; B) on cell number in PGVSMCs from WKY in the absence and presence of a RACK1-expressing plasmid. The polypeptide (PP)-fold peptides were coadministered with sitagliptin (1 μM) to block their metabolism and inactivation. P values in panels are from two-factor ANOVA, and letters above bars indicate significantly different (Fisher's LSD test) from group without RACK1-expressing plasmid and without NPY_1–36 or PYY_1–36 (a), group without RACK1-expressing plasmid but with NPY_1–36 or PYY_1–36 (b), group with RACK1-expressing plasmid but without NPY_1–36 or PYY_1–36 (c), or group with RACK1-expressing plasmid and with NPY_1–36 or PYY_1–36 (d). “Control plasmid” indicates empty plasmid not expressing RACK1. Values are expressed as means ± SE.

Effects of NPY_1–36 on [³H]thymidine incorporation in SHR PGVSMCs treated with inhibitors of the G_i/phospholipase C/PKC pathway.

The results described above suggest that RACK1 is critically involved in proliferation induced by PP-fold peptides. Since RACK1 modulates the G_i/phospholipase C/PKC pathway (10, 49), if RACK1 participates in the proliferative response to PP-fold peptides in SHR PGVSMCs, then inhibition of this pathway should attenuate the proliferative effects of PP-fold peptides. Therefore, we examined whether inhibition of this pathway would attenuate the ability of NPY_1–36 to stimulate [³H]thymidine incorporation in SHR PGVSMCs. Corresponding experiments were not performed in WKY PGVSMCs because in these cells, there was no response to NPY_1–36 and, therefore, no response to inhibit. Because the proliferative effects of NPY_1–36 and PYY_1–36 are similar, we chose to examine NPY_1–36 as representative of the PP-fold family. Also, we used [³H]thymidine incorporation as an index of cell proliferation because of the large number of experiments conducted using this protocol, and measurement of [³H]thymidine incorporation can be performed rapidly.

In SHR PGVSMCs, NPY_1–36 (10 nM) increased [³H]thymidine incorporation, and this response was attenuated by pretreatment of PGVSMCs with pertussis toxin [100 ng/ml; inhibitor of G_i-proteins (59); Fig. 6A], U73122 [10 μM; inhibitor of phospholipase C (7); Fig. 6B], and GF109203X [10 μM; inhibitor of PKC (29); Fig. 6C] The conclusion that pertussis toxin, U73122 and GF109203X attenuated the ability of NPY_1–36 to increase [³H]thymidine incorporation was supported by the highly significant interaction terms in the 2F-ANOVAs (NPY_1–36 × pertussis toxin, P < 0.0001; NPY_1–36 × U73122, P = 0.0010; NPY_1–36 × GF109203X, P < 0.0001). These findings are consistent with the concept that in SHR PGVSMCs RACK1, by organizing the G_i/phospholipase C/PKC pathway, participates in the proliferative response to PP-fold peptides.

Fig. 6.

Bar graphs illustrate the effects of neuropeptide Y_1–36 (NPY_1–36; 10 nmol/l) on [³H]thymidine incorporation in PGVSMCs from SHR in the absence and presence of pertussis toxin (A; 100 ng/ml), U73122 (B; 10 μmol/l), and GF109203X (C; 10 μmol/l). NPY_1–36 was coadministered with sitagliptin (1 μM) to block its metabolism and inactivation. P values in panels are from two-factor ANOVA, and letters above bars indicate significantly different (Fisher's LSD test) from group without inhibitor and without NPY_1–36 (a), group without inhibitor but with NPY_1–36 (b), group with inhibitor but without NPY_1–36 (c), or group with inhibitor and with NPY_1–36 (d). Values are expressed as means ± SE.

Effects of NPY_1–36 on [³H]thymidine incorporation in SHR PGVSMCs treated with inhibitors of signal transduction downstream of PKC.

PKC activates an array of downstream signal transduction pathways (either directly or indirectly), including phosphatidylinositol 3-kinases (PI3Ks; Ref. 52), extracellular signal-regulated kinases (ERKs; Ref. 61), mammalian target of rapamycin (mTOR; Refs. 58, 60), and src family kinases (40). Therefore, we also examined the effects of inhibitors of these signal transduction pathways on the ability of NPY_1–36 to increase [³H]thymidine incorporation. In SHR PGVSMCs, NPY_1–36 (10 nM) increased [³H]thymidine incorporation, and this response was attenuated by pretreatment of PGVSMCs with LY294002 [10 μM; inhibitor of PI3Ks (9); Fig. 7A], PD98059 [10 μM; inhibitor of activation of ERKs (1); Fig. 7B], rapamycin [0.2 μM; mTOR inhibitor (24); Fig. 7C], and PP1 [0.1 μM; src family kinase inhibitor (23); Fig. 7D]. The conclusion that LY294002, PD98059, rapamycin, and PPI attenuated the ability of NPY_1–36 to increase [³H]thymidine incorporation was supported by the highly significant interaction terms in the 2F-ANOVAs (NPY_1–36 × LY294002, P < 0.0001; NPY_1–36 × PD98059, P < 0.0001; NPY_1–36 × rapamycin, P < 0.0001; NPY_1–36 × PP1, P = 0.0024). These findings are consistent with the concept that RACK1, by organizing the G_i/phospholipase C/PKC pathway, participates in the proliferative response to PP-fold peptides via activation of downstream mediators of PKC.

Fig. 7.

Bar graphs illustrate the effects of neuropeptide Y_1–36 (NPY_1–36; 10 nmol/l) on [³H]thymidine incorporation in PGVSMCs from SHR in the absence and presence of LY294002 (A; 10 μmol/l), PD98059 (B; 10 μmol/l), rapamycin (C; 0.2 μmol/l), and PP1 (D; 0.1 μmol/l). NPY_1–36 was coadministered with sitagliptin (1 μM) to block its metabolism and inactivation. P values in panels are from two-factor ANOVA, and letters above bars indicate significantly different (Fisher's LSD test) from group without inhibitor and without NPY_1–36 (a), group without inhibitor but with NPY_1–36 (b), group with inhibitor but without NPY_1–36 (c), or group with inhibitor and with NPY_1–36 (d). Values are expressed as means ± SE.

Effects of NPY_1–36 on [³H]thymidine incorporation in SHR PGVSMCs treated with other inhibitors of signal transduction.

Importantly, not all inhibitors altered the ability of NPY_1–36 to increase [³H]thymidine incorporation. For example, neither diphenyleneiodonium [10 μM; inhibitor of NADPH oxidases (25)] nor SB203580 [10 μM; inhibitor p38 MAPK (12)] affected the response to NPY_1–36 (Fig. 8, A and B, respectively), and although Y27623 [10 μM; inhibitor of Rho-associated protein kinases (57)] statistically reduced the response to NPY_1–36, the attenuation was barely detectable (Fig. 8C).

Fig. 8.

Bar graphs illustrate the effects of NPY_1–36 (10 nmol/l) on [³H]thymidine incorporation in PGVSMCs from SHR in the absence and presence of diphenyleneiodonium (A; DPI, 10 μmol/l), SB203580 (B; 10 μmol/l), and Y27623 (C; 10 μmol/l). NPY_1–36 was coadministered with sitagliptin (1 μM) to block its metabolism and inactivation. P values in panels are from two-factor ANOVA, and letters above bars indicate significantly different (Fisher's LSD test) from group without inhibitor and without NPY_1–36 (a), group without inhibitor but with NPY_1–36 (b), group with inhibitor but without NPY_1–36 (c), or group with inhibitor and with NPY_1–36 (d). Values are expressed as means ± SE.

DISCUSSION

As demonstrated previously (26) and as confirmed in this study, the Y₁ receptor agonists NPY_1–36 and PYY_1–36 stimulate proliferation of SHR PGVSMCs yet do not stimulate proliferation of WKY PGVSMCs. In the present study, we focused our attention on the possible role of RACK1 as a cause for the greater effects of Y₁-receptor activation on proliferation of SHR vs. WKY PGVSMCs. The rationale for this hypothesis was that 1) Y₁ receptors signal via G_i (38); 2) G_i releases βγ subunits (46); 3) βγ subunits are well known to activate phospholipase C (46); 4) the classical effect of phospholipase C is to activate PKC via DAG and calcium (42); 5) the scaffolding protein RACK1 is well known to scaffold and localize/organize all of these signal transduction components [βγ subunits, phospholipase C and PKC (10, 49)]; and 6) our previous studies suggest a role for RACK1 in regulating the proliferation of PGVSMCs (11). The current study corroborates a role for RACK1 with regard to participating in the differential response of SHR vs. WKY PGVSMCs to Y₁ receptor agonists, and this is a novel finding.

We reasoned that increased localization of RACK1 to cell membranes could be involved in the greater response of SHR vs. WKY PGVSMCs to the proliferative effects of Y₁-receptor activation. This reasoning was based on the concept that activation of phospholipase C by βγ subunits occurs mainly in the membrane domain, and localization of PKC is known to facilitate the biological effects of PKC (42). Thus, increased localization of RACK1 to cell membranes could be involved in the greater response of SHR vs. WKY PGVSMCs to the proliferative effects of Y₁-receptor activation. Another reason to entertain the hypothesis that RACK1 localization is important is that our previous study shows that in PGVSMCs, RACK1 binds to the inositol-1,4,5-trisphosphate receptor and enhances the release of calcium from the sarcoplasmic reticulum (11), and intracellular calcium activates PKC (42).

A novel finding of the present study is that RACK1 levels in the membrane fraction are 2.5-fold higher in SHR PGVSMCs compared with WKY PGVSMCs. This is in marked contrast to the equal levels of RACK1 in the nuclear and cytosolic fractions of SHR vs. WKY PGVSMCs. Although RACK1 levels are also higher in the cytoskeletal fraction of SHR PGVSMCs, the difference between SHR and WKY PGVSMCs in this regard is not as striking compared with the increased RACK1 levels in the membrane fraction. Therefore, it appears that RACK1 expression in SHR PGVSMCs is increased in precisely the location to most efficiently influence cell proliferation.

Besides using identical amounts of total protein in our Western blot experiments, we also employed β-actin as a loading control. Although β-actin is well known to exist in the cytosolic and membrane compartments, β-actin is also connected to the cytoskeletal compartment (50) and is transported into the nucleus (14). Therefore, employing β-actin as a loading control allows for a uniform method of normalizing protein expression across cell types and between compartments. The levels of β-actin did not differ between SHR vs. WKY PGVSMCs, thus indicating that β-actin served as a reliable loading control to normalize between strains.

Our molecular studies corroborate the hypothesis that RACK1 importantly contributes to the sensitivity of SHR PGVSMCs to the proliferative enhancing effects of Y₁-receptor activation. In this regard, siRNA-based knockdown of RACK1 abolishes the ability of Y₁-receptor activation to enhance proliferation. Corresponding experiments were not performed in WKY PGVSMCs because in these cells there was no response to either NPY_1–36 or PYY_1–36 and, therefore, no response to inhibit with siRNA knockdown of RACK1.

In the current study, we used a RACK1 plasmid to determine whether overexpression of RACK1 in WKY PGVSMCs could reproduce the SHR phenotype with regard to the proliferative response to Y₁-receptor activation. Importantly, overexpression of RACK1 in WKY PGVSMCs increases the proliferation rate of WKY PGVSMCs and renders WKY PGVSMCs sensitive to the proliferative effects of Y₁-receptor activation. In control SHR PGVSMCs Y₁-receptor activation increases proliferation by 29%, and in WKY PGVSMCs pretreated with RACK1 plasmid, Y₁-receptor activation increases proliferation by 32%. Therefore, pretreatment with RACK1 plasmid reproduces the SHR phenotype with respect to sensitivity to the proliferative effects of Y₁-receptor activation.

If increased localization of RACK1 to membranes is the main reason that SHR PGVSMCs are more responsive to the proliferative effects of Y₁-receptor activation, then inhibition of the βγ/phospholipase C/PKC pathway also should attenuate the ability of Y₁-receptor activation to increase the rate of proliferation of SHR PGVSMCs. The results of the current study are in agreement with this prediction. Blocking the βγ/phospholipase C/PKC pathway at the level of any of these three components significantly attenuates the proproliferative effects of Y₁-receptor activation in SHR PGVSMCs. Thus, the involvement of the βγ/phospholipase C/PKC pathway is entirely consistent with what is known about RACK1 scaffolding. Moreover, blocking some key downstream mediators of PKC also attenuates the proliferative effects of Y₁-receptor activation in SHR PGVSMCs. Importantly, not all pathway inhibitors were effective in this regard, which suggests that the efficacy of those that were effective was specific. It is important to note that corresponding inhibitor experiments were not performed in WKY PGVSMCs because in these cells, there was no response to NPY_1–36 and, therefore, no response to inhibit.

Taken together, our results support the model illustrated in Fig. 9. In SHR PGVSMCs, Y₁-receptor activation generates the release of βγ subunits from G_i proteins and facilitates the activation of the βγ/phospholipase C/PKC pathway. Increased localization of RACK1 to membranes in SHR PGVSMCs enhances activation of the βγ/phospholipase C/PKC pathway leading to an enhanced proliferative response to Y₁-receptor activation. Several downstream mediators are also involved, including ERKs, PI3Ks, src family kinases, and mTOR, but not including ROCK, p38 MAPKs, or NADPH oxidases.

Fig. 9.

Summary of signaling pathway by which PP-fold peptides increase cell proliferation. Components in green circles indicate sites of interventions (noted in red next to the green circles) conducted in this study. DPPIV, dipeptidyl peptidase IV; NPY_1–36, neuropeptide Y_1–36; PYY_1–36, peptide YY_1–36; NPY_3–36, neuropeptide Y_3–36; PYY_3–36, peptide YY_3–36; Gi, inhibitory G-protein; βγ, βγ subunits of G protein; PLC, phospholipase C; PKC, protein kinase C; DAG, diacyglycerol; IP₃, inositol trisphosphate; Ca²⁺, calcium; RACK1, receptor for activated C kinase 1; ERKs, extracellular-signal-regulated kinases; PI3Ks, phosphatidylinositol 3-kinases; DPI, diphenyleneiodonium; SFKs, src family kinases; mTOR, mammalian target of rapamycin; ROCK, Rho-associated protein kinase; p38 MAPKs, p38 mitogen-activated protein kinases; NADPH oxidase, nicotinamide adenine dinucleotide phosphate oxidases.

What is the reason for the difference in the subcellular distributions of RACK1 between SHR and WKY PGVSMCs? The answer to this question is unknown but worth exploring because the mechanism of differential RACK1 localization could provide insights into the molecular mechanisms of genetic hypertension. In this regard, Liu et al. (33) recently identified Ser-146 in the 4th WD domain of RACK1 as a phosphorylation site that results in dimerization of RACK1 followed by binding of the RACK1 dimer to a number of other proteins (33). It is conceivable, therefore, that the phosphorylation state of RACK1 determines the localization of RACK1. Another nonmutually exclusive possibility is that some of the binding partners of RACK1 are differentially localized to membranes in SHR PGVSMCs.

Can the findings in the present study be applied to other cell types that participate in the pathophysiology of hypertension, such as smooth muscle cells in different vascular beds or mesangial cells? This is also an important question, and the answer is likely, yes, because recent studies show that RACK1 regulates proliferation of glomerular mesangial cells (11).

What is the clinical significance of the current findings? DPPIV metabolizes incretin hormones, such as gastric inhibitory polypeptide and glucagon-like peptide-1. Consequently, DPPIV inhibitors raise circulating levels of incretin hormones and, thereby, increase insulin release, which results in sustained reductions in HbA_1c in Type 2 diabetics (5, 21). However, because there are at least 35 peptide substrates for DPPIV (20, 37), inhibition of DPPIV increases levels of many other biologically active peptides, in addition to incretins, and this may result in long-term consequences. For example, studies by Brown et al. (8) demonstrate that DPPIV inhibitors increase the risk of angioedema in patients treated with angiotensin I-converting enzyme inhibitors, likely because of blockade of substance P metabolism. Because patients with Type 2 diabetes, particular those with the metabolic syndrome, have increased renal sympathetic tone (16, 17) and often ingest fatty meals (18, 36, 45, 47), they likely would have high renal levels of both NPY_1–36 and PYY_1–36. In this regard, although NPY_1–36 and PYY_1–36 are potent agonists of Y₁ receptors (6, 38), DPPIV efficiently converts PYY_1–36 to PYY_3–36 and NPY_1–36 to NPY_3–36 by cleaving two amino acids from the N-termini of PYY_1–36 and NPY_1–36 (35, 37) (Fig. 9). PYY_3–36 and NPY_3–36 are inactive at Y₁ receptors but are selective Y₂-receptor agonists (6, 38). Thus, DPPIV inhibitors chronically increase Y₁-receptor activation. DPPIV inhibitors are becoming widely employed to treat patients with Type 2 diabetes, many of whom have hypertension, vascular disease, and renal diseases as comorbidities. Studies by Kirino et al. (27) show that in DPPIV-deficient rats, more so than in DPPIV-expressing rats, streptozotocin-induced diabetes profoundly and chronically reduces creatinine clearance, and case reports are beginning to link DPPIV inhibitors to renal impairment in patients with Type 2 diabetes (30). These findings suggest that in some patients, DPPIV inhibitors may in the long term adversely influence renal structure and function. The present study suggests that the mechanism by which DPPIV inhibitors adversely affect the kidney may involve the βγ/phospholipase C/PKC pathway and that patients with increased membrane localization of RACK1 could be most at risk. Our results also suggest that inhibitors of RACK1 could be useful to prevent adverse renal effects of DPPIV inhibitors, could be antihypertensive, and could prevent or treat hypertension-induced or diabetes-induced nephropathy. Finally, some studies implicate RACK1 in the pathology of cancer (31, 41, 48), and a recent analysis suggests that patients with hypertension are at increased risk of cancer (51). It is conceivable, although speculative, that inhibitors of RACK1 could prevent the adverse effects of DPPIV inhibitors, could lower arterial blood pressure, and could decrease the risk of cancer.

GRANTS

The work was supported by the National Institutes of Health Grants HL-109002, DK-091190, HL-069846, DK-068575, and DK-079307.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: D.C., X.Z., and E.K.J. conception and design of research; D.C., X.Z., and D.G.G. performed experiments; D.C., X.Z., D.G.G., and E.K.J. analyzed data; D.C., X.Z., and E.K.J. interpreted results of experiments; D.C., X.Z., and E.K.J. prepared figures; D.C., X.Z., and E.K.J. drafted manuscript; D.C., X.Z., D.G.G., and E.K.J. edited and revised manuscript; D.C., X.Z., D.G.G., and E.K.J. approved final version of manuscript.